1

Expression and Secretion of Recombinant

Ovine Somatotropin in

Escherichia coli

A THESIS SUBMITTED TO

THE UNIVERSITY OF THE PUNJAB

IN FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY IN

BIOLOGICAL SCIENCES

By

Faiza Gul

School of Biological Sciences

University of the Punjab

Lahore, Pakistan

2012

i

In the Name of Allah, the Merciful, the Compassionate.

Read! In the Name of Thy Lord. [Quran 96:1]

ii

Dedicated to My Dear loving Parents

Mr & Mrs M.Ajmal Khan

iii

CERTIFICATE

This is to certify that the research work described in this thesis is the original work of Faiza

Gul and has been carried out under my supervision. I have personally gone through all the

data/results/materials reported in the manuscript and certify their correctness/authenticity. I further

certify that the material included in this thesis have not been used in part or full in a manuscript

already submitted or in the process of submission in partial/complete fulfillment of the award of any

other degree from any other institution. I also certify that the thesis has been prepared under my

supervision according to prescribed format and I endorse its evaluation for the award of Ph.D. degree

through the official procedures of the University.

(Prof. Dr. M. Waheed Akhtar) Research Supervisor

iv

ACKNOWLEDGEMENT

All Praise Allah Subhanahu wa Tala, Master of this Universe, and Master of

all mankind, truly without Him, man is at loss. Without His guidance there is

no light, without His protection there is no sanctuary and without His

Knowledge there is no real knowledge. Without doubt, one can not praise Al

Khaliq enough. He says “And I did not create the jinn and mankind except to

worship Me.” [Quran 51:56] Without doubt, all praises and thanks are to

Allah, the Ever-Lasting.

The writing of this dissertation has been the most significant academic

challenge I have had to face. Without the support, patience and guidance of

the following people, this study would not have been completed. It is to them

that I owe my deepest gratitude.

Above all, my sincere gratitude to my honourable supervisor Prof. Dr. M.

Waheed Akhtar, whose unsurpassed knowledge and untiring guidance have

seen this thesis through. I have heartfelt gratefulness to him for giving me the

opportunity to work in his laboratory. His profound insight, patience,

dynamic supervision and encouraging approach have granted me the

confidence to face the challenges of the Ph.D. I’m also thankful to our

Director General Dr. M. Akhtar and all the directors of School of Biological

Sciences, University of the Punjab, Lahore for their invaluable help and

guidance .

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I am deeply indebted to Dr. Saima Sadaf for her assistance and support. I also

appreciate the role Dr.Mahjabeen Saleem and Dr. Farhat Zaheer has played

in my PhD, for their moral support and help whenever I faced a problem. I

am also thankful to Dr.Naeem Rasheed for his valuable guidence specially for

primer designing.

I am thankful to (late) Dr. Mustaq Kaderbhai whose critical guidance,

suggestions and encouragement have been invaluable. It has been a great

pleasure to have guidence from such wonderful personality. His sudden

passing left me in great sorrow but his wife Dr.Naheed Kaderbhai supported

me and its all her continuous effort that I could finish the last part of my

work.as her numerous educational discussions, feedback and suggestions have

played a fundamental part in shaping my views, knowledge and

understanding. It has also been a pleasure to have worked with such

wonderful people. Sharing of ideas, thoughts and suggestions have vastly

contributed to the stance I have taken in this thesis. I thank both of them for

being a reflective and critical contributor.

My dear parents, who made me the very person I am, instilling core values of

hard-work, perseverance, resilience and patience - this PhD is really about

them and the fruits of their effort – for that I am forever indebted. My deepest

gratitude is also to my Ami Jan (Mrs.Neelam Durrani) for the support,

encouragement and comfort for when I needed it most. My loving husband

vi

Ali, without whose encouragement support and love ,this thesis could never

have continued – for the kind words, care and confidence in me, for this, my

mere expression of thanks does not suffice.

My brothers and sisters and their families have given me their unequivocal

support throughout. Rizwan, Arjumand, Rehan and Mehran, their enduring

comfort during all times is forever appreciated. The joyous times spent

together are the best memories. Special thanks to all my cousins, elders and

my in-laws in particular my new sister Sadia Durrani. Finally, to my late

dear grandparents especially my Daadi Jan, Masooma Khanum and late

father-in-law Mahmood Ahmed Khan Durrani, I know they would have been

proud of my accomplishments – thank you for the dreams and aspirations that

have enabled this work to have materialised.

I would also like to thank my dear friends, Roquyya “My twin” Gul, Sadaf

Zaidi, Nadia Azhar, Dr. Mahjabeen Saleem, Dr. Saadia Shazad Alam, Gul

Sher Muhammad Tahir , who have shown me tremendous support, above all,

they have been true friends, standing by me in all phases of my education and

career. I can never forget the tea time and walks on the jogging track.

Penultimately, I’d like to thank my little baby, Muhammad Ahmed who

taught me the skill of multi-tasking between nappy changes, feeds and my

write-up. I hope he too has learned something here.I am also appreciative of

all my other laboratory colleagues specially Annie, Hooria, Hina Farheen,

vii

Deeba, Altaf and Sajjad for the nice educational and fun time discussions. I

would also like to thank to all the technicians for their aid in anything I

needed. I am especially thankful to Muhammad “M.D. Sahib” Deen, Irfan

Sahab and Afzal for their timely support in the process of thesis submission.

I would like to end my acknowledgement with a supplication:

“My Lord! Inspire me and bestow upon me the power and ability that I may

be grateful for Your Favours which You have bestowed on me and on my

parents, and that I may do righteous good deeds that will please You and

admit me by Your Mercy among Your righteous slaves” (Quran 27:19)

FAIZA GUL

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SUMMARY

The current study involves cloning, sequence analysis, expression, secretion, purification and

different factors influencing the secretion of ovine growth hormone (oGH) gene isolated from

local ovine breed (Lohi). On the basis of conserved sequences, two forward and one reverse

primers were designed for the amplification of oGH gene. The forward primers contained NdeI,

NcoI restriction sites whereas the reverse primer contained a BamHI site at their 5’ end. Total

RNA was isolated from pituitary gland of Lohi by using Guanidium-thiocyanate-chloroform

extraction method. cDNA was synthesized by RT-PCR using gene specific primers. Moreover,

genomic DNA was isolated from the blood sample of Lohi and was amplified by using four sets

of primers designed on the basis of conserved sequence of the ovine growth hormone (oGH)

gene. These were ligated into pTZ57R/T by the dT. dA tailing technique and used to transform

into E. coli DH5α. The sequences of the DNA obtained from multiple colonies were compared

with already published ovine GH gene sequence using multiple sequence alignment software

“Clustal W”. The sequence analysis revealed only one amino acid change when compared to

previously reported OaST (Ovis aries somatotropin) or oGH gene sequences of Indian and

Australian breeds. It showed 99% homologies with bubaline, bovine and 100 percent homology

with caprine GH genes of the local breeds. The sequence of the GH of Lohi was submitted to

"Data bank of Japan" which bears an accession number AB244790.

In the present study, we report secretion of recombinant oGH into the periplasmic space and

inner membrane of E. coli under the influence of variant signal sequences. For periplasmic

translocation the recombinant proteins were expressed under the influence of pelB leader

sequence of pET 22b vector. The effect of different factors i.e., glycerol in the medium, use of E

.coli strain BL21 DE3 and pLys S ,chemical chaperon (ZnCl2) and IPTG concentration were

studied to enhance the expression while osmotic shock conditions were also optimized and

ix

studied the effect of glycerol and ZnCl2 concentration on the release of oGH by using freeze

thaw method. Best result of 22% expressed roGH on 12% SDS-PAGE was observed at 20M

(final concentration) IPTG after 4 hrs of fermentation at 370C in LB modified medium with

50µM ZnCl2 in BL21DE3 E. coli strain. The optimized freeze thaw method including 25%

glycerol with 50µM ZnCl2 enhanced the relase of oGH upto 24% in the periplasmic space of E.

coli. The oGH thus found was further purified by FPLC and authenticated by Western blot

analysis. Although the recovery of oGH was enhanced but still there was a need to enhance the

production of accurate size (22 kDa) growth hormone which was bit higher (25 kDa) by using

pelB leader sequence.

For this purpose different signal peptides i.e., DsbA, STII and natural oGH signal peptide were

utilized. Amongst the signal sequences the DsbA signal sequence was found to exhibit the best

expression, size and secretion of oGH into the inner membrane of E. coli. We further studied the

expression of oGH and targeting to the inner membrane using signal sequence (DsbA) in E. coli

cell. Factors such as temperature, IPTG induction, expression conditions were studied and

showed diverse optical density with different media compositions. The optimum expression level

of oGH in terrific broth medium was at 25ºC on induction with 20μM IPTG in early logarithmic

phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs

revealed the translocation of oGH to the inner membrane of E. coli with DsbA signal sequence

at the N terminus of roGH. The protein was easily solublized by 40% acetonitrile with ~90%

purity and was identified by Western blot and analysis on MALDI/TOF confirmed a size of

21059Da. Relatively high soluble protein yield of 65.3gm/L of oGH was obtained. The

biological function of oGH was confirmed by HeLa cell line proliferation. It was observed that

DsbA signal sequence on the basis of its hydrophobicity gave best results of 22kDa protein in

membrane bounded form as compared to pelB and reference native signal sequence of oGH

which resulted in 25kDa oGH secreted mainly into cytoplasm.

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Despite of cost effective single step purification we encountered a problem with low yield. We

developed a novel strategy for the high yield of functional recombinant ovine growth hormone

(roGH) directed to the inner membrane of E. coli. In order to enhance the yield of soluble

fraction, bacterial cells were grown under osmotic stress (4% NaCl in terrific broth medium) and

effect of compatible solutes (sorbitol, glycine betine, glycylglycine and mannitol) were studied

on the soluble expression of roGH. Other factors; temperature, induction time, induction by

IPTG and lactose were also studied. It was observed that fermentation of roGH construct with

DsbAss was best achieved with 0.6M mannitol, 50μM ZnCl2, 50mM glycylglycine at the time of

induction with 50μM IPTG in the early logarithmic phase at OD600 ~3.10 in TB medium at 25ºC

in shaking flask culture at 150rpm. These optimized conditions resulted in very high expression

~32% of soluble roGH which was recovered by ultra centrifugation (density centrifugation) from

the inner membrane of E. coli. The unbelievably high yield, 443mg/L was obtained as compared

from previos yield. The roGH was confirmed by Western blot analysis .

Furthermore the effect of amino acid substitution in the tripartite structure of DsbA signal

sequence (DsbAss) on co-translation of recombinant oGH in E. coli was studied. Six amongst

the eight constructs were designed on the basis of increasing hydrophobicity in H domain of

DsbA signal sequence to make it more efficient for the translocation of oGH through SRP (signal

recognition particle) mechanism. For this purpose all the alanines in the hydrophobic domain of

DsbA signal sequence were replaced by Isoleucine one by one, while lysine in the N terminal

and serine in the C-terminal regions were substituted by arginine and cysteine respectively. The

substitution of arginine in the N-terminal resulted in very low expression and secretion while

cysteine substitution in the C region totally impaired the expression and secretion of the

recombinant protein. it was observed that not only the hydrophobicity but the position of amino

acid in the hydrophobic core also effects the cleavage of signal sequence from recombinant

product.

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The substitution of alanine with the isoleucine residue in H domain of DsbA signal sequence

resulted in; (a) at position 11 with respect to signal peptidase site in the H domain impaired the

correct processing of oGH protein while (b) isoleucine at position 9 resulted in correctly

processed recombinant oGH protein in the inner membrane.The results showed that the

replacement of alanine amino acid at position 11 with reference to signal peptidase site in the

hydrophobic core of the DsbA ss interferes with the binding of DsbA ss hydrophobic region to

Ffh protein of SRP. This resulted in weak or no binding of Ffh with DsbA ss and consequently

oGH protein was localised in the cytoplasmic fraction rather than membrane. Thus, the gene

mutation from alanine residue to isoleucine specifically at position 11 with respect to signal

peptidase site changed the whole mechanism of protein translocation through DsbA ss. It was

hypothesized that alanine at position number 11 with respect to the signal peptidase site is crucial

for SRP routing of recombinant proteins .

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Table of Contents

INTRODUCTION & LITERATURE REVIEW ............................................................................................ 1

1.1 Somatotropin (Growth Hormone) ..................................................................................................................................... 2

1.2 Secretion of recombinant protein in E.coli ....................................................................................................................... 3

1.3 Ovine breed of Pakistan .................................................................................................................................................... 5

1.4 Impact of our study on the economy of Pakistan ............................................................................................................. 6

1.5 Review of Literature .......................................................................................................................................................... 7

1.5.1 Structural and functional aspects of GH ........................................................................................................... 7

1.5.2 Cloning and expression of GH in bacterial systems ..................................................................................... 9

1.5.3 Secretion of growth hormone in E.coli ...................................................................................................... 12

1.5.3.1 Type I secretion systems ................................................................................................................ 14

1.5.3.2 Type II secretion Mechanism ........................................................................................................ 14

1.5.3.3 SecB-dependent pathway ............................................................................................................. 14

1.5.4 Signal sequences ............................................................................................................................................ 18

1.5.5 Expression and Purification of secreted protein in E.coli. ............................................................................... 19

1.5.6. Advantages of getting soluble proteins ......................................................................................................... 24

1.6 AIMS AND OBJECTIVES .................................................................................................................................................... 26

MATERIALS AND METHODS.................................................................................................................. 28

2.1 Sample collection and storage ................................................................................................................................ 29

2.2 Chemicals and kits ................................................................................................................................................... 29

2.3 Isolation of total RNA from pituitary sample ......................................................................................................... 30

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2.4 Formaldehyde agarose gel electrophoresis ............................................................................................................ 31

2.5 cDNA Synthesis ........................................................................................................................................................ 32

2.5.1 Primer designing.............................................................................................................................................. 32

2.5.2 Reverse transcription (RT) ............................................................................................................................... 32

2.5.3 PCR Amplification ........................................................................................................................................... 33

2.6 DNA extraction from agarose gel .................................................................................................................................... 34

2.6.1 Purification of PCR product ............................................................................................................................. 34

2.7 Cloning in pTZ57R/T vector ............................................................................................................................................. 35

2.7.1 T/A cloning Kit method.................................................................................................................................... 35

2.7.2 Preparation of competent cells and transformation....................................................................................... 37

2.8 Colony PCR ...................................................................................................................................................................... 38

2.9 Sequence analysis ............................................................................................................................................................ 38

2.9.1 Q/A prep spin miniprep kit method ................................................................................................................ 38

2.9.2 Analysis of Full-Length ST Gene....................................................................................................................... 40

2.9.2.1 Extraction of genomic DNA ............................................................................................................ 40

2.9.2.2 PCR amplification of GH gene ...................................................................................................... 42

2.9.2.3 Sequencing reaction....................................................................................................................... 42

2.10 Bioinformatics tools for sequence analysis................................................................................................................... 43

2.11 Mini-preparation of plasmid DNA ................................................................................................................................. 43

2.12 Restriction analysis of pTZ-oGH clones ......................................................................................................................... 44

2.13 Restriction analysis of pET22b (+) ................................................................................................................................. 45

2.14 Ligation and transformation in DH5α and BL21 Codon + strains ................................................................................. 45

2.15 Expression of poGH clones ............................................................................................................................................ 46

xiv

2.16 SDS-Polyacrylamide Gel Electrophoresis (PAGE) .......................................................................................................... 47

2.17 Western transfer and immunoblot analysis ................................................................................................................. 49

2.18 Protein estimation ......................................................................................................................................................... 50

2.19 Primer designing for translocation of Ovine ST gene into periplasmic space.............................................................. 50

2.20 Subcellular fractionation of oGH ................................................................................................................................... 52

2.20.1 FPLC chromatography ................................................................................................................................... 54

2.20.2 MALDI-TOF .................................................................................................................................................... 54

2.21 Biological activity assessment assay ............................................................................................................................. 54

RESULTS ..................................................................................................................................................... 57

3.1 Genetic Analysis of oGH gene.......................................................................................................................................... 58

3.1.1 Extraction of genomic DNA ............................................................................................................................. 58

3.1.2 PCR amplification of oGH gene ....................................................................................................................... 58

3.1.3 Sequence analysis of oGH ............................................................................................................................... 59

3.1.3.1 Sequence comparison of oGH at amino acid level......................................................................... 61

3.1.3.2 Comparison of oGH gene at Nucleotide level ................................................................................ 64

3.1.4 Secondary structure analysis of oGH .............................................................................................................. 66

3.1.5 Hydropathy profile of oGH .............................................................................................................................. 67

3.1.6 Three Dimensional structure of oGH............................................................................................................... 68

3.2 cDNA synthesis , cloning and Periplasmic Expression of oGH ...................................................................................... 69

3.2.1 Isolation and purity of total RNA ..................................................................................................................... 69

3.2.2 RT-PCR amplification of cDNA ........................................................................................................................ 70

3.2.3 T/A cloning of oGH ......................................................................................................................................... 71

3.3 Expression of poGH ......................................................................................................................................................... 72

3.3.1 Restriction analysis of pTZ-oGH ...................................................................................................................... 72

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3.3.2 Cloning in pET22 b ........................................................................................................................................... 73

3.3.3 colony PCR of poGH......................................................................................................................................... 73

3.3.4 Shake flask fermentation of poGH-1 construct .............................................................................................. 75

3.4 Periplasmic expression of oGH ........................................................................................................................................ 75

3.4.1 Expression of poGH-2 ...................................................................................................................................... 76

3.4.2 Effect of different factors on the expression of oGH ...................................................................................... 77

3.4.2.1 Effect of ZnCl2................................................................................................................................. 77

3.4.2.2 Effect of IPTG concentration .......................................................................................................... 77

3.4.2.3 Effect of Ecoli Strain on expression of ovine growth hormone...................................................... 78

3.4.2.4 Optimization of somotic shock conditions ..................................................................................... 79

3.4.2.5 Effect of Glycerol ............................................................................................................................ 80

3.4.2 Purification of poGH-2..................................................................................................................................... 81

3.4.3 FPLC chromatography ..................................................................................................................................... 83

3.5 Effect of (DsbA,ST-11 & native oGH signal sequence ) on the expression & secretion of oGH .................................... 84

3.5.1 Primer designed for the constructs poGH-3,4 &5 ........................................................................................... 84

3.5.2 PCR amplification ............................................................................................................................................ 84

3.5.3 T/A cloning and construction of expression plasmid poGH-3,4,5 ................................................................... 85

3.5.4 Transformation and selection of high expression strains ............................................................................... 86

3.5.5 Expression of poGH-3,4 and 5 ......................................................................................................................... 87

3.5.5.1 Subcellular fractionation of poGH-3-5 constructs ......................................................................... 88

3.5.6 Computational analysis of signal sequences of poGH-2,3,4& 5 constructs .................................................... 91

3.6 Effect of medium composition on expression of poGH-3 ............................................................................................... 94

3.6.1 Effect of LB,TB & M9NG medium on the expression of poGH-3 ..................................................................... 94

3.6. 2 Effect of temperature on poGH3 construct ................................................................................................... 96

3.6.3 Effect of induction time and IPTG concentration on poGH3 construct........................................................... 96

3.7 Enhanced production of roGH ......................................................................................................................................... 97

3.7.1 Effect of compatible solute on the expression of poGH-3 construct .............................................................. 99

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3.7.1.1 Optimization of soluble roGH expression using compatible solutes(Glycylglycine , glycine

betaine,sorbitol and Mannitol ................................................................................................................... 99

3.7.2 Production of soluble roGH in TBC optimized medium................................................................................ 101

3.7.2.1 Effect of temperature .................................................................................................................. 102

3.7.2.2 Effect of IPTG and Lactose as an inducer .................................................................................... 103

3.7.2.3 Effect of induction time .............................................................................................................. 104

3.7.3 Subcellular fractionation of poGH-3 construct.............................................................................................. 104

3.8 Effect of amino acid alterations in DsbA signal sequence on poGH expression and secretion ................................... 107

3.8.1 PCR amplification and Cloning of pOaST varying constructs ......................................................................... 107

3.8.2 Construction of Expression plasmid poGH3-I-VIII ......................................................................................... 109

3.8.3 Expression of poGH-3-I-VIII ........................................................................................................................... 109

3.8.4 The expression of DsbA ss constructs with substitution of alanine with isoleucine in the H domain .......... 110

3.8.5 DsbA ss constructs with substitution of serine with cysteine in the C domain ............................................. 113

3.8.6 DsbA ss constructs with substitution of lysine with arginine in the N domain ............................................. 114

3.8.7 Purification of oGH from poGH-3-II construct............................................................................................... 115

3.8.8 MALDI TOF analysis of purified ovine growth hormone ............................................................................... 116

3.8.9 Biological activity assessment assay............................................................................................................. 117

3.8.10 Computational analysis of pOaST-3a-g constructs ...................................................................................... 118

DISCUSSION ............................................................................................................................................. 121

4.1 Characterization of oGH gene........................................................................................................................................ 122

4.2 periplasmic Expression of roGH..................................................................................................................................... 124

4.3 Secretion of oGH into the inner membrane of E.Coli. .................................................................................................. 128

4.4 Effect of medium composition on the expression and secretion of oGH in E.coli ....................................................... 131

4.5 Effect of mutation in DsbA signal sequence on the expression and secretion of OaST .............................................. 135

4.6 Purification and Biological activity Assessment............................................................................................................ 138

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4.7 Conclusion ...................................................................................................................................................................... 138

REFERENCES ........................................................................................................................................... 140

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TABLE OF FIGURES

FIGURE 1. SECONDARY STRUCTURE OF GROWTH HORMONE ........................................................... 2

FIGURE 2.SECRETION OF RECOMBINANT PROTEINS IN E.COLI. ......................................................... 3

FIGURE 3. (LOHI) OVINE BREED OF PAKISTAN....................................................................................... 5

FIGURE 4.RESTRICTION MAP,SEQUENCE AND MULTIPLE CLONING SITES OF PET 22B(+). ......... 46

FIGURE 5.GENOMIC DNA OF OGH ISOLATED FROM THE BLOOD SAMPLE OF LOCAL OVINE

BREED LOHI ................................................................................................................................... 58

FIGURE 6. PCR AMPLIFICATION ON 1% AGAROSE GEL. ..................................................................... 59

FIGURE 7. NUCLEOTIDE SEQUENCE OF OGH. ....................................................................................... 60

FIGURE 8.AMINO ACID SEQUENCE OF OGH .......................................................................................... 60

FIGURE 9.AMINO ACID SEQUENCE OF OGH. ......................................................................................... 61

FIGURE 10.COMPARISON OF GROWTH HORMONES OF OVINE CAPRICORN AND BUBALINE ..... 62

FIGURE 11,AMINO ACID SEQUENCE COMPARISON. ............................................................................ 62

FIGURE 12.COMPARISON OF OGH WITH DIFFERENT SPECIES OF CLASS MAMMALIA ................. 64

FIGURE 13.NUCLEOTIDE SEQUUENCE ALIGNMEN .............................................................................. 65

FIGURE 14OVINE GROWTH HORMONE. SECONDARY STRUCTURE .................................................. 66

FIGURE 15.THE HYDROPATHY PLOT OF OGH. ...................................................................................... 67

FIGURE 16.3D STRUCTURE OF OVINE GROWTH HORMONE............................................................... 68

FIGURE 17.ABSORPTION SPECTRA OF EXTRACTED RNA. .................................................................. 69

FIGURE 18.FORMALDEHYDE AGAROSE GEL OF RNA ......................................................................... 70

FIGURE 19.ANALYSIS OF THE RT-PCR. ANALYSIS OF THE RT-PCR AMPLIFIED PRODUCT

RESOLVED ON 1% AGAROSE GEL. LANE M, 1KB DNA LADDER USED AS MARKER; LANE ,

2, 3, 4 & 5 ~0.6KB AMPLIFIED PCR PRODUCTS. ......................................................................... 70

FIGURE 20.RESTRICTION MAP OF PTZ57R/T CLONING VECTOR ....................................................... 71

FIGURE 21.ANALYSIS OF COLONY PCR. ................................................................................................ 72

FIGURE 22.DOUBLE DIGESTION OF PTZ-OGH-1.. .................................................................................. 72

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FIGURE 23.CONSTRUCTION OF RECOMBINANT PLASMID POGH-1................................................... 73

FIGURE 24.COLONY PCR OF POGH-1....................................................................................................... 74

FIGURE 25.DOUBLE DIGESTION OF POGH-1. ......................................................................................... 74

FIGURE 26.SDS-PAGE ANALYSIS OF POGH-1 EXPRESSION................................................................. 75

FIGURE 27.CONSTRUCTION OF POGH-2 CONSTRUCT.......................................................................... 76

FIGURE 28.SDS-PAGE ANALYSIS OF POGH-2 EXPRESSION IN LB MEDIUM. .................................... 76

FIGURE 29.EFFECT OF ZNCL2................................................................................................................... 77

FIGURE 30,SDS-PAGE ANALYSIS OF EFFECT OF IPTG. ........................................................................ 78

FIGURE 31.EFFECT OF E.COLI STRAINS ON THE PERIPLASMIC EXPRESSION OF POGH-2. ........... 78

FIGURE 32. GRAPHICAL REPRESENTATION OF DIFFERENT OSMOTIC SHOCK CONDITIONS ON

OGH. ................................................................................................................................................. 79

FIGURE 33.SDS-PAGE ANALYSIS OF SUBCELLULAR FRACTIONS OF POGH. 2................................ 80

FIGURE 34. SDS-PAGE ANALYSIS OF POGH-2 IN LBMODIFIED MEDIUM. ........................................ 80

FIGURE 35. EFFECT OF GLYCEROL. ........................................................................................................ 81

FIGURE 36.SDS-PAGE ANALYSIS OF SUBCELLULAR FRACTIONS OF ROGH-2& WESTERN BLOT

ANALYSIS. ...................................................................................................................................... 82

FIGURE 37.FPLC PEAK OF PURIFIED OGH .............................................................................................. 83

FIGURE 38.AGAROSE GEL ANALYSIS OF PCR. ...................................................................................... 85

FIGURE 39.COLONY PCR ANALYSIS OF POGH-3-4-5. ........................................................................... 85

FIGURE 40.CONSTRUCTION OF EXPRESSION PLASMIC POGH-3,4&5 ................................................ 86

FIGURE 41.COLONY PCR ANALYSIS. ...................................................................................................... 86

FIGURE 42.DOUBLE DIGESTION OF RECOMBINANT CLONES. ........................................................... 87

FIGURE 43.SDS-PAGE ANALYSIS OF PROTEIN EXPRESSION OF CONSTRUCT POGH-3,4&5. ......... 88

FIGURE 44.SCHEMATIC REPRESENTATION OF SUBCELLULAR FRACTIONATION OF CELLS. ..... 89

FIGURE 45.SDS-PAGE ANALYSIS OF SUBCELLULAR FRACTIONATIONS OF POGH-3,4 & 5

CONSTRUCTSSDS PAGE . ............................................................................................................. 91

FIGURE 46.KYTEDOOLITTLE ANALYSIS OF HYDROPHOBICITY OF ALL FOUR SIGNAL

SEQUENCES. ................................................................................................................................... 92

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FIGURE 47.SECONDARY STRUCTURE ANALYSISOF POGH2,3,4&5.. .................................................. 93

FIGURE 48.SDS-PAGE ANALYSIS AND GRAPHICAL REPRESENTATION OF EFFECT OF MEDIUM

ON POGH-3 ...................................................................................................................................... 95

FIGURE 49.EFFECT OF TEMPERATURE,INDUCTION TIME AND IPTG CONCN. ON POGH-3. .......... 97

FIGURE 50. GROWTH OF POGH-3 IN DIFFERENT MEDIUM.................................................................. 98

FIGURE 51.GRAPHICAL REPRESENTATION OF THE EFFECT OF 2 SETS OF COMPATIBLE

SOLUTES ON THE GROWTH OF POGH-3 IN TB MEDIUM ....................................................... 100

FIGURE 52.EFFECT OF COMPATIBLE SOLUTES ON THE SOLUBLR EXPRESSION OF POGH-3 IN TB

MEDIUM . ...................................................................................................................................... 101

FIGURE 53.SDS-PAGE ANALYSIS OF OPTIMIZED COMPATIBLE SOLTE IN TB MEDIUM ON

EXPRESSION OF POGH-3............................................................................................................. 102

FIGURE 54.EFFECT OF TEMPERATURE.. ............................................................................................... 103

FIGURE 55.EFFECT OF IPTG AND LACTOSE.. ....................................................................................... 104

FIGURE 56.SUBCELLULAR FRACTIONATION OF POGH-3. ................................................................ 105

.FIGURE 57.PCR AMPLIFICATION OF POGH-3-I-VIII............................................................................ 108

FIGURE 58.COLONY PCR AND DOUBLE DIGESTION OF POGH-3-I-VIII. .......................................... 108

FIGURE 59.CONSTRUCTION OF RECOMBINANT PET FOR POGH-3-I-VIII CONSTRUCTS . ............ 109

FIGURE 60.SDS-PAGE ANALYSIS OF POGH-3-I-VIII CONSTRUCTS IN LB MEDIUM. ...................... 110

FIGURE 61.SDS-PAGE ANALYSIS OF POGH-3-II-VI&I ......................................................................... 112

.FIGURE 62.SDS-PAGE ANALYSIS OF POGH-3-III&V. .......................................................................... 113

FIGURE 63,SDS-PAGE ANALYIS OF POGH-3 VIII. ................................................................................ 114

FIGURE 64.SDS-PAGE ANALYSIS OF POGH-3-VII. ............................................................................... 114

FIGURE 65.SUBCELLULAR FRACTIONATION OF POGH-3II AND WESTERN BLOT ANALYSIS. ... 115

FIGURE 66.MALADI-TOF ANALYSIS OF PURIFIED OVINE GROWTH HORMONE ........................... 116

FIGURE 67.BIOLOGICAL ACTIVITY OF OGH IN THE PRESENCE OG HELA CELL LINES............... 117

FIGURE 68.MINNOU SERVER PREDICTION RESULTS. ........................................................................ 119

FIGURE 69.HOMOLOGY MODEL OF DSBA SS WITH ALTERED ALANINE. ...................................... 120

FIGURE 70.PHYLOGENETIC TREE OF OVINE GROWTH HORMONE ................................................. 124

xxi

FIGURE 71.MODEL REPRESENTING THE MECHANISM OF DSBA SIGNAL SEQUENCE WITH

ALTERED AMINO ACID WITH SRP MECHANISM.................................................................... 137

xxii

LIST OF TABLES

TABLE 1. SEQUENCE OF PRIMERS USED FOR CONSTRUCTION OF POGH-3-I-VIII PLASMIDS......................... 51

TABLE 2.PRIMERS DESIGNED FOR THE POGH-3,4 & 5 CONSTRUCTS ................................................................... 84

TABLE 3.HYDROPATHIES OF POGHCONSTRUCTS .................................................................................................... 92

TABLE 4.COMPOSITION OF DIFFERENT MEDIUMS USED ........................................................................................ 94

TABLE 5.EFFECT OF MEDIUM COMPOSITION ON PRODUCTION OF OGH............................................................ 96

TABLE 6. EFFECT OF COMPATIBLE SOLUTES INTB MEDIUM ON THE GROWTH OF POGH-3 ........................ 100

TABLE 7.EFFECT OF COMPATIBLE SOLUTE IN TB MEDIUM ON YIELD OF SOLUBLE OGH ........................... 106

TABLE 8.HYDROPATHY INDICES OF MODIFIED DSBA SS IN POGH-3-I-VIII CONSTRUCTS ........................... 118

xxiii

ABBREVIATIONS

APS Ammonium persulphate

oGH ovine growth hormone

BSA bovine serum albumin

CaCl2 Calcium chloride

ZnCl2 Zinc Chloride

cDNA complementary deoxy ribonucleic acid

DMEM Dulbecco’s Modified Eagle’s Medium

DMSO dimethyl sulfoxide

dNTP deoxyribonucleoside triphosphate

DTT dithiothreitol

EDTA ethylene diamine tetracetate

FBS fetal bovine serum

FPLC Fast protein liquid chromatography

GdCl Guanidinium chloride

GH growth hormone

hGH human growth hormone

HRP horse radish peroxidase

IPTG isopropylthio-β-galactoside

Kb kilo base pairs

kDa kilo dalton

LB Luria-Bertani

TB Terrific broth

xxiv

M-MuLV Molony Murine Leukemia Virus

OD Optical density

PBS phosphate buffer saline

PCR polymerase chain reaction

PEG polyethylene glycol

PMSF phenylmethyl sulfonyl fluoride

rcGH recombinant caprine growth hormone

rGH recombinant growth hormone

RNA ribonucleic acid

roGH recombinant ovine growth hormone

RT room temperature

SDS sodium dodecyl sulphate

Taq Thermus aquaticus

TEMED N, N, N’, N’ tetra methyl ethylene diamine

X-Gal 5-bromo-4-chloro-3-indolyl β-D-galactoside

ST Somatotropin

RP-HPLC Reversed phase high performance liquid chromatography

HPLC High performance liquid chromatography

TRI Trizol

FPLC Fast protein liquid chromatography

MALDI/TOF Matrix assisted laser desorption ionization/time of flight

xxv

1

Introduction & Literature Review

2

1.1 Somatotropin (Growth Hormone)

Somatotropin commonly known as Growth hormone (GH) a protein of 22 kDa, is naturally

synthesized as a pre-hormone with an extension of 26 amino acid signal peptide which directs

the release of GH into the blood stream (Paladini et al., 1983). GH consists of 190 or 191 amino

acids with two disulfide bridges.

Figure 1. Secondary structure of Growth hormone

Source;www.endotext.org

Pituitary growth hormone is a protein hormone which regulates somatic growth in most

vertebrates and has effects on various metabolic activities (Wallis, 1988). It is a polypeptide that

controls the differentiation, growth and metabolism of many cell types, and is secreted from the

hypophysis of all vertebrate species tested so far. Despite the overlapping evolutionary,

structural, immunological and biological properties, it is well-known that growth hormones

from distinct mammalian species have significant species-specific characteristics.

3

1.2 Secretion of recombinant protein in E.coli

Various animal growth hormones such as bovine (George et al., 1985; Klein et al., 1991) ovine

(Wallis and Wallis, 1989) and porcine (Vize and Wells, 1987) have already been produced

through recombinant DNA technology. E. coli has been used extensively for the expression and

production of recombinant proteins. In E. coli., proteins are synthesized in the cytoplasm, but

many must be targeted to different destinations within the cell to perform their ultimate

functions. Towards this end, a number of active systems exist which recognize proteins destined

for various destinations and catalyze their insertion or export to these specific locations. All these

routs, cytoplasm, periplasm or inner membrane and in the culture are shown in figure below.

Figure 2.secretion of recombinant proteins in E.coli.

Source; www.slideshare.net

The production of many recombinant proteins in the bacterial cytoplasm is frequently limited by

their tendency to form inclusion bodies. These inclusion bodies can, however, in some cases ease

the isolation of the recombinant proteins but may not yield functional renatured molecules.

Correctly folded, functional recombinant proteins with a required amino terminus can be

conveniently produced by means of secretion or export into the periplasmic space where there is

4

provision of a less harsh environment compared with that of the cytoplasm. Protein misfolding in

the periplasmic space can be countered by a slower and controlled folding rate imposed by the

signal sequence and polypeptide threading through the Sec translocon.

The availability in the periplasmic space of many of the essential post-translational modification

enzymes catalyzing signal peptide processing, disulphide bridging and molecular chaperones

such as cytochrome c maturation factors can ensure generation of post-translationally modified,

bioactive heterologous bioproducts . Moreover, in vitro permeabilization of the Escherichia coli

cell wall (Kaderbhai et al., 1997) can facilitate selective discharge of the periplasmic contents

into the growth medium, easing recovery of highly pure recombinant proteins.

Several groups have reported the expression of growth hormone or its derived fusion protein in

E. coli cytoplasm (Khan et al., 1998; Wallis et al., 1995; Patra et al., 2000; Sadaf et al., 2007a).

However, the cytoplasmic production of a protein has certain disadvantages: high level

accumulation often leads to insoluble protein aggregates that can be difficult to refold and

solubilize, a refolding step is frequently required to obtain the native conformation and to form

the correct disulfide bonds (Becker and Hsiung, 1986). Alternative expression systems have been

based on the secretion of the protein into the E. coli periplasmic space, which not only allow a

greater chance to obtain the protein in a folded and soluble form but a lower load of

contaminating proteins in the periplasmic fluid makes purification process easier. Either

premature cytoplasmic protein folding or incorrect disulfide bond formation in the bacteria

periplasm are two known limitations in the overproduction of secreted proteins. Secretion

process in the periplasmic space of E. coli cells, which mimics the natural process of

somatotropic cells in the pituitary gland has been reported for the human GH (Deoliveira et al.,

1999; Soares et al., 2003). However, the secretory expression of recombinant ovine growth

hormone has not been reported to our knowledge.

5

1.3 Ovine breed of Pakistan

More than 50% of sheep are reared in the western dry mountains, western dry plateau and

northern dry mountains. Of the 31 breeds of sheep, the most important are Baltistani, Bibrik,

Cholistani, Kachhi, Kajli, Lohi and Lati, or Salt Range. The local ovine breed Lohi belongs to

family bovidae, subfamily caprinae and genus ovis. Genus ovis constitute 6species, one of them

is ovis aries commonly known as sheep. This study is specifically on the growth hormone taken

from pituatry tissues of slaughtered local ovine breed Lohi. Sheep breeds are classified into thin

tail and fat tail breeds. Lohi sheep are one of the important thin tail mutton breeds available in

the central districts of Punjab province (Pakistan). The breed exhibits an excellent capacity to

adapt to these areas. Lohi is one of the massive and highly productive breeds which comprise

some 40% of the Punjab and 15% of the national sheep population production and reproduction

performance (Economic survey of Pakistan, 1997). The Lohi is one of the best sheep breeds of

Pakistan. This breed is found in the central districts of Punjab. Its rapid body growth, coupled

with good quality meat is the main characteristics of this breed. Lohi sheep are large, having

deep and massive body, weighing on average 45-62 kg. The general body colour is white with a

.large reddish brown head having .long and drooping ears. The tail is short, thick and stumpy.

The average body weight at birth, 3, 6, 9 and 12 months of age is 3.59::!:0.69, 15±0.20,

26.5±3.56, 30.4±0.40 and 33.4±0.46 kg respectively.

Figure 3. (Lohi) ovine breed of Pakistan Livestock production research institute, Bahadur nagar, okara, Punjab.

6

The Lohi breed expressed a significant innate resistance to artificial infection of H. contortus.

Although Pakistan has been placed at tenth amongst sheep producing countries, a huge amount

of foreign exchange have to spend on the import of milk, wool and dairy products in order to

meet the increasing demands of urban population.

1.4 Impact of our study on the economy of Pakistan

The application of roGH to farm animals is of great importance for the enhanced

production of milk, meat and wool which have in turn a great role in the economy of country like

Pakistan. The enhanced milk, meat and wool production would not only reduce the import of

milk and wool products but would also encourage the production of competitive value added

quality products for exporting to the gulf states and middle eastern countries along with meeting

the local demands of milk meat and wool products. Therefore, there is tremendous scope and

certainly a compelling need for improving the growth rate of these animals.

In this context, the use of recombinant growth hormone (rGH) which is a well-known

animal productivity booster both for milk (Bauman, 1999; Walli and Samanta, 2000) and meat (

Bonneau et al., 1999) could provide an answer. Either exogenous administration of

recombinantly produced homologous GH in the immediate future or, possibly the transgenesis

and cloning approaches in the relative long-term, may provide the keys for boosting the

productivity of these animals, and help to meet the future nutritional challenges of several south

Asian countries.

Our study investigated the cloning, expression, secretion and purification of recombinant ovine

growth hormone isolated from local ovine breed Lohi .

7

1.5 Review of Literature

During the last 30 years, the gene encoding ST has been isolated and characterized from over 50

vertebrate species including cattle, sheep, goat, pig and human. In the following sections,

different aspects of ST relating to structure, function, purification, characterization, recombinant

protein production ,secretion and advantages of getting protein in soluble forms are been

reviewed.

1.5.1 Structural and functional aspects of GH

GH, in most vertebrate species is synthesized by the anterior part of pituitary gland and its

gene is composed of four introns and five exons of varying length. In mammals, only one gene

codes for ST. An exception, however, occurs in higher primates like human who have five GH-

related genes, all clustered on chromosome 17. One of these codes for pituitary ST, while the

other four for genes expressed in placenta, including two genes for placental lactogen, one for

placental lactogen-like protein and one for a GH-variant (Chen et al., 1989). A cluster of eight

GH-like genes has recently been identified in marmoset, a new world monkey (Wallis and

Wallis, 2001; Wallis and Wallis, 2002). In 1995, Wallis and Wallis demonstrated that this gene

cluster is confined to primates only and have arisen by a series of gene duplications during

evolution. However, a number of reports appeared later indicating that there are duplicate GH

genes in some caprine ruminants as well (Lacroix et al., 1996; Wallis et al., 1998). Despite the

presence of duplicate genes in primates and certain other mammals, ST is mainly secreted by the

somatotrophs and its exon-intron structure is well conserved between different taxa (Forsyth and

Wallis, 2002) Studies undertaken to elucidate the structural aspects of mammalian GHs have

shown that gene encoding GH, more specifically bST, has a primary transcript of 1793

nucleotides with five exons (I-V) interrupted by four intervening introns. The gene is mapped to

19q chromosome at 1-7qter location. Like ovine, the TATAAA sequence which is involved in

transcription initiation is also present in the 5′-flanking region of bST gene .Exon I and a part of

8

exon II code for 26 amino acids long N-terminal signal peptide, which is not retained in mature

bST (Chen et al., 1990). Presence of 28 kDa N-terminal signal sequence has been reported in

feline and other mammalian STs, as well (Castro-Peralta and Barrera-Saldana 1995; Secchi and

Borromeo, 1997). The main function ascribed to this signal sequence is to direct the maturation

of the nascent protein by a very specific protease, which recognizes and cleaves the signal

sequence, once the sorting process is completed.

While the details of this sorting and cleavage process remain unclear, it is believed that

this post-translational modification influences the protein maturation and contributes to the

structural diversity of the STs (Martoglio and Dobberstein, 1998). Ovine growth hormone gene

was isolated (Byrne, 1987) isolated and sequenced the ovine growth hormone gene. The

structure of the gene was found to be similar for other growth hormone genes, particularly the

bovine gene, which constitute five exons and 4 introns with variying sizes of 264 bp, 231 bp,

227 bp and 273 bp (Robert, 1983). The purified bovine growth hormone, consisting of a single

polypeptide chain, was found to contain NH2-terminal alanine, phenylalanine, and methionine in

nearly equal amounts. The minimum molecular weight calculated from its amino acid

composition was 20,846. It was also demonstrated that mature bST is comprised of 190 or 191

amino acids due to the presumed ambiguity in the removal of this signal peptide. The bST

amino-terminus is heterogeneous having either alanine (Ala-Phe-Pro-Ala) or the adjacent

phenylalanine (Phe-Pro-Ala) as the N-terminal residue. There is no known genetic element

responsible for the variability in this cleavage event (Bauman, 1992; 1999).

Despite the observed structural variations, biological effects of STs were found similar

amongst the different vertebrate groups. In mammals, ST is involved in a variety of metabolic

activities like protein and nucleic acids synthesis, increased secretion of insulin and glucagon

from the pancreas and lipid-mobilization (Etherton and Bauman, 1998). oGH was tested for its

effects on lipolysis of rat and ovine adipose tissue in vitro. In Australia, the gene for oGH has

been over-expressed in sheep under the control of the metallothionein promoter. The transgenic

9

animals showed improved performance in growth, body fat and wool production. More recently,

transgenic rams from this experiment have been bred and the progeny assessed (Adams et al.,

2002). Associated with the increased expression of growth hormone was a reduction in fat depth,

increase in wool yield and increase in liveweight of the progeny animals. The somatogenic and

galactopoietic effects of recombinant ovine placental lactogen (oPL) were compared with the

effects of oGH in post-weaned growing lambs and in lactating ewes. It is concluded that oPL and

oGH have similar somatogenic effects in lambs. Both hormones exhibited galactopoietic effects,

but oGH was considerably more potent than oPL ( Leibovich et al., 2000). The growth

promoting effects of oGH on both whole-body and tissue protein turnover were generally

accompanied with no change in the efficiency of deposition of newly synthesized protein

(Foster, 1991). For the same ratio size, the oGH group showed higher retentions of ingested

nitrogen. They concluded that oGH significantly enhances whole-body growth rates as a result of

the stimulatory effect on protein synthesis rates with little effect on protein degradation (Adams

et al., 2002). In birds, functions of ST, however, appear to differ from mammals. For example, in

mammals, ST plays an important role in post-natal body development, while in birds; it exhibits

no effect on growth during the post-hatch growing period (Zhao et al., 2004). Moreover, in

birds, ST signal peptide may be involved in post-translational modifications while no such

function is ascribed to mammalian ST signal peptide.

1.5.2 Cloning and expression of GH in bacterial systems

Development of successful cloning techniques led to the cloning of cDNA for GHs from a

large number of species. Molecular cloning of DNA complementary to bGH mRNA was carried

out by (Miller and co-workers, 1980). The DNA complementary to bGH mRNA coding for

cloned into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E.

coli x 1776. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal

10

peptide and confirmed the published amino acid sequence of the secreted hormone. The

nucleotide sequence of bGH mRNA showed 83.9% and 76.5% homology with rat and human

GH mRNA respectively (Miller et al., 1980). In recent research cDNAs prepared using poly

(A) mRNA from pituitaries and containing the coding sequences for bovine and porcine growth

hormones bGH and pGH were cloned in bacteria. 90% homology was found in their primary

structure. Ovine genomic library from thymus DNA was constructed by (Seeburg, 2009; Orian et

al., 1988). From this library, gene encoding oGH was isolated using the cDNA clone for bGH as

a probe. The exon-intron junctions in the oGH sequence were also determined by analogy with

the bST genomic sequence. Analysis revealed that ovine and bGH genes are 97.5 % homologous

in the coding regions. The coding sequence of oGH was found similar to the previously

published cDNA sequence for oGH (Warwick and Wallis, 1984) except for one base at position

1047 where an A was found instead of a G. This difference, however, did not result in a

difference in amino acid sequence”. mRNA from pituitary tissues of caprine was isolated and

used to construct cDNA library (Yamano et al., 1988).

After cloning, an important step was to achieve a high-level expression of these animal

GHs in bacterial systems. The high level expression of cloned gene in E. coli generally requires a

strong promoter, a properly spaced Shine-Delgarno (SD) sequence and an effective ribosome

binding site for efficient translation of the mRNA (Das, 1990).

A recombinant plasmid expression vector was constructed with six histidine residues

(His6) at the amino-terminus under the control of a T5 promoter. Upon induction with isopropyl-

β- -thiogalactopyranoside, the recombinant protein was synthesized and accumulated in the

cytoplasm in the form of inclusion bodies, at levels of approximately 18% of the total cellular

protein. The recombinant ovine growth hormone containing His tag was recovered and purified

to >95% homogeneity in a single step by immobilized metal-ion chromatography with a special

affinity Ni2+·NTA. The purified roGH after refolding was found to be functionally active in

terms of its receptor binding and antigenicity as analyzed by radio receptor assay and radio

11

immunoassay. Yields of the purified expressed protein were found to be 32 μg/ml at a shake-

flask level ( Appa Rao et al., 1997).

oGH was cloned into plasmid poGHe101, based on pUC8 and found very high

expression (up to 25% of total cell protein) after induction. oGH was found in the form of

inclusion bodies. They described purification of oGH1 by 6 M guanidinium chloride containing

dithiothreitol. ion- exchange and gel filtration chromatography. Purified oGH1 had a Mr of 22

000, an isoelectric point of about 6·7 and an N-terminal sequence corresponding to that of oGH.

It was concluded that conclude that oGH behaved similarly to authentic bovine GH in a

radioimmunoassay, a radioreceptor assay and a weight-gain assay in hypophysectomized rats.

Thus the renatured hormone appeared to be correctly folded (Wallis and Wallis, 1990). By

feeding yeast extract along with glucose during fed-batch operation, high cell growth with very

little accumulation of acetic acid was observed. Use of yeast extract helped in maintaining high

specific cellular protein yield which resulted in high volumetric productivity of r-oGH (Panda,

1999; Ronald, 1985) got high-level expression of bovine growth hormone in Escherichia coli

resulted in the formation of distinct cytoplasmic granules that were visible with the phase-

contrast microscope, now known as inclusion bodies. They were isolated from crude cell lysates

by differential centrifugation and were further purified by a simple washing procedure that yields

nearly homogeneous bGH.

The oGH was expressed in Escherichia coli in the form of inclusion bodies using the pQE-

30 expression vector. In a simple fed-batch fermentation, 800 mg/L of recombinant ovine growth

hormone (roGH) was produced at a cell concentration of 12 g dry cell weight/L. Inclusion bodies

were isolated from cells with >95% purity by extensive washing using detergent, and the r -oGH

from the purified inclusion bodies was solubilized in 2 M Tris-HCl buffer at pH 12 containing 2

M urea. The roGH solubilized in the above conditions exhibited considerable secondary structure

as determined by circular dichroism spectra and was immunologically active. Solubilization of

the inclusion body protein with retention of native-like secondary structure gave higher yields

12

during refolding. To suppress protein aggregation, refolding was carried out in gel filtration

column. Refolding, buffer exchange, and the purification of monomeric roGH from aggregated

complex was achieved in a single step using gel filtration chromatography. More than 60% of

the initial inclusion body protein was refolded into a native-like conformation by the use of this

procedure. The refolded protein was characterized by circular dichroism, fluorescence, SDS-

PAGE, Western blotting, and radio receptor binding assay and found to be similar to native,

pituitary-derived, ovine growth hormone. (R.H.Khan,1998). High level expression of the

somatotropin gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis (BbST)

was obtained by synthesizing a codon-optimized ST gene through the introduction of silent and

non-silent mutations involving single and multiple base substitutions in +2 and subsequent

codons of BbST. The clones were expressed in the form of inclusion bodies. The inclusion

bodies represented over 20% of the E. coli cellular proteins (Sadaf et al., 2007a; Sadaf et al.,

2007b; Sadaf et al., 2008). High-level of expression~30 % of the total cell protein in a T7-

promoter based pET expression system of caprine growth hormone was achieved by making

silent base substitutions which are likely to minimize secondary structure formation at the 5'-end

of the cGH transcript. The over expressed cGH was in the form of inclusion bodies Inclusion

bodies were solubilized in 100mM Tris buffer containing 2M urea at pH 12.5. Solubilized protein

was refolded by pulsatile renaturation process in refolding buffer and purified using DEAE

sephadex anion exchange chromatography. The cGH was found to be biologically active on rat

lymphoma Nb2 cell bioassay (Khan, 2007).

1.5.3 Secretion of growth hormone in E.coli

Several groups have reported the expression of growth hormone or its derived fusion

protein in E. coli cytoplasm (Khan et al., 1998; Wallis et al., 1995; Patra et al., 2000; Sadaf et

al., 2007a). Secretion process in the periplasmic space of E. coli cells, which mimics the natural

13

process of somatotropic cells in the pituitary gland was achieved by linking the signal peptide

sequence to the human GH (Deoliveira et al., 1999; Teresa et al., 2000; Soares et al., 2003). By

fusing the human ST cDNA with an outer membrane protein A (ompA) signal peptide of E. coli,

achieved an even higher concentration (15 mg/L) of ST in the periplasm was achieved (Becker

and Hsiung, 1986). N-terminal sequence analysis of protein demonstrated that the fusion protein

was correctly processed to authentic 22 kDa human GH. Their results also showed that E. coli

periplasm provides an appropriate environment for proper folding of the expressed proteins.

In 1987, Chang and co-workers fused the human ST cDNA to E. coli heat-stable

enterotoxin II (ST-II) signal peptide and expressed it under the control of phoA promoter. E.

coli produced 15-25 µg of human ST/ml/A550, 90 % of which was exported to the periplasmic

space. Later, several research groups reported the periplasmic expression of recombinant STs in

E. coli up to a concentration of 10-25 µg ST/ml/OD).

The influence of different factors acting on Escherichia coli periplasmic expression of

recombinant hGH in shake flask cultures was investigated (Soares et al., 2003) .bacterial

vectors containing the phage lPL promoter, which is temperature activated, were utilized. Four

different signal peptides were compared: DsbA, npr, STII and one derived from the natural hGH

signal peptide, this last used as a reference. The expression level was affected by the signal

peptide and by the induction conditions, being more effective when activation started in the early

logarithmic phase which, however, exhibited remarkably different optical density (OD)

according to medium composition. Our results thus indicate that 6 hrs activation at 40±42°C,

starting with an OD (A600) of ~3 in a very rich medium, were conditions capable of providing

the maximum secretion level for a vector utilizing the DsbA signal sequence and E. coli W3110.

Multiple pathways direct protein translocation across the bacterial membranes but most of

the periplasmic pre-proteins are routed via the Sec-export-dependent pathways (Mori and Ito,

2001; Berks et al., 2000). In Gram-negative bacteria, secreted proteins have to cross the two

membranes of the cell envelope, which differ substantially in both composition and function

14

(Koebnik et al., 2000). There are five secretion pathways known as( type I, II, III, IV, and V)

but first three are most widely used (Koster et al., 2000).

1.5.3.1 Type I secretion systems

Transport proteins in one step across the two cellular membranes, without a periplasmic

intermediate (Binet et al., 1997). E. coli normally uses this pathway for the secretion of high-

molecular-weight toxins and exoenzymes. Although the type I secretion mechanism is capable of

exporting the target protein to the culture medium, it has two significant drawbacks. Firstly, the

secreted peptide remains attached to the signal sequence and therefore an additional cleavage

step is required to obtain the intact native protein (Blight and Holland, 1994). Secondly,

coexpression of the components of this system is often necessary to increase transport

capacity(Shokri et al., 2003).

1.5.3.2 Type II secretion Mechanism

The general secretory pathway is a two-step process for the extracellular secretion of

proteins mediated by periplasmic translocation (Koster et al., 2000). Three pathways can be used

for secretion across the bacterial cytoplasmic membrane: : the SecB-dependent pathway, the

signal recognition particle (SRP), and the twin-arginine translocation (TAT) pathways.

1.5.3.3 SecB-dependent pathway

Secreted proteins targeted to the SecB-dependent pathway contain an amino-terminal

signal peptide that functions as a targeting and recognition signal. These signal peptides are

usually 18–30 amino acid residues long and are composed of a positively charged amino

terminus (N-region), a central hydrophobic core (H-region), and a polar cleavage region (C-

15

region) (Choi and Lee, 2004; Fekkes and Driessen, 1999). The N-region is believed to be

involved in targeting the preprotein to the translocase and binding to the negatively charged

surface of the membrane lipid bilayer. Increasing the positive charge in this region has been

shown to enhance translocation rates, probably by increasing the interaction of the preprotein

with SecA (Fekkes and Driessen, 1999; Wang et al., 2000). The H-region varies in length from 7

to 15 amino acids. Translocation efficiency increases with the length and hydrophobicity of the

h-region, and a minimum hydrophobicity is required for function (Wang et al., 2000). Although

this pathway has been used extensively for recombinant protein production, it has one serious

drawback. This system is not able to transport folded proteins and, since transport is largely

posttranslational, the secretion of proteins that fold rapidly in the cytoplasm may not be possible.

In these cases the protein should be targeted to the SRP or the TAT pathways.

1.5.3.3.1 TAT pathway

A Sec-independent pathway was reported to be functional in E. coli (Santini et al., 1998;

Sargent et al., 1999). This pathway has been termed the TAT (twin-arginine translocation)

system because preproteins transported by it contain two consecutive and highly conserved

arginine residues in their leader peptides. The TAT pathway is capable of transporting folded

proteins across the inner membrane (Stanley et al., 2000) independently of ATP (Yahr and

Wickner, 2001) using the transmembrane PMF (DeLeeuw et al., 2002). The TAT pathway has

been used in the secretion of several recombinant proteins including antibody fragments (DeLisa

et al., 2003) glucose–fructose oxireductase(Blaudeck et al., 2001), ribose binding protein (Pradel

et al., 2003)alkaline phosphatase (Masip et al., 2004), and green fluorescent protein (Barrett et

al., 2003; De Lisa et al.,2002; Santini et al., 2001; Thomas et al., 2001).

The H-region of TAT signal peptides is usually less hydrophobic than that of Sec leader

peptides. The C-region contains the cleavage site and shows a strong bias towards basic amino

acid residues (Berks et al., 2000). It has been shown that transport via the TAT pathway is less

16

efficient (DeLisa et al., 2004) and slower than the Sec pathway with transit half-times in the

order of a few minutes (Santini et al., 1998; Sargent et al., 1998) instead of a few seconds

(Berks et al.,2000)Despite these disadvantages, the TAT pathway is capable of transporting

folded protein across the inner membrane, unlike the SecB or the SRP pathways (De Lisa et al.,

2003).

1.5.3.3.2 SRP pathway

The signal recognition particle (SRP) pathway is used by E. coli primarily for the

targeting of inner membrane proteins (Economou, 1999). This system has been exploited in the

secretion of several recombinant proteins including Mtla–OmpA fusions (Neumann-Haefelin et

al., 2000) MalF–LacZ fusions (Tian et al., 2000) maltose binding protein, chloramphenicol

acetyl transferase (Lee and Bernstein, 2001; Peterson et al., 2003) and haemoglobin protease

(Sijbrandi et al., 2003). The system consists of several proteins and one RNA molecule. SRP

recognizes its substrates by the presence of a hydrophobic signal sequence (hence the name

signal recognition particle). The presence of an N-terminal signal sequence with a highly

hydrophobic core, combined with a lack of a trigger factor binding site (Patzelt et al., 2001)

results in cotranslational binding of the nascent chain to Ffh (Beck et al., 2000).

For a productive interaction between the preprotein and Ffh, 4.5S RNA is required

(Herskovits et al., 2000). It has been suggested (Fekkes and Driessen, 1999) that the interaction

between SRP and the signal sequence is dependent on the hydrophobicity of the nascent chain

since preproteins with more hydrophobic signal sequences are translocated with higher

efficiency. It has been shown (Gu et al., 2003) that SRP binds the ribosome at a site that overlaps

the binding site of trigger factor. A discriminating process has been proposed in which SRP and

trigger factor alternate in transient binding to the ribosome until a nascent peptide emerges.

Depending on the characteristics of the nascent peptide, the binding of either SRP or trigger

17

factor is stabilised, thus determining whether the peptide is targeted to the membrane via the

SRP pathway, or posttranslationally by the SecB pathway (Gu et al., 2003). FtsY is found both

in the cytoplasm and at the membrane (Herskovits et al., 2000) and can interact with ribosomal

nascent chain–SRP complexes in the cytosol. Upon interaction with membrane lipids, the

GTPase activities of FtsY and Ffh are stimulated, thus releasing the nascent chain to the

translocation site (Nagai et al., 2003). This site may be the SecYEG translocon (Koch et al.,

1999; Valent et al., 1998; Zito and Oliver, 2003), although it has been demonstrated that

membrane insertion can occur independently of SecYEG (Cristobal et al., 1999b). Insertion of

transmembrane segments can occur in the absence of SecA (Scotti et al., 1999) while

translocation of large periplasmic loops is SecA-dependent (Neumann-Haefelin et al., 2000; Qi

and Bernstein, 1999; Tian et al., 2000). The protein YidC was also identified as a translocase-

associated component during insertion (Scotti et al., 2000). It has been proposed that this protein

facilitates the diffusion of transmembrane segments into the lipid phase (van der Laan et al.,

2001).

For recombinant protein production, SRP targeting can be achieved by engineering the

hydrophobicity of the signal sequence (Bowers et al., 2003; de Gier et al., 1998; Peterson et al.,

2003). SRP system is advantageous if for instance the target protein folds too quickly in the

cytoplasm, adopting a conformation incompatible with secretion by the SecB-dependent system

(Lee and Bernstein, 2001; Schierle et al., 2003). Various studies have shown the SRP

mechanism in E. coli with special reference to translation arrest of recombinant protein N, G and

M domains of Ffh.

The DsbA signal sequence works with SRP targeting mechanism and has been the best

choice for translocation of recombinant proteins to the inner membrane of E. coli. The extra-

cytoplasmic expression of human growth hormone under the influence of DsbA signal sequence

has been reported. However, the secretory expression of the recombinant ovine growth hormone

has not been reported to our knowledge.

18

1.5.4 Signal sequences

Proteins destined for translocation across the cytoplasmic membrane are synthesized as

precursors carrying an amino-terminal signal sequence that direct polypeptides into the secretory

pathways (Economou, 1999). Although variable in primary structures (Izard and Kendall, 1994)

signal sequences contain a conserved and ordered structure (Von Heiinr, 1999) that channels the

passenger portion into the export pathway (Thanassi and Hultgren, 2000). The amino-terminal

positively charged end, together with the central hydrophobic core, directs Sec-independent and

proton-motive force (PMF)-dependent signal peptide translocation across the membrane (van

voorst and de kruiff, 2000) and substitutions of the hydrophobic residues with charged ones

diminish or abolish export competency of signal sequences (silhavey et al., 1983). The

efficiency of preprotein translocation is independent of the structure of the cleavage region. This

region can accommodate varying hydrophobicities with the exception of bulky residues at −1, −3

positions. By reducing the signal peptide to simplified, idealized segments it has been shown

that a largely polymeric sequence with retention of the early consensus sequence and a central

hydrophobic core, MKQST(L10)−(A6), can function equivalently to the wild-type alkaline

phosphatase signal peptide (Laforet and Kendall, 1991). It has been shown that the positive

charge in the N-terminal region of signal peptide plays an important role in the function of the

eukaryotic signal peptide as well as that of prokaryote. Using signal sequence containing

additional Arginine residues, secretion levels of HLY in yeast were notably increased (Yoshinori

Tsuchiya, 2000).

The rate of inversion increases with more positive N-terminal charge and is reduced with

increasing hydrophobicity of the signal. Inversion may proceed for up to 50s, when it is

terminated by a signal-independent process. These findings provide a mechanism for the

topogenic effects of flanking charges as well as of signal hydrophobicity. It was also suggested

that translational kinetics and signal sequences act in concert to modulate the export process. The

19

expression of the natural and optimized gene sequence was compared in order to produce leech

carboxypeptidase inhibitor (LCI) in the bacterial periplasm, and evaluated export efficiency of

LCI fused to different signal sequences. The best combination of these factors acting on

translation and export was obtained when the signal sequence of DsbA was fused to an E. coli

codon-optimized mature LCI sequence (Juan et al., 2009). Proinsulin fused to the C-terminus of

the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site. As DsbA is the main

catalyst of disulfide bond formation in E. coli, expected increased yields of proinsulin by intra-

or intermolecular catalysis of disulfide bond formation. In the context of the fusion protein,

proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide

bond formation (J. Winter, 2000). The E. coli cytoplasmic protein thioredoxin 1 can be

efficiently exported to the periplasmic space by the signal sequence of the DsbA protein

(DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding

protein (MBP) (Clark, 2003). The best combination of these factors acting on translation and

export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized

mature LCI sequence (Veit goder and Martin spiess, 2003).

1.5.5 Expression and Purification of secreted protein in E.coli.

The heterologous expression and production of recombinant GH from various species of

bovidae family including bovine (Klein et al., 1991), porcine (Seeburg et al., 1983) and ovine

(Rao et al., 1997) in E. coli has been reported. E. coli constitutes cytoplasm, periplasm, inner

membrane and outer membrane spaces. All proteins in E. coli are synthesized in cytoplasm and

translocated to their defined destinations. About 71, 21, 6, and 2 % of the proteins are found in

the cytoplasm, inner membrane, periplasmic space and outer membrane respectively. Several

groups have reported the expression of GH or its derived fusion protein in E. coli cytoplasm

(Wallis et al., 1995; Khan et al., 1998; Patra et al., 2000; Sadaf et al., 2007). However, the

cytoplasmic production of a protein has certain disadvantages: high level accumulation often leads

to insoluble protein aggregates that can be difficult to refold and solubilize and a refolding step is

20

frequently required to obtain the native conformation to form the correct disulfide bonds (Becker

et al., 1986). Alternative expression systems have been based on the secretion of the protein into

the E. coli periplasmic space, which not only allows a greater chance to obtain the protein in a

folded and soluble form but also lower load of contaminating proteins in the periplasmic fluid

makes purification process easier. Secretion process in the periplasmic space of E. coli cells

mimics the natural process of somatotropic cells in the pituitary gland and has been achieved by

linking the signal peptide sequence to the human GH (de oliveira et al., 1999; Teresa et al., 2000;

Soares et al., 2003). It has been reported that types of culture media and medium additives

(compatiblesolutes, chemicalchaperon) can affect the yield of recombinant protein production

(kaushik, 2003). The spectrum of compatible solutes used by microorganisms comprises only a

limited number of compounds; sugars (e.g.trehalose), polyols (e.g. glycerol and glucosylglycerol),

free amino acids (e.g. proline and glutamate) derivatives there of (e.g. proline, betaine and

ectoine) quaternary amines and their sulfonium analogues (e.g. glycine betaine, carnitine and

dimethylsulfoniopropionate) sulfate esters (e.g. choline-O-sulfate) and N-acetylated diamino acids

and small peptides. In general, compatible solutes are highly soluble molecules and do not carry

a net charge at physiological pH. In contrast to inorganic salts, they can reach high intracellular

concentrations without disturbing vital cellular functions such as DNA replication, DNA-protein

interactions and the cellular metabolic compatible solutes also serve as stabilizers of proteins and

cell components against the denaturing effects of high ionic strength (Leibly et al., 2012).

A positive effect of low molecular weight additives supplemented in the culture medium

were being observed in various studies in terms of yields of periplasmic expressed proteins

(Diamant et al., 2001). Sorbitol addition to the culture medium resulted in higher accumulation of

a functional single chain Fv (Huston et al., 1991; Hashimoto et al., 1999) glycine betaine and

sucrose were also seen to be beneficial for the folding of immunotoxin and cytochrome c550 (Ou,

2002) While L-arginine and ethanol increased the yields of human pro-insulin (Winter et al.,

2001) plasminogen activator and a single chain Fv (Huston et al., 1991). Also the supply of

21

reduced glutathione, alone or in combination with DsbC over-expression, increased the

accumulation of disulfide-dependent proteins (Zapun et al., 1995; Missiakas et al., 1995). Use of

compatible solute in the medium has shown remarkable results (Barth et al., 2000). Besides

those strategies, one of the methods for soluble expression of recombinant protein is the Dsb co-

expression system (Sandee et al., 2005).

Several groups have described the purification of recombinant STs, expressed either in E.

coli cytoplasm, periplasm or even culture medium, using either affinity chromatography or

reversed phase HPLC (RP-HPLC). The 98 % purity of human ST was achieved from the E. coli

culture medium, using RP-HPLC (Hsiung et al., 1989). RP-HPLC was used to obtain 95 % pure

oGH from E. coli (Adams et al., 1992). The recombinant oGH in this study, however, was in the

form of inclusion bodies, which were solubilized in a cationic surfactant cetyltrimethyl

ammonium chloride (CTAC) prior to application on column. The purification of an ovine ST

variant to homogeneity using ion-exchange chromatography and gel filtration, after solubilization

and renaturation of oST from inclusion bodies was reported (Wallis and Wallis, 1990). The

purification of recombinant human ST to near homogeneity using ammonium sulfate

precipitation, ion-exchange chromatography and gel-filtration on Sephacryl S-200 column.

Similarly was also studied (Igout et al., 1993). FPLC (Mono Q anion exchanger) and RP-HPLC

were also used to achieve highest purification of excreted recombinant human GH. By exploiting

metal-protein binding affinity, (Mukhija et al., 1995) described the purification of His6-tagged

human ST to virtual homogeneity in a single-step. Their strategy involved selective binding of

His6-tagged recombinant protein to Ni+2-nitrilotriacetic acid (NTA) column followed by elution

with increasing concentration of imidazole. The purified protein was obtained at a level of 30

mg/L of the culture. Using similar strategy (Appa rao et al., 1997) purified the recombinant ovine

ST to greater than 95 % homogeneity on Ni+2-NTA resin, in a single-step. Yield of the purified

protein was around 62 % of the total expressed ST and was quite significant in comparison to the

other methods of purification.

22

Single-step purification of recombinant oST using gel-filtration column was reported

(Khan et al., 1998). In a simple fed-batch fermentation, 800 mg/L of recombinant oST was

produced, but all as in the form of inclusion bodies. The inclusion bodies were isolated from E.

coli with >95 % purity by extensive washing in the presence of detergent followed by

solubilization in urea at alkaline pH. Since, sufficient purity was achieved at the stage of inclusion

bodies, gel filtration served only as a polishing step which purified the monomeric recombinant

ovine ST from aggregated complex dimers or oligomers. Instead of using conventional

chromatographic techniques, (Jeh et al., 1998) employed a somewhat different strategy for

purifying ST from E. coli cytoplasm. They found that most of the E. coli proteins contaminating

the recombinant-ST are readily precipitated with the addition of two volumes of secondary

butanol (an organic solvent), while the flounder ST remained soluble. After the secondary butanol

treatment, the purity of recombinant flounder ST was more than 98 % and the recovery yield was

around 47%.

Ni+2-chelate affinity chromatography was used as a first step while purifying His10-tagged

human ST from E. coli cytoplasm (Shin et al., 1998). Further, by employing ion-exchange

chromatography (Q-Sepharose fast flow), a purity of greater than 99 % was achieved. A five-step

purification scheme involving precipitation, gel-filtration, ion-exchange, and hydrophobic

interaction chromatography (HIC) was applied to obtain a highly purified pharmaceutical grade

recombinant human GH for clinical use (Deoliveira et al., 1999). In contrast, a single-step high

performance liquid chromatography based on size exclusion was reported to obtain human GH for

use in radioimmunoassay as standard and tracer . Although the final product was not 100 % pure,

yet it was adequate for its intended use as a chemical reagent in immunoassay. The labeling

reaction presented a yield of about 65% and the purified tracer exhibited an antibody binding of

~50%. The values were very similar to those obtained by radioiodinating the highly-purified

clinical-grade recombinant human ST which was obtained after the regular six-step purification

process.

23

A two-step procedure involving ion-exchange chromatography on DEAE-sepharose

column and gel-filtration on sephacryl S-200 was used to get purified preparation of recombinant

hGH from E. coli. Recovery of recombinant protein from ion-exchange matrix was around 65 %

while overall yield of the purified refolded GH from the E. coli inclusion bodies, was ~50 %.

(Patra et al., 2000). While a combination of hydrophobic and ion-exchange chromatographies for

the purification of bubaline and caprine GHs which expressed in E. coli cytoplasm as inclusion

bodies. Prior to application on decyl agarose column, the inclusion bodies were solubilized in

guanidinium chloride and air-oxidized to get properly folded recombinant GHs for column

applications. Nearly 90 % purity was achieved at the end of HIC purification. An additional step

of ion-exchange chromatography using fast-flow DEAE-Sepharose was then employed for

purification to near homogeneity. The overall yield of recombinant GHs at the end of two-step

purification scheme was about 40 % of the starting material and the purity was > 98

%”(Mukhopadhyay and Sahni, 2002c).

The purity of protein as judged by RP-HPLC was greater than 95 %. Purification of hGH

from the periplasmic fraction by anion exchange and size exclusion gave hGH of greater than 90%

purity. Characterization by SDS-PAGE, amino terminal analysis, trypsin mapping, and circular

dichroism demonstrated that the fusion protein was correctly processed to authentic hGH and that

the E. coli periplasm provided an appropriate environment for proper folding of hGH and disulfide

bond formation (Becker and Hsiung, 1986). High-yield purification procedure for the preparation

of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic

space was investigated. The accuracy of hGH and total protein quantification, especially in the

early steps of the process, and the maximum elimination of hGH-related forms were also studied

in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely

valuable analytical tools (Deoliveira et al., 1999). The influence of four different signal peptides

(DsbA, npr, STII and one derived from the natural hGH) acting on E. coli periplasmic expression

of hGH in shake flask cultures were described, and concluded that DsbA signal sequence gave

24

best results (Soares et al., 2003). It has been reported that types of culture media and medium

additives (compatible solutes,chemical chaperon)can affect the yield of recombinant protein

production (kaushik, 2003). The spectrum of compatible solutes used by microorganisms

comprises only a limited number of compounds; sugars (e.g. trehalose), polyols (e.g. glycerol and

glucosylglycerol), free amino acids (e.g. proline and glutamate), derivatives thereof (e.g. proline,

betaine and ectoine), quaternary amines and their sulfonium analogues (e.g. glycine betaine,

carnitine and dimethylsulfoniopropionate), sulfate esters (e.g. choline-O-sulfate), and N-acetylated

diamino acids and small peptides. In general, compatible solutes are highly soluble molecules and

do not carry a net charge at physiological pH. In contrast to inorganic salts, they can reach high

intracellular concentrations without disturbing vital cellular functions such as DNA replication,

DNA-protein interactions, and the cellular metabolic Compatible solutes also serve as stabilizers

of proteins and cell components against the denaturing effects of high ionic strength (Leibly DJ,

2012).Use of compatible solute in the medium has shown remarkable results (Barth et al. 2000).

Besides those strategies, one of the methods for soluble expression of recombinant protein is the

Dsb co-expression system (Sandee et al., 2005 ).

Periplasmic proteins are recovered from transformed gram negative bacteria by a process

comprising freezing and thawing the cells. Advantages are obtained by culturing the cells in

phosphate limiting media and by killing the cells prior to separation of periplasmic proteins . The

compatible solute supported enhanced periplasmic expression of recombinant proteins under

stress conditions. Periplasmic protein in the shock fluid was purified by combinations of metal ion

affinity and size exclusion chromatography. This was substantially stabilized in the presence of

1M hydroxyectoine after several rounds of freeze-thawing even at low protein concentration

(Barth et al., 2000).

1.5.6. Advantages of getting soluble proteins

25

Periplasmic or extracellular secretion can increase the solubility of a gene product as

exemplified by the production of bacterial PNGaseF and human granulocyte colony-stimulating

factor (Loo et al., 2002; Jeong and Lee, 2001). Obtaining a soluble protein often constitutes a

bottleneck in the production of proteins for structural studies or proteomics (Goulding and

Jeanne Perry, 2003; Pedelacq et al., 2002; Yokoyama, 2003). Product secretion can provide a

way to guarantee the N-terminal authenticity of the expressed polypeptide because it often

involves the cleavage of a signal sequence (Mergulha et al., 2000) thus avoiding the presence of

an unwanted initial methionine on a protein that does not normally contain it. This extra

methionine can reduce the biological activity and stability of the product (Liao et al., 2004) or

even elicit an immunogenic response in the case of therapeutic proteins Protein secretion can

increase the stability of cloned gene products. For instance it was shown that the half-life of

recombinant proinsulin is increased 10-fold when the protein is secreted to the periplasmic space

(Talmadge and Gilbert, 1982). Secretion was also useful in the production of penicillin amidase

from E. coli as intracellular product degradation was a severe problem (Ignatova et al., 2003).

The increased stability of gene products on the periplasm and in the culture medium probably

results from the lower levels of E. coli proteases that can be found in these locations (Gottesman,

1996; Mergulha et al., 2004).

Additionally, if the product is secreted to the culture medium cell disruption is not required

for recovery and even in the case of periplasmic translocation, a simple osmotic shock or cell

wall permeabilization can be used to obtain the product without the release of cytoplasmic

protein contaminants (Mergulha et al., 2004; Shokri et al., 2003).

Biological activity is dependent on protein folding and, particularly if disulfide bonds must

be formed, proper folding is unlikely in the reducing environment of the cytoplasm.

Additionally, the correct pair bonding of cysteines contributes to the thermodynamic stability of

the proteins (Kadokura et al., 2003; Maskos et al., 2003; Raina and Missiakas, 1997).

26

1.6 AIMS AND OBJECTIVES

The objectives we planned to be achieved were as follows:

1. Isolation of total RNA from ovine pituitary gland.

2. cDNA synthesis by reverse transcription and PCR amplification using suitable primers and cloning

into suitable expression vector.

3. Coding and non coding Sequence analysis of cloned gene and comparison with other reported

sequences.

4. Expression and secretion of oGH gene in a suitable bacterial system.

5. Effect of different factors (signal nucleotide variation, medium composition and bacterial strain,

etc) on secretion of ovine growth hormone gene.

6. Purification of oGH by anion exchange, size exclusion chromatography.

7. Biological activity assesment of recombinant oGH.

27

28

Materials and Methods

29

2.1 Sample collection and storage

Pituitary glands from the freshly slaughtered ovine were collected from a local abattoir. The

collected samples were carefully dissected to remove the anterior part of the pituitary gland (site

for the production of GH), weighed and either stored at 80C or in liquid nitrogen for further

use in RNA isolation. Approximately 10 ml blood from the subject ovine was sampled into

vaccutainer tube containing 10.5 mg ethylene diamine tetra acetate (EDTA). The blood was

mixed gently and frozen at 20C until analysis.

2.2 Chemicals and kits

All chemicals used in this study were of the highest purity grade commercially available.

Polymerase chain reactions (PCR) were performed using GC-RICH PCR amplification system of

Roche Applied Science (Mannheim, Germany). For DNA extraction and plasmid mini-

preparation, QIAquick gel extraction and QIAprep spin miniprep kits of QIAGEN Inc.(CA,

USA) were respectively used. RNeasy Mini and Midi kits used to isolate RNA from bacterial

cultures and animal tissues were also acquired from QIAGEN. RevertAidTM Moloney Murine

Leukemia Virus (M-MuLV) reverse transcriptase, T4 DNA ligase, restriction endonucleases and

DNA and protein size markers, IPTG, 6 x loading dye were either purchased from New England

Biolabs (MA, USA) or MBI Fermentas (MD, USA)

E. coli strain BL21 CodonPlus (DE3) RIPL (Stratagene, CA) was used for expression studies.

pET22b(+) expression plasmid was obtained from Novagen Inc. Trizol reagent for total RNA

isolation from pituitary gland was purchased from Invitrogen. QIAquick gel extraction for DNA

extraction from agarose gels and QIAprep Spin Miniprep kit for plasmid preparation were

procured from Qiagen. InsT/A clone PCR product cloning kit, MMLV-RTase, Taq DNA

polymerase, restriction enzymes, IPTG and T4 DNA ligase were purchased from MBI

Fermentas. For western blot analysis, rabbit anti-bovine GH and commercially bovine GH were

purchased from US Biological (USA), goat anti-rabbit IgG conjugated with alkaline phosphatase

30

was from BioRad (USA). NaCl, Trypton and yeast extract (Oxoid, England) were used in Luria -

Bertani (LB) medium for the growth of Escherichia coli strains.All other cultivation media and

bulk chemicals were purchased from Difco laboratories, or US Biological (CA, USA), unless

stated otherwise.. 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium

(NBT), used as color development substrates, were purchased from MBI Fermentas and US

Biological. The HeLa cell lines were a gift from Agha Khan University Hospital, Karachi,

Pakistan. DMEM (Dulbecco’s Modified Eagle’s Medium) mammalian cell culture medium and

fetal bovine serum (FBS) was purchased from PAA Laboratories GmbH (THE CELL

CULTURE COMPANY).

2.3 Isolation of total RNA from pituitary sample

The RNA was isolated from the pituitary tissue of freshly slaughtered young sheep breed

(Lohi) by using Guanidium- thiocyanate- chloroform extraction method (Chomczynski et al.,

1987).

Mortar and pestal were washed with DEPC treated water (diethyle Pyrocarbonate) weighted

pituitary tissue 100mg, put it in mortar and covered it with aluminium foil, kept it for 40

minutes at -80oC. Then crusedh it while covering the mortar with aluminium foil from one side,

crushed it until it became fine powder.

Added 1ml TRI reagent in it and mixed it well with the help of pestal. Poured it into eppendorff,

centrifuged at 12,000g for 10 min at 4oC. Transferred the clear supernatant to a fresh tube and

proceeded with phase separation. This step was to remove insoluble material from the

homogenate, the resulting pellet contained extracellular membrane, polysaccharides, high

molecular weight DNA, while the supernatant contained RNA.

Supernatant recovered in fresh tube was 850 ml, left it at room temperature for 5 minutes to

permit complete dissociation of nucleoprotein.

31

Added 0.28 ml chloroform for phase separation as 0.2 ml required for 0.75 TRI reagent.Left it

for 15 minutes at room temperature centrifuged it at 12,000 g for 15 minutes at 4o C. Following

centrifugation, the mixture separated into a lower red, phenal chloroform phase, interphased and

the colourless aquaus upper phase. Transferred the aquaus phase which was 600ml to the fresh

tube.

Added 1ml Isopropanal and left it at room temperature for 10 min, centrifuged it at 12,000g for

8 minutes at 4oC. Discarted the supernatant solublized the pellet in 100ml H20. Results were

taken at 280mm absorbance.

2.4 Formaldehyde agarose gel electrophoresis

Since, RNA has a high degree of secondary structure; the samples were first denatured by

treatment with formamide and then separated by electrophoresis through the formaldehyde

containing agarose gel. Formaldehyde in the gel kept the RNA in denatured state by preventing

intra-strand Watson-Crick base paring.

1.2 % formaldehyde agarose (FA) gel was prepared by mixing 0.6g agarose with 50ml 1x

FA gel buffer (20mM MOPS, 5mM sodium acetate, 1mM EDTA, pH 7.0) in an

Erlenmeyer flask. The mixture was heated to dissolve agarose, cooled to 60-70C by

gentle swirling and poured onto the gel casting tray (in a fume hood) after adding 900 l

of 12.3 M formaldehyde and 0.5 l of 10 mg/ml ethidium bromide (a dye that intercalates

into the grooves of double-stranded DNA/RNA and fluoresces in UV-light).

Comb was inserted immediately after pouring to form wells for sample loading. Gel was

polymerized at room temperature for 30-40 minutes and then equilibrated in 1x FA

running buffer (1x FA gel buffer, 2.5 M formaldehyde) for additional 30-40 minutes, prior

to electrophoresis. RNA samples were denatured by mixing 4 volumes of RNA with 1

volume 5x RNA loading buffer (0.25% bromophenol blue, 4mM EDTA, 0.9M

32

formaldehyde, 20% glycerol, 30.1% formamide and 4x FA-gel buffer), chilled on ice and

then electrophoresed at 5-7 V/cm until the bromophenol blue dye migrated approximately

2/3 of the way through the gel.

RNA bands were visualized by illuminating the gel under UV light followed by image

recording using gel documentation system.

2.5 cDNA Synthesis

2.5.1 Primer designing

cDNA was synthesized by RT-PCR. On the basis of conserved sequences, two forward and one

reverse designed primers were used in amplification of ovine growth hormone gene. Forward

primers PBGH1 (5`CAT ATG ATC CAT GGC CTT CCC AGC CAT G3`) PBGH2 (5`CAT

CAT ATG GCC TTC CCA GCC ATG 3`) and reverse primer PBGH3 (5`TAG GAT CCG CAA

CTA GAA GGC AGC 3`). The forward primers contained Nde1, Nco1 restriction sites at their

5’end whereas the reverse primer contained a BamH1 site.

2.5.2 Reverse transcription (RT)

Reverse transcription was done to synthesize cDNA. Reaction was prepared in the PCR tube by

adding 5 μl (0.1-5μg) of isolated RNA, 1 μl (200 U) of RevertAid Molony Murine Leukemia

Virus (M-MuLV) Reverse Transcriptase, 1 μl of 50 μM antisense primer [OaST], 4 μl of 5 x RT

buffer, 2 μl of 10 mM dNTPs, 1 μl of (20 U) ribonuclease inhibitor and 6 μl of RNase free water

to make up the volume up till 20 μl. The reaction was carried out in Applied Biosystems 2720

Thermo Cycler for 60 minutes at 42°C, reaction was stopped by increasing the temperature at

70°C for 10 minutes and then chilled on ice.

33

2.5.3 PCR Amplification

Polymerase chain reaction (PCR) is a repetitive bidirectional and exponential DNA synthesis by

primer extension of a nucleic acid region. Amplification of cDNA was done and reaction mixture

was prepared by adding two oligonucleotide

primers of the required gene to be amplified (50 pmol of each primer),

four deoxynucleotide triphosphates (dNTPs) each of 0.4 mM,

Taq polymerase (Fermentas #EP0402), 2.5U

10 x PCR buffer containing ammonium sulphate 5 μl

MgCl2 1.5-4 mM,

of DNA template 0.1-1 μg

plasmid DNA 50 pg-1.0 ng

was required for total reaction mixture of 50 μl.

Reaction was carried out in 0.5 ml PCR tubes in duplicates. As magnesium ions make complexes

with the primer, dNTPs and DNA template, hence concentration of magnesium ions should be

optimized. Low concentration of Mg ions results in low yield, while high concentration results in

non specific products.

Amplification was carried out in a Thermalyne DNA thermalcycler programmed as:

one cycle of initial denaturation at 94C for 3 minutes,

followed by 35 cycles of denaturation, annealing and extension (94, 55, 72C

respectively) each with a hold time of 1 minute

and a final extension at 72C for 20 minutes.

34

After amplification, the reaction product was either stored at 4C or subjected to agarose gel

electrophoresis as described by Sambrook and Russell (2001).

The PCR products were analyzed by loading 5 μl of the samples on 1 % agarose gel

electrophoresis. Gel was visualized on UV transilluminator and photographed by using gel

documentation system (Dolphin, WEALTEC, USA).

2.6 DNA extraction from agarose gel

The DNA fragments or PCR products which were needed to be analyzed were purified from

agarose gel by Vivantis GF-1 Gel DNA Recovery Kit (Cat # VSGF-GP-100). The agarose gel

portion containing the required DNA fragment or PCR product was excised by using a sterilized

sharp scalpel. The gel slice was weighed, placed in the microcentrifuge tube and dissolved in the

binding and solubilization buffer GB (1 vol. GB: 1 vol. gel). The gel slice was dissolved at 50°C

for 5-10 minutes until gel had melted completely. The dissolved gel slice was transferred to the

spin column and centrifuged at 10,000 g for 1 minute. Flow-through was discarded and 750 μl of

washing buffer was added into the column and again centrifuged at 10,000 g for 1 minute. The

residual washing buffer was removed by doing centrifugation at 10,000 g for 1 minute. Column

was then shifted and placed on a fresh 1.5 ml microcentrifuge tube. 30 μl of elution buffer51was

added on to the column and waited for 2 minutes at RT. Then centrifugation was done at 10,000

g for 1 minute and elution step was repeated to get better yield of the purified product. Hence,

total 60 μl was obtained at the end of the extraction process. The purified sample was then

visualized on 1 % agarose gel electrophoresis and stored at -20°C for further use.

2.6.1 Purification of PCR product

The PCR products were excised from the agarose gel and purified by using Vivantis GF-1 Gel

DNA Recovery Kit (Cat # VSGF-GP-100). Protocol has been mentioned earlier in section

35

2.2.2.4. The purified PCR product was again run on 1 % agarose gel to quantify its concentration

after purification and to confirm its purity.

2.7 Cloning in pTZ57R/T vector

2.7.1 T/A cloning Kit method

The amplified product was ligated into the pTZ57RT by T/A cloning technique to produce the

recombinant pTZOaST1 construct which was then transformed into E. coli DH5 .

Ligation

Autoclaved the medium, allowed to cool at 55̊ C. Then, added 50 µl of Ampicillin in 50 m l LB

medium from stock solution (50,000 mg/ 1000ml). Pipette 40 µl of 2 percent X-Gal solution and 7

µl of 20% IPTG on the centre of ampicillin plate. Used a sterile spreader to spread it over the

entire surface of the plate. Incubated the plate at 37o C until all the solution or fluid has

disappeared The plate is incubated for 3- 4 hours.

Took out the plates from incubator in which E.Coli DH5α was spreaded. Round single colonies

will be observed. From T/A kit took C_medium and added 1.5 ml of it into two test tubes 1.5 ml

each. Pre warmed culture tubes containing a required amount (1.5ml) of transformed C_ medium

at 37o C. Moved a small portion of bacterial culture (4x4 mm size for each 1.5 ml of C_medium)

of DH5α and from the overnight LB plate using on inoculating loop into the pre warmed

C_medium. Suspended the culture by gently mixing and incubating the tubes in a shaker at 37o C

for 2hrs. The colonies on LB plates can be stored at 4o C and used for inoculating fresh culture

within 10 days.

Prepared Transform Aid T- solution by mixing equal volume of T solution (A) and T solution (B).

Took 430 ml of T(A) and 430 ml of T (B) and mixed them, then put on ice. Dispensed 1.5 ml of

fresh culture into a microfuge tube (eppendorffe) and spinned at maximum speed for 1minute at

room temperature or at 4o C. Discarded the supernatant and resuspended the pellet in 300 ml

36

transform aid T solution ( mix of T(A) and T(B) ). Incubated tubes on ice for 5 min. Spin down

the cells again for 1 min at RT and remove the supernatant. Resuspended the cells in 120 ml of (T

solution mixture) and incubated on ice for 5 min.

Prepared the DNA for transformation by dispensing 1ml of supercooled DNA (10000pg) or 2.5 ml

of ligation mixture (10-20 mg of vector DNA) into a new microfuged tubes and sat them on ice

for 2 min. Added 50 µl of resuspended cells to each tube containing DNA and incubated on ice for

5 min. Plated the cells on pre warmed LB ampicillin agar plates. Incubated the plates overnight at

37o C.

For transformation used 2ml (10mg of DNA) of the ligation mixture with PCR DNA fragment.

Incubated overnight at 37o C usually result in 200- 1000 colonies per plate (about 90% of which

were white). Now prepeared again LB Ampicillin plates. Made LB medium and added ampicillin

after autoclaviing the medium and then solidifying the plates. Picked colonies from the

transformation (white colinies) and spot them on LB ampicullin plates in duplicates. Incubated at

37o C overnight.

More than 90 % colonies appeared to be white on LB agar plates (containing ampicillin 100

μg/ml, 2 % X-gal and 0.1 mM IPTG) after transformation, as genes were inserted in the

pTZ57R/T vector containing lacZ region. While blue colonies showed the absence of insert, as

lacZ is functional in E. coli and encodes β-galactosidase that hydrolyzes X-gal into a colorless

galactose and 4-chloro-3-brom-indigo giving blue color to the colony. Thus blue/white screening

was done and single white colony was picked from the plates and inoculated in 10 ml of LB

medium (100 μg/ml ampicillin) for plasmid preparation. If clear, round colonies on duplicate

plates were found on plates. It meant that gene is being inserted. To amplify the gene inserted in

the plasmid colony PCR will be done.

Plasmid extraction was done by using Vivantis GF-1 Plasmid DNA Extraction Kit (Cat # VSGF-

PL-100) as described in sectio.2 and run on 1 % agarose gel electrophoresis.

37

2.7.2 Preparation of competent cells and transformation

Picked a single bacterial colony (2-3mm in diameter) from a DH5α plate that has been incubated

for 16 to 20hrs at 37o C. Transferred the colony into 100 ml of LB broth in a 1 litre flask.

Incubated the culture for 3hrs at 37o C with vigorous agitation, monitoring the growth of the

culture. (For efficient transformation, it is essential that the number of whole cells not exceed 10-

8 cells/ml which for most strains of E_coli is equivalent to an OD600 of 0.4. To ensure that the

culture does not grow to a higher density, measured the OD600 of the culture every 15- 20min.

Began to harvest the culture when the OD600 reaches 0.35). Transferred the bacterial cells to

sterile, disposable ice cold 50ml polyprylene tubes. Cooled the cultures to 0oC by storing the

tubes on ice for 10 min. Recovered the cells by centrifugation at 2700 g (41000rpm) for 10min at

4o C. Decanted the medium. Stood the tubes in an inverted position to allow the last traces of

media to drain away.

Resuspended each pellet by gentle vortexing in 30ml of ice cold MgCl2 , CaCl2 solution (80 mM,

MgCl2, 20 mMCaCl2). Recovered the cells by centrifugation at 2700g for 10min at 4o C.

Decanted the medium from the cell pellets. Stood the tubes in an inverted position to allow the

medium to draw away. Resuspended the pellet by swirling or gentle vortexing in 2ml of ice cold

0.1 M CaCl2 for each 50ml of original culture. At this point, either use the cells directly for

transformation as dispensed into alliquots and freezed at -70o C. (The cells may be stored at 4o C

in CaCl2 solution for 24- 48 hrs. The efficiency of transformation increases 4- 6 folds during the

first 12- 24 hrs of storage and thereafter decreases to the original level). Dispensed a sample of

the competent cells (200ml) into a sterile precooled microcentrifuge tube.

Added plasmid DNA (approx 0.5mg) mixed gently and left on ice for 40min. Quickly transferred

the tube to a 42o C water bath for 2 min then returned it to ice for 5min. Added LB medium

(0.8ml) to the tube, mixed and incubated it for 2 hrs at 37o C without shaking. Spread samples of

38

the transformed cells (50 -200ml) on prewarmed, dried LB plates containing the appropriate

antibiotic to select for the plasmid.

2.8 Colony PCR

The transformation resulted in the production of colonies containing the plasmids. One of

the methods to screen the colonies whether containing the desired plasmid or not is colony PCR.

For colony PCR, a single colony was picked from the LB agar plate by using a sterile tooth pick

or by pipette (P200, Gilson) tips and transferred to the PCR tube containing 30 μl of sterile

distilled water. The colony was mashed, mixed thoroughly and denatured at 95°C for 10 minutes,

25°C for 10 minutes and then the denaturation reaction was stopped by holding at 4°C. The PCR

tubes were centrifuged for 1 minute at 12,000 g and from the supernatant 5 μl were taken as a

template in the PCR reaction. PCR reaction mixture was prepared by taking two oligonucleotide

primers of the required gene present in the plasmid (50 pmol of each primer), four

deoxynucleotide triphosphates (dNTPs) each of 0.4 mM, 2.5 U Taq polymerase (Fermentas #

EP0402), 5 μl of 10 x PCR buffer containing ammonium sulphate, 5 μl of template plasmid and

MgCl2 (1.5-4 mM) was added to make total reaction mixture of 50 μl. The amplification was

done in a Thermocycler (Applied Biosystem 2720 Thermo Cycler) using similar conditions as

used to amplify the required gene.

2.9 Sequence analysis

2.9.1 Q/A prep spin miniprep kit method

This protocol is designed for purification of up to 20mg of high copy plasmid DNA from 1-5 ml

overnight cultures of Ecoli in LB medium.

39

Resuspended pelleted bacterial cells in 250ml. Buffered P1 and transferred to a microcentrifuge.

Ensured that RNase A has been added. Buffered P1. No cell clumps should be visible after

resuspension of the pellet. Added 250ml Buffer P2 and gently inverted the tube 4-6 times to mix.

Mixed gently by inverting the tube. Did not vortex as this would result in snearing of genomic

DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly

clear. Did not allow the lysus reaction to proceed for more than 5min. Added 350ml. Buffered N3

and inverted the tube immediately but gently 4-6 times. To avoid localized precipitation, mixed

the solution gently but thoroughly, immediately after addiction of buffer N3. The solution should

become cloudy.

Centrifuged for 10min at 13,000 rpm (-17, 900xg) in a table top microcentrifuge. A compact

white pellet will form. Applied the supernatant from step 4 to the Q/A prep spin column by

decanting or pipetting. Centrifuged for 30-60s. Discarded the flow through. (Optional): washed

the Q/A prep spin column by adding 0.5ml. Buffered PB and centrifuging for 30-60s.

Washed Q/A prep spin column by adding 0.75ml. Buffered PE and centrifuging for 30-60s.

Discarded the flaw through and centrifuged for an additional 1 min to remove residual wash

buffer. Important: Residual wash buffer will not be completely removed unless the flaw- through

is discarded before this additional centrifugation. Residual ethanol from buffer PE may inhibit

subsequent enzymatic reactions. Placed the Q/A prep column in a clear 1.5 ml microcentrifuge

tube. To elude DNA, added 50ml. Buffer EB (10mM Tris.Cl2, ph 8.5) or water to the centre of

each Q/A prep spin column, let stood for 1 min and centrifuged for 1 min.

Ethanol precipitation for sequencing reaction:Prepared a labeled, sterile 0.5ml microfuge tube for

each sample. Prepared fresh stop solution/ Glycogen mixture as follows (per sequencing reaction):

2µl of 3M Sodium Acetate (pH 5.2)

2µl of 100m M Na2 EDTA (pH 8.0)

1µl of 20 mg/ml of glycogen supplied with the kit.

40

To each of labeled tubes, added 5µl of the stop solution/ glycogen mixture. Transferred the

sequencing reaction to the appropriately labeled 0.5ml microfuge tube and mixed thoroughly.

Added 60ml of cold absolute ethanol from -20o C freezer and mixed thoroughly. Kept it on ice for

20 minutes. Centrifuged it on 12,000 rpm at 4o C for 20 minutes. Rinsed the pellet 2 times with

70% cold ethanol from -20o C 100µl each. For each rinsed, centrifuged immediatelyat 12,000 rpm

at 4o C for 2minutes. After centrifugation carefully remove all of the supernatant by micropipette.

Vacuumed dry for 10 minutes (or until dry). Resuspended the sample in 40µl of sample loading

solution.Stored it at -20o C.The reverse Primer used was M13/PUC reverse sequencing Primer (-

46), 24-mer 5’-d (GAGCGGATAACAATTTCACACAGG)-3’ .which had Tm: 70o C .CG

content: 46%. This primer is a synthetic single-stranded 24-mer oligonucleotide with free 5’ and

3’- hydroxyl ends. The primer anneals to the 5’- terminus of the lac Z gene such that its 5’- end

lies 46 nucleotides upstream of the ECOR l site of M13 mP18. The DNA polymerase extension

products grew in the direction that coincides with the transcription of lacZ gene. The primer was

supplied as an aqueous solution and can be used for DNA sequencing of all phages, phogennides

and plasmids carrying lacZ gene. Each lot of M13/PUC reverse sequencing Primer was assayed in

the PUC 19 DNA dideoxy sequencing reaction.

2.9.2 Analysis of Full-Length ST Gene

2.9.2.1 Extraction of genomic DNA

DNA was isolated from the blood samples of local ovine breed (Lohi) by using chloroform

phenol extraction method.

10ml blood was drawn to falcon tubes having 400ml of 0.5M EDTA, mixed gently and frozen the

samples. After few days the blood samples were taken from the freezer and proceeded the

extraction method.

TE buffer was added to all tubes up to the level of 45ml (22.5ml for 5ml blood). Samples were

shaken vigorously to resuspend all the cells evenly in TE buffer (this Hcl pH 8.00 10 m M, EDTA

pH 8.00 : 2mM). Then samples were centrifuged on table top centrifuge at 3000 rpm for 25 min at

41

25o C. Upper layer of the buffer was removed leaving 25ml in the tube ( 12.5ml in tube for 5ml of

blood) and the remaining layer was shaken vigorously to resuspend the pellet. TE buffer was

added to each tube up to (22.5) 45ml and centrifuged at 3000 rpm for 25min at 25o C. Upper layer

was poured off leaving 15ml in tubes. Pellet was shaken to resuspend evenly. TE buffer was

added to make the volume up to 45ml (22.5ml) and centrifuged at 3000 rpm for 25min at 25o C.

Again upper layer was poured off leaving 5ml of samples containing pellet. Pellets were

resuspended and volumes were roused up to (22.5) 45ml of TE buffer.

Samples were centrifuged at 3000 rpm for 25min at 25o C. After centrifugation, liquid layer were

poured off. In this way all the red blood cells were washed away. TNE buffer was added to falcon

tubes containing pellets of white blood cells according to blood volume (for 10ml blood = 6ml

buffer) ( for 5ml = 3ml buffer). Resuspended the pellet in it. 10% SDS was added to each tube

according to amount of blood (200ml SDS = 10ml blood) ( 100ml SDS = 5ml blood). Then

proteimase K (10mg/ml stock) was added according to blood volume (50ml for 10ml blood)

(25ml for 5ml blood) incubated at 37o C for 14 hours (overnight) at continuous shaking. Next day

1ml of GM Nacl (1ml for 10ml blood) (0.5ml for 5ml) was added and shaken vigourously until it

became foamy. Tubes were placed on ice for 15min. 2ml (1ml for 5ml) phenal chloroform

solution was added tilted 20 times gently and centrifuged at 3000 rpm for 20min. Upper clear

layer were taken to labeled falcon tubes (labeled with pedigrees & names). Isopropanal was added

in equal volume & centrifuged at 3000 rpm for 15min.

Supernatants were discarded & washed the DNA with 5ml (2.5ml) ethanol 70% and centrifuged

at 3000 rpm for 15min. DNA pellets were dried by inverting the tubes on the table top after

discarding the supernatant. TE buffer was added according to the amount of blood ( added 1.5ml

TE buffer (This Hcl pH 8.00: 10mM, EDTA pH 8.00: 0.1mM) for 7.5, 8, 9, 10ml blood, 1ml TE

buffer for 4, 5, 6ml blood and 20ml TE buffer for ml blood). Then the DNA samples were

incubated by placing the tubes at 37o C for overnight at continuous shaking. Next day, samples

42

were given heat shock at 70o C for 1hour to inactivate any remaining nucleases and poured to

labeled screw cap tubes.

DNA concentrations were determined by agarase gel electrophonesis or by spectrophotometery.

Working DNA concentrationn was kept at 25mg /ml & 2ml was used per PCR reaction.

2.9.2.2 PCR amplification of GH gene

On the basis of conserved sequences 4 reverse and 4 forward designed primers were used in the

amplification of growth hormone gene.

P1F(CCAGTTCACCAGACGACTCAG), P1R(TTGAAGGTGTCAGCAGCCAG)

P2F(CCCTGGACTCAGGTGGTG), P2R(GATGGTTTCGGAGAAGCAGA)

P3F(GGGACAGAGATACTCCATCCAG) P3R(CCTTCAGCTTCTCATAGACACG)

P4F(TCGCATCTCACTGCTCCTTATC ) P4R(CAGATGGCTGGCAACTAGAAC)

PCR amplification of the first strand was carried by fermentas Taq DNA polymerase in applied

biosystem ‘s2720 thermalcycler. Denaturation, annealing and extension were carried out,

respectively at 94, 60 and 72̊C with a hold time of I minute each for 35 cycles.

2.9.2.3 Sequencing reaction

Purified PCR products of DNA and cDNA were T/A cloned to pTZ57R/T vector by using dT.

dA tailing technique. Five constructs, OST1(for coding sequence analysis) and OST 2, OST 3,

OST 4, OST 5(for genomic DNA analysis) were developed. The recombinant clones were

identified by blue /white screening and confirmed by colony PCR. The nucleotide sequencing

was performed by using CEQ-800 sequencing analyzer.

43

2.10 Bioinformatics tools for sequence analysis

The nucleotide and amino acid sequences of GH were obtained by database homology

programs BLASTN and BLASTP available at www.expasy.org. The sequences were aligned and

phylogenetic tree was constructed by using neighbor joining method on MUSCLE alignment

software (http://www.phylogeny.fr/). The secondary and 3-D structures were determined by

using SWISS-MODEL (www.swissmodel.expasy.org) and the structures were visualized on

VMD version 1.8.7.

2.11 Mini-preparation of plasmid DNA

Preparation of cells

o Inoculated 2ml of rich medium (LB, YT or terrific broth) containing the appropriate

antibiotic (Ampicillum) with a single colony of transformed bacteria. Incubated the culture

overnight at 37o C with vigorous shaking. Poured 1.5ml of the culture into a microfuge

tube. Centrifuged it at maximum speed for 1 min at 4o C in a microfuge. Stored the unused

portion of the original culture at 4o C. When centrifugation was completed, removed the

medium by pupetting, leaving the bacterial pellet as dry as possible.

Lysis of cells

o Resuspended the bacterial pellet in 100ml of ice cold alkaline lysis solution I by vigorous

vortexing. Added 200 ml of freshly prepared lysis solution II to each bacterial suspension,

closed the tube tightly and mixed the content by inverting the tube rapidly 5 times. Did not

vortex, stored the tube on ice. Added 150ml of ice cold alkaline lysis solution III through

the viscous bacterial Lysate by inverting the tube several times. Stored the tube on ice for

3-5 min. Centrifuged the bacterial Lysate at maximum speed for 5 min at 4o C in a

microfuge. Transferred the supernatant to a fresh tube. Added on equal volume of phenal:

Chloroform mixed with organic and aquaus phases by vortexing and then centrifuged the

44

emulsion at maximum speed for 2 min at 4o C in a microfuge. Transferred the aquouss

upper layer to a fresh tube.

Recovery of plasmid DNA

o Precupitated nucleuc acids from the supernatant by adding 2 volumes of ethanol at room

temperature, mixed the solution by vortexing and then allowed the mixture to stand for

2min at room temperature. Cancelled the precupitated nucleuc acids by centrifugation at

maximum speed for 5min at 4o C in a microfuge. Removed the supernatant by gentle

aspiration, stood the tube in an inverted position on a papertowel to allow all of the fluid to

draw away. Used disposable pipette tip to remove any drops of fluid adhering to the walls

of the tube. Added 1ml of 70% ethanol to the pellet and inverted the closed tube several

times. Recovered the DNA by centrifugation at maximum speed for 2min at 4o C in a

microfuge. Again removed all of the supernatant by gentle pipetting. Removed any beads

of ethanol that formed on the sides of the tube. Stored the open tube at room temperature

until the ethanol had evaporated and no fluid was visible in the tube (5-10min). Dissolved

nucleuc acid in 50ml of TE (PH 8.0) store at 4o C. Vortexed the solution gently for few

secondes. Stored DNA solution at – 20o C.

2.12 Restriction analysis of pTZ-oGH clones

The plasmids were screened and confirmed by restriction analysis and reactions were prepared in

1.5 ml eppendorf tubes. Recombinant plasmid i.e. pTZoGH was digested with NcoI/BamHI and

NdeI/BamHI. The reaction was made as follows; 25 μlof recombinant plasmid (10 μg), 1 μl of

(10 U/μl) each enzyme, 10 μl of 10 x Orangebuffer [50 mM Tris-Cl (pH 7.5 at 37°C), 10 mM

MgCl2, 10 mM NaCl and 0.1 mg/mlBSA] Volume was made up to 100 μl using autoclave

distilled water and tubes were then incubated at 37°C for 16 hrs.Restriction analysis was

checked on 1 % agarose gel electrophoresisand required gene was gel excised and purified by

using Vivantis GF-1 Gel DNARecovery Kit (Cat # VSGF-GP-100) illustrated in section 2.2.2.4.

45

2.13 Restriction analysis of pET22b (+)

For restriction analysis of pET22b (+) vector, 100 μl reaction was made by adding 20 μl of

pET22b vector (4 μg), 10 μl of 10 x Red buffer, 1 μl each of (NcoI/BamHI and NdeI/BamHI

)enzyme in respective construct and volume was made up to 100 μl with distilled water. Tube

was incubated for 5 hrs at 37°C. Second restriction of pET22b was made by using 1 μl each

enzyme, (NcoI/BamHI and NdeI/BamHI ) 10μl of 10 x Orange buffer, 20 μl of pET22b (+)

vector (4 μg) and water was added to make up the volume to 100 μl. Tube was incubated at 37°C

for 16 hrs. Restrictions were confirmed by running 1 % agarose gel electrophoresis and restricted

bands were excised from the gel and gel purified by using Vivantis GF-1 Gel DNA Recovery Kit

(Cat # VSGF-GP-100) described earlier.

2.14 Ligation and transformation in DH5α and BL21 Codon + strains

Ligation of oGH gene fragments were ligated in the pET22b (+) vector .The restricted purified

fragment oGH was ligated in pET22b (+) restricted and purified]. Ligation mixture was made in

1.5 ml eppendorf tube by adding, 2 μl of 10 x ligation buffer (400 mM Tris-Cl, 100 mM MgCl2,

100 mM DTT, 5 mM ATP, pH 7.8 at 25°C), 2 μl of restricted and purified pET22b vector, 5 μl of

restricted and purified OaST product, 1 μl of T4 DNA ligase (5 U) and water was added to make

up the volume up to 20 μl. Ligation mixture was incubated at 22°C for 18hrs.

The ligated plasmid was then transformed in DH5α as stated before.

The bacterial colonies were confirmed by colony PCR and restriction analysis (using restriction

enzymes i.e. NdeI/BamHI, NcoI/BamHI ). The positive transformants were then transformed in

BL21 Codon + strain to obtain expression of the oGH gene.

46

2.15 Expression of poGH clones

The pET system is one of the most commonly used systems for cloning and expression of genes

in E. coli host. The target genes in this system are under the control of the strong bacteriophage

T7 transcription signal. The T7 RNA polymerase promoter is one of the strong promoters, when

it is fully induced it uses most of the host resources to synthesize the target protein. At the same

time the expression level can be controlled by the use of inducer. The target protein expression

can be initiated either by infecting the host cells with λCE6, a phage carrying the T7 RNA

polymerase gene or by transferring the plasmid into an expression host which carries a

chromosomal copy of T7 RNA polymerase gene under the control of lacUV5 and for this IPTG

is required as inducer. In this study the genes were cloned and expressed in pET22b (+) vector

which has a T7 lac promoter .

Figure 4.Restriction map,sequence and multiple cloning sites of pET 22b(+). Vector was used for expression of all poGH constructs

47

In this vector the lac operator sequence exists just downstream the T7 promoter region in the

plasmid and carries the natural promoter and lacI (lac repressor). The T7 RNA polymerase gene

and lacI are diverging. The lac repressor acts at the lacUV5 promoter in the host chromosome to

repress the T7 RNA polymerase gene and at the T7 lac promoter in the vector to prevent the

transcription of the target gene. The plasmid has 5493 bp size and carries an N-terminal pelB

signal sequence for potential periplasmic localization, His- Tag at C-terminal and ampicillin

resistant marker gene for selection.

For expression studies a single colony from poGH clones in BL21 Codon Plus was picked

and inoculated in 10 ml LB medium (100 μg/ml ampicillin) in 100 ml Erlenmeyer flask.

The flasks were incubated in shaking incubator (Irmeco GmbH, Germany) at 37°C with

100 rpm. Next morning one percent of cultures were refreshed in 25 ml LB medium (100

μg/ml ampicillin) and grown under same conditions as mentioned above.

The OD was checked at 600 nm after 2 1/2 hrs, when OD reached at 0.5-0.6 cells were

induced with 0.5 mM IPTG. After 4 hrs of post induction 1 ml sample was drawn from

each culture and centrifuged for 3 minutes at 12,000 g. The supernatant was discarded and

pellet was resuspended in 100 μl of lysis buffer (50 mM Tris-Cl pH 8.5 + 5 mM EDTA + 1

mM PMSF + 100 mM NaCl)

Samples were properly mixed with 3cc syringe to reduce the viscosity and denatured at

85°C for 5 minutes. Samples were then short spin and loaded on to the 12 % SDS gel, 5 μl

of protein marker (Fermentas SM0661) was also loaded for protein molecular weight

determination.

2.16 SDS-Polyacrylamide Gel Electrophoresis (PAGE)

To analyze the total cell protein and molecular size of the expressed proteins, one

dimension SDS-PAGE was done (Laemmli, 1970).

48

By adjusting the SDS-PAGE assembly (Bio-Rad), 12 % resolving gel [1.6 ml H2O, 2.0 ml of 30

% acrylamide (Bio- Rad), 1.3 ml of 1.5 M Tris (pH 8.8), 0.1 ml of 10 % SDS, 0.05 ml of 10 %

APS (freshly prepared) and 0.002 ml of N, N, N’, N’ tetra methyl ethylene diamine (TEMED)]

was prepared and poured in the assembly leaving 0.5 inch vacant at the top. Approximately 1ml of

distill water was poured on to the resolving gel to get the flat surface and to remove air bubbles.

Gel was left to polymerize for half an hour.

After polymerization, water was removed and 5 % stacking gel [3.4 ml H2O, 0.83 ml of 30 %

acrylamide (Bio-Rad), 0.63 ml of 1 M Tris (pH 6.8), 0.1 ml of 10 % SDS, 0.05 ml of 10 % APS

(freshly prepared) and 0.005 ml of N, N, N’, N’ tetra methyl ethylene diamine (TEMED)] was

prepared and poured on to the polymerized resolving gel and immediately comb was inserted so

that wells should be formed for sample loading. Gel was again left for 30 minutes for proper

polymerization, after that comb was removed carefully from the gel. The gel was assembled in a

SDS-PAGE apparatus (Mini-PROTEAN 3, BIO-RAD) and 1 x Trisglycine buffer (25 mM Tris-

Cl, 250 mM glycine and 0.1 % SDS) was poured in theelectrophoresis chamber.

Wells were washed with the above mentioned buffer and ~ 9 μl samples (5-15 μg protein) were

prepared by mixing equal volume with 2 x loading dye [100 mM Tris (pH 6.8), 20 % (v/v)

glycerol, 4 % (w/v) SDS, 200 mM dithiothrietol(DTT) and 0.2 % (w/v) bromophenol blue].

Samples were then properly mixed with the loading dye by using 3cc syringe and denatured by

boiling at 85°C for 5 minutes.

Samples were short spin and then loaded on to the gel and 5 μl of protein marker (Fermentas

SM0661) was also loaded for protein molecular weight determination. The gel was

electrophoresed at 120 volts for 1 hr and 30 minutes at RT. Electrophoresis was stopped when the

dye seemed to reach 1 mm close to the bottom of the gel.

The gel assembly was taken out of the chamber and plates were separated carefully by using

spatula. Gel was then put in a staining solution (50 mg Coomassie Brilliant Blue R250, 450 ml

methanol, 90 ml acetic acid and 450 ml H2O) for 20 minutes with gentle rotation.

49

Gel was then removed from staining solution and placed in destaining solution (50 ml methanol,

70 ml acetic acid and volume made up to 1 L) to get protein bands prominent and background

becomes transparent.

Densitometry was carried out on gels using Dolphin imager (WEALTEC, USA). The gels were

placed inside an image and illuminated with white light exposure. After that, automatic

background subtraction identical areas around the protein bands were selected for analysis. The

internal OD of all the proteins in a gel was calculated and %age of the target protein band in all

the expressed proteins was calculated by the formula as follows:

%age of expressed protein = Internal OD of the target protein x 100/

Sum of internal OD of all the proteins.

2.17 Western transfer and immunoblot analysis

The protein samples resolved by 13% SDS-PAGE were transferred to a 0.45m

nitrocellulose membrane using mini transfer-blot electrophoretic transfer cell at 50V for one

hour. The transfer buffer was 25mM Tris-Cl (pH 8.3), 192mM glycine and 20% (v/v) methanol.

To block the non-specific membrane sites, the blot was incubated in TBS-T buffer [20mM Tris

(pH 7.6), 13 mM NaCl, 0.1% (v/v) Tween 20] containing 2% (v/v) gelatin and 0.4% (v/v)

sodium azide, for 30 minutes. After blocking, the membrane was successively incubated with

rabbit anti-bovine growth hormone (1:2,000 dilutions) and IgG alkaline phosphatase conjugate

(1:6,000 dilutions) for 1 hour at room temperature with gentle shaking on a rotatory shaker. Each

successive incubation was followed by 3x10 minutes washings with TBS-T buffer. Finally, the

membrane was stained with chromogenic detection substrates [100l of 75mg/ml NBT and

150l of 25mg/ml BCIP in 25ml carbonate buffer (8.4 g/L NaHCO3, 0.203 g/L MgCl2.6H2O, pH

9.8)] until the signal was clearly visible (30-60 seconds). Chromogenic reaction was stopped by

rinsing the membrane twice with distilled water followed by image scanning of dried membrane.

50

2.18 Protein estimation

Protein concentration was determined either by absorption measurements at λ280 or dye-

binding method of Bradford (1976) using bovine serum albumin (BSA) as standard. The dye-

binding reagent was prepared by dissolving 100mg of Coomassie brilliant blue G250 in 50ml

ethanol and 100ml ortho-phosphoric acid. Volume was made up to 1liter with distilled water and

the reagent was filtered twice before use. For assay, appropriately diluted protein sample was

mixed with 5 ml dye-binding reagent and incubated at room temperature for 10-15 minutes prior

to absorbance measurements at A595. A standard curve was thereafter plotted with known

concentrations of BSA and used to calculate the concentration of protein in test samples

2.19 Primer designing for translocation of Ovine ST gene into periplasmic space

Eight constructs based on the DsbAss with amino acid variations upstream of the 5'- start codon

of OaST gene were produced. The primers (FP1-FP8) were designed as shown in Table 1.The

forward primer had NdeI site at the 5'-end, and the reverse primer had BamHI site at the 5'-end.

The wild-type growth hormone sequence in the construct pTZoGH-1 was amplified using each

of the forward and reverse primers (FP-1 to FP_8). These PCR products were then digested with

NdeI and BamHI and ligated into the NdeI/BamHI site of pET22b thus generating a series of

recombinant plasmids (poGH-3-I-VIII) as shown in Table 1. These constructs were transformed

into E. coli DH5 .and then into E. coli BL21 codon plus cells for the expression studies.

51

Table 1. sequence of primers used for construction of poGH-3-I-VIII plasmids

Primer Name *Nucleotide Sequence (5ʹ→ 3ʹ)

FP-1

CAT ATG AAA AAG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA GCG

TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC

FP-2

CAT ATG AAA AAG ATT TGG CTG ATT CTG GCT GGT TTA GTT TTA GCG

TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC

FP-3

CAT ATG AAA AAG ATT TGG CTG ATT CTG ATT GGT TTA GTT TTA ATT

TTT AGC ATT TCG GCG GCC TTC CCA GCC ATG TCC

FP-4 CAT ATG AAA AAG ATT TGG CTG ATT CTG ATT GGT TTA GTT TTA GCG

TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC

FP-5

CAT ATG AAA AAG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA ATT

TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC

FP-6

CAT ATG AAA AAG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA ATT

TTT AGC ATT TCG GCG GCC TTC CCA GCC ATG TCC

FP-7

CAT ATG AGA AGG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA GCG

TTT AGC GCA TCG GCG GCC TTC CCA GCC ATG TCC

FP-8 CAT ATG AAA AAG ATT TGG CTG GCG CTG GCT GGT TTA GTT TTA GCG

TTT TGT GCA TGT GCG GCC TTC CCA GCC ATG TCC

*The sequence shown in bold and italics represents the OaST cDNA sequence whereas the underlined sequence is the site for

NdeI restriction enzyme. Nucleotide variation incorporated in DsbA signal sequence are highlighted in grey

These PCR products were then digested with NdeI and BamHI and ligated at the NdeI/

BamHI site of pET22b thus generating a series of recombinant plasmid pEToGH2-11.While the

reference constructpTZ-oGH1 was double digested with Nco I and BamHI and ligated at the

NcoI/ BamHI site of pET22b thus generating a reference vector poGH-1.These constructs were

transformed into E. coli DH5 and recombinant plasmids were isolated. The reverse primer

;PBGH3 (5`TAG GAT CCG CAA CTA GAA GGC AGC 3`)was used. PCR amplification of the

first strand was carried by fermentas Taq DNA polymerase in applied biosystem ‘s2720

thermalcycler. Denaturation,annealing and extension were carried out, respectively at 94̊,60̊ and

72̊ C with a hold time of 1 minute each for 35 cycles.

52

2.20 Subcellular fractionation of oGH

E. coli BL21 codon plus was transformed with different constructs, and cells from a single

colony of each of the transformant were transferred to LB medium containing 100 g/mL

ampicillin and grown overnight at 25 C in an orbital incubator shaker. LB medium (10 mL) was

inoculated with 0.25 mL of overnight culture of each construct and induced with 1mM IPTG

when the OD600 reached 0.6. After 5h of induction, 1.5 mL culture of cells carrying each

construct was centrifuged, and the pellet was suspended in 100 L of PBS buffer (137 mM

NaCl2, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) and 100 L of reducing buffer [100

mM Tris-Cl pH 6.8, 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 200mM

dithiothreitol] was added. Total cell protein analysis of the lysate was done by SDS-PAGE using

15% gel according to the method of (Laemmli, 1970). Resolved proteins were visualized by

staining with commassie brilliant blue. The protein profile of the samples after SDS-PAGE was

analyzed densitometrically using Dolphin-ID gel analysis software (Wealtec).

Freeze thaw

cells were harvested by centrifugation at 3,700 × g for 10 min at 4°C. For all the following steps,

tubes were kept on ice. The bacterial pellet was centrifuged, and its wet weight was

determined.Cells were frozen at −196°C. After thawing, the cells were resuspended in 75 mM

Tris-HCl (pH 8)–300 mM NaCl–1 capsule of protease inhibitors/50 ml (Complete; Roche

Diagnostics, Mannheim, Germany)–5 mM dithiothreitol–10 mM EDTA–10% (vol/vol) glycerol

and were sonicated six times for 30 s at 200 W. The periplasmic fraction was recovered after

centrifugation at 21,000 × g for 30 min at 4°C and was transferred to 75 mM Tris-HCl (pH 8)–1

M NaCl–10% (vol/vol) glycerol using Hitrap desalting columns (Pharmacia).

53

Osmotic Shock method ( a )

Periplasmic osmotic shock fluid was obtained by the method of( Koshland and Botstein, 1980).

Briefly, a volume of fermentation broth corresponding to 100 A600 was harvested by

centrifugation at 3000 g for 5 min. All subsequent steps were carried out at 4°C in an ice bath.

Pellets were resuspended in 1.0 ml of ice‐cold 10 mM Tris–HCl, pH 7.5, containing 20% (w/v)

sucrose. Then, 33 µl of 0.5 M EDTA, pH 8.0, were added and incubation on ice continued for 10

min. The cells were then centrifuged and the pellet rapidly resuspended by vigorous agitation in

1.0 ml of a cold 1 mM Tris–HCl, pH 7.5, solution. The suspension was then incubated for 10

min on ice and centrifuged again for 5 min. The supernatant was removed and saved as the

periplasmic fraction, also called osmotic shock fluid.

Osmotic shock method ( b ) We followed the protocol by late Dr.Mustak kaderbhai (Kaderbhai

et al., 2008).100ml culture of grown cells for 6 hours in LB medium at 25°C,150rpm were

harvested by centrifugation at 5,000g for 5minutes.Discarded the supernatant and resuspended

pellet in 20ml of STE (20%sucrose in 0.33M tris,1mM EDTA pH8.0).Left the resuspended

solution at room temperature for 10 minutes and then centrifuged at 7,500 g for 10minutes at 4°

C,discarded the supernatant while leaving behind just 250µl and resuspend pellet thoughrouly by

adding 10ml of chilled 0.5mM MgCl2.After resuspension left it on ice for 10minutes and then

centrifuged at 5,000g for 5min,at 4° C.Collected the supernatant as a shock fluid and used the

pellet for further fractions.

Sonication;Resuspended the pellet in 20ml TE (0.3M Tris, 1mM EDTA pH 8.0)and sonicated for

6 bursts of 30seconds each with interval of 3 minutes.Centrifuged the sonicated sample at 5,000

rpm for 20 minutes at 4o C. Used the supernatant as cytoplasmic fraction and pellete for further

fractionation.

Ultracentrifugation ;Washed the pellet with 5ml0.3 M sucrose and centrifuged at 11,000rpm for

10minutes with(0.3M sucrose,0.3M Tris,pH8.0).Took supernatant and ultracentrifuged it at

100,000g for 1hour,collected the pellet and resuspended it in 5ml 0.3M tris pH8.0.

54

2.20.1 FPLC chromatography

Anion exchange Resource-Q column (1.6 x 3.0 cm) was used and was pre-equilibrated with 2

column volumes of 20 mM Tris-HCl at pH 8.0. The filtered protein was then loaded and

separated using Amersham Biosciences FPLC (Amersham Pharmacia Biotech). The bound

protein was eluted with a continuous NaCl gradient (0.1-1 M) at a flow rate of 1ml/min and

dialyzed against 20 mM Tris-HCl (pH 8.0) to remove the salt.

2.20.2 MALDI-TOF

The mass spectrometry was performed on Bruker Autoflex III smart beam MALDI-TOF/TOF

(Bruker Daltonics GmbH, Fehrenheit str.-4, Bermen). 1 µg/µl of purified OaST-2 protein was

mixed with 3, 5 dimethoxy-4-hydroxycinnamic acid and 1 µl of the protein was deposited on

stainless steel target plate of the mass spectrometer. The machine was calibrated against the

standard protein BSA

2.21 Biological activity assessment assay

To analyze the biological activity of the purified roGH, HeLa cell lines were used. BSA and

commercially available bovine GH were used as negative and positive control, respectively. Prior to

the assay, HeLa cells were arrested at G0/G1 phase.Then, 30,000 HeLa cells/200 μl/well was taken;

500 μl of DMEM and 2 % fetal bovine serum (FBS) were added. BSA, bovine GH, and roGH each

of about 10 μg were added in duplicate wells (having 30,000 HeLa cells, 500 μl DMEM, and

2%FBS). The cells were then incubated for 24 h at 37 °C, and after incubation, cells were counted by

using inverted microscope (OLYMPUS IX51). By microscope, five boxes/ well were visualized and

cells of each box were counted and average number of cells in a well was calculated. The number of

cells per milliliter and in 200 μl was calculated as follows:

55

Average no. of cells x 10,000 = per ml (1000 μl)

&

No. of cells in 200 μl = average no. of cells x 10,000 x 200

1000

56

57

RESULTS

58

3.1 Genetic Analysis of oGH gene

3.1.1 Extraction of genomic DNA

The genomic DNA was isolated from the blood sample of local ovine breed Lohi by

chloroform phenol extraction method as explained in materials and method (Fig.5 ).

Figure 5.Genomic DNA of oGH isolated from the blood sample of local ovine breed lohi Lane 1, genomic DNA; lane 2, DNA Marker.

3.1.2 PCR amplification of oGH gene

The coding and non-coding regions of oGH were amplified by using five sets of gene-

specific primers as described under materials and methods. PCR amplification yielded single

products of expected length i.e. 459-, 422-, 462-, 639- and 640-bp as shown in Fig.6.

59

Figure 6. PCR amplification on 1% agarose gel.

M, 1 kb DNA ladder; Lane 1, PCR amplified product 422, lane 2, 3, 4 & 5 PCR amplified product 459, 639, 462 and 640

respectively

The best amplification of 422-,459, 462-bp long DNA fragments were achieved at an

annealing temperature of 53C while that of 640- and 639-bp DNA fragments, at 55C.

Following analysis, the amplicons were gel purified and their sequence determined.

3.1.3 Sequence analysis of oGH

The sequencing reaction was performed both in the forward and reverse directions to

resolve discrepancies, if any. Ovine growth hormone full gene sequence, capital letters (exons)

small letters (introns), mature peptide in green colour, red colour shows signal nucleotide region

and purple shows stop codon. The oGH shows 5 exons and 4 introns.as shown in Fig.7.

60

ATGATGGCTGCAggtaagctcacgaaaatcccctccattagcgtgtcctaagggggtgatgcggggggccctgccgatggatg

tgtccacagctttgggttttagggcttctgaatgtgaacatgggtatctgcacccgacatttggccaagtttgaaatattctcagtccctggagg

gaagggcaggcggggctggcaggagatcaggcgtctagctctctgggcccctccgtcgcggccctcctggtctctccctagGCCCC

CGGACCTCCCTGCTCCTGGCTTTCACCCTGCTCTGCCTGCCCTGGACTCAGGTGGTGG

GCGCCTTCCCAGCCATGTCCTTGTCCGGCCTGTTTGCCAACGCTGTGCTCCGGGCTCA

GCACCTGCATCAACTGGCTGCTgacaccttcaaagagtttgtaagctccccagagatgtgtcctagaggtggggaggc

aggaagggatgaatccgcaccccctccacacaatgggagggaactgaggacctcagtggtattttatccaagtaaggatgtggtcagggg

agtagaagtgggggtgtgtggggtggggagggtccgantaggcagtgaggggaaccccgcaccagttgagacctgtgtgggtgtgtcct

ccccccaggagcgcacctacttcccggaGGGACAGAGATACTCCATCCAGAACACCCAGGTTGCCT

TCTGCTTCTCCGAAACCATcccagcccccacgggcaagaatgaggcccagcagaaatcagtgagtggccacctagga

ccgaggagcaggggacctccttcatcctaagtaggctgccccagctctctgcaccgggcctggggcgtccttctccccgaggtggcagag

ggtgttggatggcagtggaggatgatggttggtggtggtggcaggaggtcctcgggcagaggccgaccttgcagggctgccccgagcc

cgcagcaccgaccaaccacccatctgccagcaggacttggagctgctTCGCATCTCACTGCTCCTTATCCAGTC

GTGGCTTGGGCCCCTGCAGTTCCTCAGCAGAGTCTTCACCAACAGCCTGGTGTTTGG

CACCTCGGACCGTGTCTATGAGAAGCTGAAGGacctggaggaaggcatcctggccctgatgcgggtgagg

atggcattgttgggtcccttccatgctgagggccatgctcaccctctcctggcttagccaggagaacacacgtgggctgggggagagagat

ccctgctctctctctctttctagcagcccagccttgacccaggagaaacctcttccccttttgaaacctccttcctcgcccttctccaagcctata

ggggagggtggaaaatggagcgggcaggagggagccgctcctcagggcccttcggcctctctgtctctccctcccttggcaaGAGC

TGGAAGATGTTACCCCCCGGGCTGGGCAGATCCTCAAGCAGACCTATGACAAATTT

GACACAAACATGCGCAGTGATGATGCGCTGCTCAAGAACTACGGTCTGCTCTCCTGC

TTCCGGAAGGACCTGCACAAGACGGAGACGTACCTGAGGGTCATGAAGTGTCGCCG

CTTCGGGGAGGCCAGCTGCGCCTTCTAG

Figure 7. Nucleotide sequence of oGH.

Capital letters (exons) small letters (introns), mature peptide in green colour, red colour shows signal nucleotide region

and purple shows stop codon. It shows 5 exons and 4 introns

All the exons were united to get the amino acid sequence of oGH gene.which comprises of

217 amino acid in which first 26 are of signal sequence and rest of 191 are mRNA of oGH.

Figure 8.Amino acid sequence of oGH Amino acid sequence .oGH shows that it comprises of 217 amino acid.While mature hormone comprises 191 amino acids start

from AFPAM in first row.

61

(a)

(b)

Figure 9.amino acid sequence of oGH. (a) Amino acid sequence of oGH isolated from local ovine breed Lohi. (b) Nucleotide sequence of oGH.

3.1.3.1 Sequence comparison of oGH at amino acid level

Amino acid sequence of ovine growth hormone isolated from local breed (Lohi) showed

that it is comprises of 191 amino acids, calculated molecular weight of 21.85kDa while

isoelectric point was 7.86 when analyzed on( web.expasy.org/protparam/). This sequence was

than aligned with locally isolated growth hormone sequences of caprine and bubaline breeds of

Pakistan. It was analyzed that amino acid sequence of ovine Lohi breed is same for GH gene

isolated from local breed of caprine and has difference with local breed of bubaline at positions

9, 130 and 140 as shown in Fig.10.

62

Figure 10.Comparison of growth hormones of ovine capricorn and bubaline

Comparison of 3 locally isolated growth hormones of ovine, caprine and bubaline at amino acid level.

The amino acid sequence of Pakistani ovine breeds (Lohi) accession no. AB244790

showed difference with the amino acid sequence of growth hormone isolated from Australian

and Indian breeds when compared on Clustal W 1.81 for sequence alignment

(www.clustal.org/clustal2/). It showed variation of one amino acid at position 147 where

threonine is replaced with arginine as shown in Fig.11.

Figure 11,Amino acid sequence comparison. .Amino acid sequence comparison of Lohi (row 1) with Indian (row 2) accession number NM-001009315 and Australian accession

number S50877 (row3) breeds

63

Ovine growth hormone sequence was compared with the growth hormone sequence of the

species of family Bovidae and other species of class mammalian.

Homosapiens AFPTIPLSRLFDNASLRAHRLHQLAFDTYQEFEE 34

Pan AFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEE 34

Canis NAVLRAQHLHQLAADTYKEFER 22

Felis FPAMPLSSLFANAVLRAQHLHQLAADTYKEFER 34

Equus FPAMPLSSLFANAVLRAQHLHQLAADTYKEFER 33

Mustela AFPAMPLSSLFANAVLRAQHLHQLAADTYKDFER 34

Hippopotamus AFPAMPLSSLFANAVLRAQHLHQLAADTYKEFER 34

Camelus AFPAMPLSSLFANAVLRAQHLHQLAADTYKEFER 34

Ovis AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFER 34

Lohi AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFER 34

Bos AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFER 34

Capra AFPAMSLSSLFANAVLRAQHLHQLAADTFKEFER 34

Bubalus AFPAMSLSSLFANAVLRAQHLHQLAADTFKEFER 34

Monodelphis AFPAMPLSSLFANAVLRAQHLHQLVADTYKEFER 34

Crocodylus FPAMPLSNLFANAVLRAQHLYLLAAETYKEFER 33

Rana FPQMSLSNLFTNAVIRAQHLHQMVADTYRDYER 33

Ambystoma AYPAAPLSSLFNHAVARARRLHQIAMDIYTDFEG 34

Cynops AFPGVSLTNLFNNAVIRAQHLHFLAADIYQEFER 34

Amia AYPSIPLYNLFTNAVIRAEHLLQLATDIYKDFER 34

Homosapiens AYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFL 94

Pan AYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFL 94

Canis AYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFL 93

Felis AYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFL 93

Equus AYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDMELLRFSLLLIQSWLGPVQLL 93

Mustela AYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDMELLRFSLLLIQSWLGPVQFL 93

Hippopotamus AYIPEGQRYS-IQNTQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFL 93

Camelus TYIPEGQRYS-IQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFL 93

Ovis TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93

Lohi TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93

Bos TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93

Capra TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93

Bubalus TYIPEGQRYS-IQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFL 93

Monodelphis TYIPEAQRHS-IQSTQTAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLSPVQFL 93

Crocodylus SYIPEEQRHS-NKNSQSAFCYSETIPAPTGKDDAQQKSDMELLRFSLVLVQSWLNPVQFL 93

Rana TYIPEDQRFS-NKHSYSVYCYSETIPAPTDKDNTHQKSDIELLRFSLLLLQSWMNPIQIV 93

Ambystoma TYISDEQRQS-SRIYQAAFCCSETIPAPTGKDDAQQRSDMELLRFSLTLIRSWLTPVQFL 93

Cynops TYIPNEQRHT-SRNSQTAFCCSETIPAPTGKDDAQQRSDIELLRFSLTLIRSWLTPVQAL 93

Amia TYVPDEQRQS-SKSSPLAGCYSESIPAPTGKDEAQQRSDVELLGFSFTLIQSWISPLQTL 93

:::.. *: : : * **:**:*:.:::::*:*::*** :*: *::**: *:* :

Homosapiens RSVFA-NSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHN 152

Pan RSVFA-NSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHN 152

Canis SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRS 151

Felis SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRGGQILKQTYDKFDTNLRS 151

Equus SRVFT-NSLVFGTSDR-VYEKLRDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRS 151

Mustela SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRAGPILKQTYDKFDTNLRS 151

Hippopotamus SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNMRS 151

Camelus SRVFT-NSLVFGTSDR-VYEKLKDLEEGIQALMRELEDGSPRAGQILRQTYDKFDTNLRS 151

Ovis SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDRNMRS 151

Lohi SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRS 151

Bos SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRS 151

Capra SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRS 151

Bubalus SRVFT-NSLVFGTSDR-VYEKLKDLEEGILALMRELEDGTPRAGQILKRTYDKFDTNMRS 151

Monodelphis SRVFT-NSLVFGTSDR-VYEKLRDLEEGIQALMQELEDGSSRGGLVLKTTYDKFDTNLRS 151

Crocodylus SRVFT-NSLVFGTSDR-VFEKLRDLEEGIQALMRELEDGSHRGPQILKPTYEKFDINLRN 151

Rana NRVFG-NNQVFGNIDR-VYDRLRDLDEGLHILIRELDDGNVRNYGVLTFTYDKFDVNLRS 151

Ambystoma SSVLT-NSFVFGSSDK-VYERLKDLEEGIQTLIRELDDGSPRGSSLLKLTYDNFDANQRN 151

Cynops SNVFFPNSFVFGTSER-VYERLKDLEEGIQTLIKELDDGSPRGFSLLKLTYDGFDANQRN 152

Amia SRAFS-NSLVFGTSDR-IFEKLKDLEEGIMVLMRGLDEGNPRLLGAQTLTYEKFDINLRN 151

.: *. *:. : ::: *:**:**: :: *: * **. ** * .

Homosapiens DDALLKNYGLLYCFRKDMDKVETFLRIVQCR-SVEGSCGF 190

Pan DDALLKNYGLLYCFRKDMDKVETFLRIVQCR-SVEGSCGF 190

Canis DDAL------------------------------------ 155

Felis DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191

Equus DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191

Mustela DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191

Hippopotamus DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191

Camelus DDALLKNYGLLSCFKKDLHKAETYLRVMKCRRFVESSCAF 191

Ovis DDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF 191

64

Lohi DDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF 191

Bos DDALLKNYGLFSCFRKDLHKTETYLRVMKCRRFGEASCAF 191

Capra DDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF 191

Bubalus DDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF 191

Monodelphis DEALLKNYGLLSCFKKDLHKAETYLRVMECRRFVESSCAF 191

Crocodylus EDALLKNYGLLSCFKKDLHKVETYLKLMKCRRFGESNCSI 191

Rana EEGRAKNYGLLSCFKKDMHKVETYLKVMKCRRFVESNCTF 191

Ambystoma EDALFRNYGLLSCFKKDMHKVETYLKVMKCRRILENNCTI 191

Cynops EDALFRNYGLLSCFKKDMHKVETYLKVMKCRRMLDNNCTI 191

Amia DDALMKNYGLLACFKKDMHKVKTYLKVMKCRRFVESNCTL 191

: .

Figure 12.comparison of oGH with different species of class mammalia

The sequence of the growth hormone(GH) gene or GH cDNA of Cetartiodactyla species

were downloaded from GeneBank . The Clustal W 1.81 was employed to align all the sequences

with the default option .Multiple amino acid sequence alignment of various animals (shown in

Fig. 13) was done by MUSCLE software. The amino acid sequence of growth hormone of

following species was downloaded [(Homo sapiens, accession number; AAT11509), (Pan

troglodytes, Accession number; AAL72284), (Canislupus, Accessionnumber; CAA80601),

(Felis, Accessionnumber; NP_001009337), (Hippopotamus amphibius, Accession number;

NP_001009337), (Equus caballus, Accession number; AAA21027), (Mustela vison, Accession

number; CAA42448), (Camelus bactrianus, Accession number; CAE01391),(Ovis aries,

Accession number; BAE66634), (Ovis aries (lohi), Accession number;AB24479), (Bos taurus,

Accession number; AAX0971344 ), (Capra hircus, Accession number; AAX35770), (Bubalus

bubalis, Accession number; CAA09679)]. The phylogenetic tree of multiple aligned amino acid

sequences of mammals was plotted by using neighbor joining method on

http://www.phylogeny.fr/ as shown in Fig. 13.

3.1.3.2 Comparison of oGH gene at Nucleotide level

The alignment of these three locally isolated growth hormones at nucleotide level reveal that

ovine growth hormone of Lohi is different at 16 points with locally isolated growth hormone of

Ovis aries some of them are important variation as shown by difference at amino acid level

while variation with cGH is just on 3 points and it is silent variation as shown in Fig.13.

65

ovine GCCTTCCCAGCCATGTCCTTGTCCGGCCTGTTTGCCAACGCTGTGCTCCGGGCTCAGCAC 60

caprine GCCTTCCCAGCCATGTCCTTGTCCGGCCTGTTTGCCAACGCTGTGCTCCGGGCTCAGCAC 60

bubaline GCCTTCCCAGCCATGTCCTTGTCCAGCCTGTTTGCCAACGCTGTGCTCCGGGCTCAGCAC 60

************************ ***********************************

ovine CTGCATCAACTGGCTGCTGACACCTTCAAAGAGTTTGAGCGCACCTACATCCCGGAGGGA 120

caprine CTGCATCAACTGGCTGCTGACACCTTCAAAGAGTTTGAGCGCACCTACATCCCGGAGGGA 120

bubaline CTGCATCAGCTGGCTGCTGACACCTTCAAAGAGTTTGAACGCACCTACATCCCGGAGGGA 120

******** ***************************** *********************

ovine CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTTCTCCGAAACCATCCCAGCC 180

caprine CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTTCTCTGAAACCATCCCGGCC 180

bubaline CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTTCTCCGAAACCATCCCGGCC 180

******************************************** *********** ***

ovine CCCACGGGCAAGAATGAGGCCCAGCAGAAATCAGACTTGGAGCTGCTTCGCATCTCACTG 240

caprine CCCACGGGCAAGAATGAGGCCCAGCAGAAATCAGACTTGGAGCTGCTTCGCATCTCACTG 240

bubaline CCCACAGGCAAGAACGAGGCCCAGCAGAAATCGGACTTGGAGCTGCTTCGCATCTCACTG 240

***** ******** ***************** ***************************

ovine CTCCTTATCCAGTCGTGGCTTGGGCCCCTGCAGTTCCTCAGCAGAGTCTTCACCAACAGC 300

caprine CTCCTTATCCAGTCGTGGCTTGGGCCCCTGCAGTTCCTCAGCAGAGTCTTCACCAACAGC 300

bubaline CTCCTCATCCAGTCGTGGCTTGGGCCCCTGCAGTTCCTCAGCAGAGTCTTCACCAACAGC 300

***** ******************************************************

ovine CTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAAGGACCTGGAGGAAGGCATC 360

caprine CTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAAGGACCTGGAGGAAGGCATC 360

bubaline TTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAAGGACCTGGAGGAAGGCATC 360

***********************************************************

ovine CTGGCCCTGATGCGGGAGCTGGAAGATGTTACCCCCCGGGCTGGGCAGATCCTCAAGCAG 420

caprine CTGGCCCTGATGCGGGAGCTGGAAGATGTTACCCCCCGGGCTGGGCAGATCCTCAAGCAG 420

bubaline CTGGCCCTGATGCGGGAGCTGGAAGACGGCACCCCCCGGGCTGGGCAGATCCTCAAGCGG 420

************************** * **************************** *

ovine ACCTATGACAAATTTGACACAAACATGCGCAGTGATGATGCGCTGCTCAAGAACTACGGT 480

caprine ACCTATGACAAATTTGACACAAACATGCGCAGTGACGACGCGCTGCTCAAGAACTACGGT 480

bubaline ACCTATGACAAATTTGACACAAACATGCGCAGTGACGACGCGCTGCTCAAGAACTACGGT 480

*********************************** ** *********************

ovine CTGCTCTCCTGCTTCCGGAAGGACCTGCACAAGACGGAGACGTACCTGAGGGTCATGAAG 540

caprine CTGCTCTCCTGCTTCCGGAAGGACCTGCACAAGACGGAGACGTACCTGAGGGTCATGAAG 540

bubaline CTGCTCTCCTGCTTCCGGAAGGACCTGCACAAAACGGAGACGTACCTGCGGGTCATGAAG 540

******************************** *************** ***********

ovine TGTCGCCGCTTCGGGGAGGCCAGCTGTGCCTTCTAG 576

caprine TGTCGCCGCTTCGGGGAGGCCAGCTGTGCCTTCTAG 576

bubaline TGCCGCCGCTTCGGGGAGGCCAGCTGTGCCTTCTAG 576

** *********************************

Figure 13.nucleotide sequuence alignmen

Nucleotide sequence alignment of Lohi growth hormone with locally isolated growth hormone of caprine and bubalus

bubalis. The variation is shown in form of blue color for bubalus bubalis, green for LOHI and pink for caprine.

The sequence analysis of Ovine growth hormone isolated from local ovine breed Lohi suggests

that it is in complete accordance with the already published sequence of Ovine growth hormone.

The comparison also shows that it has four to five nucleotide variations which do not affect the

amino acid sequence. The length of introns and exons of growth hormone gene of local ovine

breed (Lohi) when compared with other members of the family Mammalia showed slight

variation in the length of initial intron shown in Fig.12.

66

3.1.4 Secondary structure analysis of oGH

The secondary structure of ovine growth hormone was obtained by MINNOU server.It

showed that around 60 % of amino acid residues involved in the formation of α-helices (Fig.14).

Amongst the four key α-helices, N- and C-terminal helices were longer than the other two

helices. Most of the amino acid residues involved in helix formation was hydrophobic.

Figure 14ovine growth hormone. secondary structure

The secondary structure of ovine growth hormone predicted by chou-fasman rule. H is alpha helix, E is beta sheet and C

is coil

67

3.1.5 Hydropathy profile of oGH

The hydropathy profile of ovine growth hormone was analyzed by using kyte Doolittle

hydropathy plot . It showed that 60 % of the growth hormone is hydrophobic, while the rest were

those containing either a charged or an uncharged polar side chain as shown in Fig.15.

Figure 15.The hydropathy plot of oGH. The hydropathy plot of OST using window size 9, each peak above the central line shows that part of the hormone is

hydrophobic.

68

3.1.6 Three Dimensional structure of oGH

Further, predicted three-dimensional (3-D) structure of oGH showed the presence of four

α-helices, anti-parallel and tightly packed to form a four-helix bundle a structure (Fig.16). This

peculiar structure is very similar to known structures of ovine, caprine, bovine, porcine and

human STs.

Figure 16.3D structure of ovine growth hormone. 3D structure of ovine growth hormone was predicted by using Phyre server and taking human growth hormone as a basic

reference source. 1, 2, 3 & 4 depicts the helix and N & C terminals.

N

C

69

3.2 cDNA synthesis , cloning and Periplasmic Expression of oGH

3.2.1 Isolation and purity of total RNA

Total RNA was extracted from the anterior pituitary tissue of local ovine breed (Lohi) by

guanidium thiocyanate chloroform extraction method (Chomezynski and Sacchi, 1987). The

concentration of extracted RNA was found to be 1.94µg/mg of the pituitary tissue when

measured at λ260. The A260/A280 ratio for isolated RNA was found to be 1.82 (Fig.17), which

indicates sufficiently good purity of extracted RNA (Sambrook and Russell, 2001).

Figure 17.Absorption spectra of extracted RNA. Absorption spectra of extracted RNA from pituitary of local ovine "Lohi" at λ220 - λ300.

70

The extracted RNA was further analyzed on denaturing 1.2% formaldehyde agarose gel. Two

prominent bands of 18S and 28S ribosomal RNA could be seen on the gel indicating that the

extracted RNA is intact and has suffered no major degradations (Fig. 18).

Figure 18.Formaldehyde agarose gel of RNA Formaldehyde agarose gel of total RNA isolated from ovine pituitary tissue.

3.2.2 RT-PCR amplification of cDNA

The purified RNA was subjected to reverse transcription in order to get cDNA which was

amplified by PCR using gene specific primers as described in Materials and Methods. The RT-

PCR yielded a single product of approximately 0.6 kb which was expected size of OaST gene

(Fig. 19)

Figure 19.Analysis of the RT-PCR. Analysis of the RT-PCR amplified product resolved on 1% agarose gel. Lane M,

1kb DNA ladder used as marker; lane , 2, 3, 4 & 5 ~0.6kb amplified PCR products.

71

3.2.3 T/A cloning of oGH

The gel purified PCR products were cloned by using InsTAcloneTM PCR Product Cloning kit.

pTZ57R/T vector was designed for cloning of Taq DNA polymerase amplified PCR products, as the

enzyme adds up extra adenine residues to the 3’end of the PCR products. These single stranded A-

overhangs are required for the base pairing with the 5’-T overhangs in the pTZ57R/T vectors .(Fig.

20).

Figure 20.Restriction map of pTZ57R/T cloning vector .

The amplified oGH cDNA was purified, T/A cloned in pTZ57R/T vector and recombinant

plasmid (pTZ-oGH) thus obtained was used to transform in E. coli strain DH5α. The

recombinant clones were identified by blue/white screening, as vector is genetically marked

with LacZ gene. Several white colonies along with blue colonies appeared on LB-agar plates

supplemented with ampicillin, X-gal and IPTG. These white colonies were further analyzed by

colony PCR using gene specific primers. Six white colonies were picked up from the plate and

five of them gave positive result while one proved as false colony as shown in Fig.22. The four

72

recombinant pTZ-oGH colonies were selected for plasmid preparation and then subjected to

sequence analysis.

Figure 21.Analysis of colony PCR. Analysis of positive transformants by colony PCR. lane 1, DNA marker; lane 2, 3, 5, 6, 7 products of different colony

PCR reactions; Lane 4, colony no 4 indicates negative clone

3.3 Expression of poGH

3.3.1 Restriction analysis of pTZ-oGH

The single colony of recombinant pTZ-oGH construct confirmed by sequencing was used for the

further analysis. For this purpose the pTZ-oGH was amplified and double digested by Nde I and

BamH I restriction enzymes (Fig.22).

Figure 22.Double digestion of pTZ-oGH-1..

Double digestion of pTZ-oGH-1.M, DNA size markers;Lane1,plasmid pTZ-oGH-1after double digestion with

NdeI/BamHIrestriction endonucleases

73

3.3.2 Cloning in pET22 b

The amplified product was gel purified and cloned between Nde I and BamH I sites of pET-

22b(+) using restriction enzyme digestion and ligase mediated cloning. This generated an

expression plasmid designated as, poGH-1 (Fig.23.). The construct contained the native

sequence of OST mRNA and was predicted to encode a 191 amino acid oGH (MW ~22 kDa) in

frame with the translational initiator codon under the control of T7lac promoter.

Figure 23.Construction of recombinant plasmid poGH-1. Construction of recombinant plasmid poGH-1 by cloning a 0.6 kb long oGH cDNA in pET-22b(+) expression vector.

pT7lac, T7lac promoter; rbs, ribosome binding site; ori, origin of replication; fi ori, F1 origin of replication; lacI, Lac

repressor gene; ampr, ampicillin resistance gene.

3.3.3 colony PCR of poGH

poGH-expression plasmid was transformed into E. coli DH5α (cloning host) for vector

propagation and clone selection. Efficiency of transformation reaction was very high; almost 100

% of the screened colonies were positive for the insert as confirmed by colony PCR and/or

restriction digestion. The results obtained from a representative plasmid are presented as Fig. 25.

When resolved on 1 % agarose gel, colony PCR amplification products yielded a single band of

~0.6 kb length (Fig.24)

74

Figure 24.Colony PCR of poGH-1 Colony PCR,lane M,marker;lane 1,2,,3,4 recombinant clone s of poGH-1

In order to confirm the in frame cloning of oGH in poGH-1 construct the clone was double

digested again with NdeI and BamHI and hence showed 573kb band of oGHand 5.4kb band of

pET expression vector when analyzed on 1% agarose gel (Fig.25) which showed successful

cloning of oGH in pET expression vector. Four colonies were picked and spotted on the plate

.The desired oGH band of approximately 0.6kb was confirmed with the colony PCR of poGH -1

clone.

Figure 25.Double digestion of poGH-1.

M, DNA size markers;Lane1,plasmid poGH-1 after double digestion with NdeI/BamHIrestriction endonucleases

75

3.3.4 Shake flask fermentation of poGH-1 construct

The E. coli BL21 were transformed with recombinant plasmid poGH-1. The transformants in set

of four were grown in LB medium at 37 ̊C, induced with 0.2mM IPTG when growth of the cell

reached 0.6 at OD600. The cells were collected from each transformants and were treated with

lysis buffer to check total cell protein as explained in material and methods. The SDS-PAGE

analysis of total cell protein of poGH-1 construct revealed no visible expression (Fig. 26)

Figure 26.SDS-PAGE analysis of poGH-1 expression.. SDS-PAGE analyses of poGH-01 plasmid ..M is commercially available bovine growth hormone used as a marker .lane

1,induce pOaST-1 plasmids total cell protein after 4 hrs of 0.5mM induction of IPTG in LB medium

3.4 Periplasmic expression of oGH

The very low levels of expression of GH gene have been treated with several strategies

(secondary structure changes, bicistrone methods, changes at N terminal of GH gene and lot

more). We tried to use leader sequence of pET vector in order to get expression of oGH gene.

For this purpose a plasmid was constructed with oGH gene at NcoI and BamHI sites of pET 22b

vector so that to attach leader sequence at N terminal site of oGH gene as shown in Fig. 27.

76

Figure 27.construction of poGH-2 construct

3.4.1 Expression of poGH-2

When the leader sequence of pET22b was used in reference construct (poGH-1) and

expressed in E. coli BL21, the construct (poGH-2) showed visible expression of oGH but at

25kDa. The expected size of ovine growth hormone calculated from its amino acid sequence is

approximately 22kDa, while the band appeared on SDS-PAGE showed extra 3kDa.

Figure 28.SDS-PAGE analysis of poGh-2 expression in LB medium. SDS PAGE analysis of poGH-2 expression in LB medium.. Lane 1, induced post-2colony no1; lane 2, induced poGH-2colony no2;

lane 3, induced poGH-2colony no1; Lane 4, uninduced poGH-2colony no2; Lane 5, induced poGH -2 colony no2; lane M,Marker.

pelB leader

77

3.4.2 Effect of different factors on the expression of oGH

3.4.2.1 Effect of ZnCl2

The ZnCl2 is being used to reduce the proteolytic degradation of periplasmic protein.The

different combination of ZnCl2 were used in the pre-culture in order to see its effect on the

production of recombinant oGH protein . For this purpose 0.1mM,0.5mM,1mM,5mM , 10mM

and 50mM concentrations of ZnCl2 were used and it was observed that by increasing the amount

of ZnCl2 from 0.1 to 1mM the cell growth enhances while increasing it upto 50 mM reduces

the cell growth. The best selected concentration was observed at 0.5 mM as shown in graph.

Figure 29.effect of ZnCl2. A graph representing the effect of ZnCl2 on the protein content of expressed cells in LBmedium

3.4.2.2 Effect of IPTG concentration

The IPTG as an inducer was used in the shake flask fermentation of roGH-2 construct

with above optimized conditions. a study of the effect of IPTG concentration (10uM to 1mM)

showed that beyond 20uM there was no increase in the expression levels (Fig. 30).We observed

a constant expression of roGH by adding IPTG 20uM,40,60,80uM,0.1mM,0.5,1mM A

progressive decrease in expression levels was observed below 20uM IPTG concentration as

shown in fig.

78

( a ) ( b )

Figure 30,SDS-PAGE analysis of effect of IPTG. SDS-PAGE analysis of effect of IPTG concentration on the expression of oGH-2(a).:lane M ;marker,lane C,control pET22b ,lane

U,uninduced,lane 1-&,IPTG concn 20,40,60,80,100,1000 and 2000uM (b) effect of IPTG concentrattion less than 10uM on the

expression;lane M,markaer,U uninduced,lane 1-3,IPTG concn 2,5 and 10 uM respectively

3.4.2.3 Effect of Ecoli Strain on expression of ovine growth hormone

OGH1-pET22b construct was transformed into BL21 DE3 and P Lysis strains of E.coli. The

protein expression was observed on 15% SDS-PAGE.It was observed that BL21-DE3 results in

better expression of roGH as shown in fig.We observed the subcellular fractions in both strains

as well .The cytoplasmic fraction in the case of Plysis appeared to have more roGH as compared

to BL21 DE3,but the periplasmic fraction in both strains appeared to be same 10% as shown in

fig 31.

Figure 31.Effect of E.Coli strains on the periplasmic expression of poGH-2. SDS-PAGE analysis of expressed protein of poGH-2(lane1-4,expression in BL21DE3 strain & lane6-8 exspression in PLysis) Lane 1, sonicated sample for cytoplasmic fraction cf; lane 2,shock fluid for periplasmic fraction pf; lane 3,induced poGH-2 ; lane 4,un- induced

pET 22b; lane 5,SDS protein marker, lane 6, cytoplasmic fraction cf , lane7 shock fluid for periplasmic fraction pf, lane,8 induced

poGH-2,

79

3.4.2.4 Optimization of somotic shock conditions

The construct poGH-2 was used for further analysis.As this constitute PelB leader sequence

of PET22b which translocate recombinant protein into the periplasmic space. The destination of

recombinant protein was checked by the analysis of periplasmic and cytoplasmic fractions. For

this purpose 10ml sample was taken after the above optimized fermentation conditions in LB

medium. Already optimized osmotic conditions for the release of oGH into the periplasmic space

were used. I used 3ml each for the each osmotic shock procedure in seperate falcon tubes. These

were proceeded for subcellular fractionations as explained in the material and methods. The

protein content in the periplasmic and cytoplasmic samples was analyzed by Bradford method

explained earlier.The samples were loaded on 15% SDS-PAGE and analysed the result(fig 32 ).

The graphical representation shown (fig 32 a ,b and c) that we obtained the best release of oGH

in osmotic shock when treated with our optimized freeze thaw method as the release was

3.12ug/ml as compared to other protocols where it was achieved much lower.

Figure 32. graphical representation of effect of different osmotic shock conditions on oGH.

.

The SDS-PAGE analysis of above three osmotic shock procedures showed the clear difference

on the release of oGH in periplasmic fraction.Fig a and b showed negligible oGH when treated

80

with osmotic shock conditions stated in graphs a and b (fig 33 ) while the periplasmic and

cytoplasmic fraction in freeze thaw method showed a visible 20% molecular weight band.

Figure 33.SDS-PAGE analysis of subcellular fractions of poGH. 2 SDS-PAGE analysis of cytoplasmic and periplasmic fraction of roGH by using different osmotic shock methods .Cf is

cytoplasmic fraction,Pf is periplasmic fraction and Tcp is total cell protein.(a) osmotic shock conditions described

by(Koshland ,1980) and its effect on release of oGH( b ) osmotic shock conditions narrated by( Ramakrishnan et al., 2010) and its effect on release of oGH( c ) Freeze thaw conditions explained by (Barth et al., 2000) and its effect on

release of oGH.

3.4.2.5 Effect of Glycerol

We studied the effect of glycerol addition in the growth medium and in the osmotic shock

procedure. In LB medium the addition of glycerol enhanced the expression level of oGH from

18% to 22% when observed a on SDS-PAGE .

Figure 34. SDS-PAGE analysis of poGH-2 in LBmodified medium. Lane 1, M; lane 2,uninduced poGH-2; lane 3 induced poGH-2 after 3 hrs of induction, Lane 4, induced poGH-2 after 4

hrs of induction ;lane 5, uninduced poGH-2;lane 6, induced poGH-2 after 6 hrs of induction ;lane 7, induced poGH-2

after 8 hrs of induction

81

As freeze thaw method resulted in best release of oGH (20%) as shown (fig 32& 33). We further

optimized its condition in order to enhance the roGH release in periplasmic space. For this

purpose we used 9 different falcon tubes with 5ml each sample of fermented cells in above stated

optimized conditions.The glycerol was added in a range of (10,15,20,25,30,35,40,45,50%).The

protein content after osmotic shock was calculated by Bradford method.It was observed that the

protein content was highest 110ug/ml when treated with 25% glycerol in freeze thaw

method.The release of oGh in the specific sample was analysed on SDS-PAge and observed

24%band as shown in fig 35.

Figure 35. effect of Glycerol. A graphical representation of the effect of glycerol on the release of oGH into the shock fluid

3.4.2 Purification of poGH-2

The oGHT band was also confirmed by western blot by using specific antibody. The gene linked

with pelB signal peptide is destined for the secretion into periplasmic space of E. coli. In order to

see the fate of this expressed oGHgene with an extra 3kDa, total cell protein was proceeded for

cytoplasmic and periplasmic fractions as explained in the materials and methods. The fractions

were analyzed on SDS-PAGE and showed 25kDa oGH gene band both in cytoplasmic and

periplasmic fraction. Fig. 36 a. The use of glycerol in the osmotic shock procedure enhanced the

release of oGH in shock fluid as shown in fig below .we recovered 24% roGH from the shock

fluid which was aunthenticated by western blot analysis ( fig 36 b )

82

( a ) ( b ) Figure 36.SDS-PAGE analysis of subcellular fractions of roGH-2& western blot analysis. (a)SDS-PAGe analysis of periplasmic and cytoplasmic fraction of roGH by using optimized freeze thaw method ,Tcp is total cell protein,Pf is periplasmic fraction,Cf is cytoplasmic fraction.(b) western blot analysis of roGH

83

3.4.3 FPLC chromatography

The 80% purified ovine growth hormone was further purified by anion exchange,Q sepharose

FPLC chromatography. For this purpose 2M NaCl2 gradient was established and the peak was

observed at 0.5M concentration .The tube were collected with absorbance at 280 attached with

FPLC equipment. The purified product resulted in single sharp peak as shown in figure 37.

Pooled fractions were run on 12 % SDS-PAGE to visualize the purified protein. Further, they were

dialyzed against 20 mM Tris-Cl (pH 8.3) to remove salt traces

resQ004( FAiza ) 001:10_UV1_280nm resQ004( FAiza ) 001:10_UV2_0nm resQ004( FAiza ) 001:10_UV3_0nm resQ004( FAiza ) 001:10_Conc resQ004( FAiza ) 001:10_Flow resQ004( FAiza ) 001:10_Fractions resQ004( FAiza ) 001:10_Inject resQ004( FAiza ) 001:10_Logbook

0

500

1000

1500

mAU

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 ml

A1 A2 A3 A4 Waste A1 Waste A2 A3 A4 A5 A6 A7 A8 A9 A10 A12 A14 B1 Waste

Figure 37.FPLC peak of purified oGH

84

3.5 Effect of (DsbA,ST-II & native oGH signal sequence ) on the expression &

secretion of oGH

On the basis of inefficiency of leader sequence to translocate exact size (22kDa)

recombinant ovine growth hormone, a new strategy of using different signal sequences in pET

22b expression vector was applied. For this purpose three different signal sequences (DsbA,

ST11, native signal sequence of ovine growth hormone gene) were introduced in pET22b

expression vector in place of pelB leader sequence as described in materials and methods

.

3.5.1 Primer designed for the constructs poGH-3,4 &5

The following primers were designed for the introduction of signal sequence prior to N

terminus of OaST gene.The reverse primer was same as previously used with BamH1 siteas

explained in materials and methods

Table 2.primers designed for the poGH-3,4 & 5 constructs

3.5.2 PCR amplification

The DNA purified previously was amplified with the primers as described above and got

the PCR products of approximately 0.6kb when observed on agarose gel as shown in (Fig.38)

primer Primer sequence Features

pF-2 CATATGCATGCCCCCGGACCTCCCTGCTCCTGGCTTTCA

oGH signal

sequence

pF-3 CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGT

TTAGCGCATCGGCGGCCTTCCCAGCCATGTCC

DsbA signal

sequence

pF-4 CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGT

TTAGCGCATCGGCGGCCTTCCCAGCCATGTCC ST-11,signal

sequence

85

Figure 38.Agarose gel analysis of PCR. .Agarose gel analysis of amplified PCR products of primers ,pF2,pF3,pF4. Lane M, DNA marker; lane 2 PCR product of

primer set 2; lane 3, PCR product of primer set 3; lane 4, PCR product of primer set 3

3.5.3 T/A cloning and construction of expression plasmid poGH-3,4,5

These PCR products were than T/A cloned to pTZ57RT vector as explained in material

and methods. The recombinant clones were confirmed by colony PCR as shown in (Fig.39)

Figure 39.Colony PCR analysis of poGH-3-4-5. Agarose gel analysis of Colony PCR of pTZoGH-3,4 and 5 .M,marker; Lane 1, pTZ-oGH-3construct; Lane 3, pTZ-oGH-4 construct; lane 4, pTZ-oGH-5 construct

The amplified products were gel purified and cloned between Nde I and BamH I sites of

pET-22b(+) using restriction enzyme digestion and ligase mediated cloning. This generated

series of expression plasmid designated as, pOaST-3,4 and 5 (Fig.40). The construct contained

86

the native sequence of oGH mRNA with the signal sequence of DsbA, native signal sequence of

ovine growth hormone and signal sequence of ST-II and was predicted to encode a 191 amino

acid oGH (MW ~22 kDa) in frame with the translational initiator codon under the control of

T7lac promoter.

Figure 40.construction of expression plasmic poGH-3,4&5

3.5.4 Transformation and selection of high expression strains

All poGH-series expression plasmids were transformed into E. coli DH5α (cloning host)

for vector propagation and clone selection. Efficiency of transformation reaction was very high;

almost 100 % of the screened colonies were positive for the insert as confirmed by colony PCR

and by restriction digestion.

Figure 41.colony PCR analysis. Colony PCR of recombinant clones. lane M, DNA marker, lane 1, poGH-3 construct, lane 2,poGH-4 construct;lane 3, poGH-5 construct.

87

The results obtained from a representative plasmid are presented (Fig. 34). When resolved

on 1 % agarose gel, colony PCR amplification products yielded a single band of ~0.6 kb length,

while restriction digestion products generated two bands corresponding to 5.5 kb vector and 0.6

kb insert DNA (Fig. 42). The data thus confirmed the presence of insert in all poGH-series

plasmids.

Figure 42.Double digestion of recombinant clones. Agarose gel analysis showing double digestion of recombinant clones, M, DNA marker, lane 2, uncut plasmid pET22b

with insert, , lane 3,4 & 5, double digested pOST-3,4,5 constructs respectively.

3.5.5 Expression of poGH-3,4 and 5

E. coli transformed with poGH-series vectors (poGH-3 to -5) were grown in LB-ampicillin

broth and induced with 0.5 mM IPTG at an OD600 of 0.6. After 4 hours of induction, equal

amounts of cells (based on OD600 values) were lysed and the protein expression was analyzed by

15% SDS-PAGE as shown in Fig. 43.

88

Figure 43.SDS-PAGE analysis of protein expression of construct poGH-3,4&5. SDS PAGE analysis of total cell proteins of poGHconstructs. lane 1, poGH-5; lane 2 uninduced poGHT-5; lane 3, poGH-3; lane 4,

poGH-4; M , protein marker.

3.5.5.1 Subcellular fractionation of poGH-3-5 constructs

The fate of the expressed oGH was analysed by subcellular fractionation of the fermented

cells.The scheme of subcellular fractionation was as explained in materials and method.To

devise an appropriate strategy for downstream processing, the relative distribution of the

expressed oGH was examined in soluble and insoluble fractions. Cells expressing oGH were

lysed and then centrifuged as described under materials and methods. The supernatant and pellet

fractions thus obtained were analyzed by 15 % SDS-PAGE. When the cells were lysed under

native conditions, virtually all the expressed oGH was found in the supernatant representing the

soluble fraction while no or very little traces were found associated with the pellet .The total

cells were analyzed for the periplasmic, cytoplasmic and membrane fractions of the cell as

explained in materials and method. The subcellular fractionation was proceeded as shown in

following flow chart.

89

Figure 44.Schematic representation of subcellular fractionation of cells.

The destination of the expressed roGH was analyzed by subcellular fractionation of the

bacterial cells of each poGH3-5construct. The scheme of subcellular fractionation as shown in

Fig. 44 was devised for downstream processing and relative distribution of the expressed roGH

in soluble and insoluble fractions. Cells expressing roGH in each construct (poGH3-5)was lysed,

centrifuged and then TCP, P (Periplasmic), C (Cytoplasmic) and MF (Membrane fraction) were

analyzed on 12% SDS-PAGE (Fig. 45a,b,c,d).

90

The results of subcellular fractions of all four constructs showed variable outcomes; in construct

poGH1, roGH was expressed at 25kD though expected molecular weight for GH was 22kD.

However, western blot analysis of the gel showed the authenticity of roGH (data not shown). It

was assessed that the additional 3 kD was due to attached signal peptide as the approximate

molecular weight of pelB leader sequence is ~3 kD. During the secretion process, this signal

sequence did not get cleaved from the roGH. The sub-cellular fractions of this construct showed

that half of the roGH was found in soluble form as in the periplasmic fraction while half was

detected in the cytoplasmic fraction and no trace was found in membrane fraction (Fig. 44a).

Similar results were observed for constructs poGH2 and poGH4 showing roGH of 25 kD with

the attached signal sequence.

However, construct poGH2 showed roGH protein in the cytoplasmic fraction while very little

amount was found in periplasmic fraction(Fig. 44b). Construct poGH4 showed no trace of roGH,

suggesting lack of expression and/or protein degradation(Fig. 44d).Importantly, the subcellular

fractionation of the construct poGH3 showed complete translocation of roGH into the inner

membrane (MF) of E. coli. Here the molecular weight of the expressed roGH was 22kD and was

in complete accordance with the molecular weight of the other GH reported (Paladini et al.,

1983). . Furthermore, it was observed that none or very minute traces of roGH were found in the

C or P fractions as shown in Fig. 44C. Construct poGH3 was used for optimization studies and

the roGH production.

91

Figure 45.SDS-PAGE analysis of subcellular fractionations of poGH-3,4 & 5 constructsSDS PAGE . 12% of sub-cellular fractionation of poGH1-4 constructs. Rectangle box showing expression in all the constructs. (A) SDS PAGE

analysis of subcellular fractions of poGH-5. Lane MF, Membrane fraction; lane C, Cytoplasmic fraction; lane P, Periplasmic fraction; lane TCP, Total cell protein. (B) SDS PAGE analysis of subcellular fractions of poGH-4. Lane C, Cytoplasmic fraction; lane P,

Periplasmic fraction; lane TCP, Total cell protein; lane MF, Membrane fraction. (C) SDS PAGE analysis of subcellular fractions of

poGH-3. Lane MF, Membrane fraction; lane C, Cytoplasmic fraction; lane P, Periplasmic fraction; lane TCP, Total cell protein. (D)

SDS PAGE analysis of subcellular fractions of poGH-2. Lane MF, Membrane fraction; lane C, Cytoplasmic fraction; lane TCP, Total cell protein; lane P, Periplasmic fraction; lane M, Protein marker.

3.5.6 Computational analysis of signal sequences of poGH-2,3,4& 5 constructs

In order to understand the reason of varying behavior of these four signal sequences. Each

signal sequence used in this study; DsbA ss ,ovine growth hormone signal sequence, STII and

pelB leader sequence were compared on the basis of probability of signal sequence by signalp3.0

server (results attached in appendix), hydropathy plot by kytedoolittle method and their

secondary structure by polyview prediction server. The data showed that all of them are good

structured signal sequences. However the hydropathies of these signal sequences gave variable

results as shown below (Fig 46).

92

Figure 46.Kytedoolittle analysis of hydrophobicity of all four signal sequences. (a) pET signal sequence (b) DsbA signal sequence (c) ST11 signal sequence (d) ovine growth hormone signal sequence.

Table 3.Hydropathies of poGHconstructs

Constructs

(signal

sequence)

Nucleotide Sequence (5'-3') Hydropa

thy

poGH2 (pelB) CATATGAAATACCTGCTGCCGACCGCTGCTG

CTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCG

ATGGCCATGGCCTTCCCAGCCATGTCC

1.157

poGH3 (DsbA) CATATGAAAAAGATTTGGCTGGCGCTGGCTG

GTTTAGTTTTAGCGTTTAGCGCATCGGCGGCC

TTCCCAGCCATGTCC

1.389

poGH4 (oGH) CATATGCATGCCCCCGGACCTCCCTGCTCCT

GGCTTTCA

1.840

poGH5 (STII) CATATGAAAAAGATTTGGCTGGCGCTGGCTG

GTTTAGTTTTAGCGTTTAGCGCATCGGCGGCC

TTCCCAGCCATGTCC

0.986

The above table shows that there is an optimal range of hydropathy value, above or below which

the signal sequence does not function properly .The secondry structure of these signal sequence

(DsbA, ST-11, pelB and ovine growth hormone signal sequence)were also analysed.They

93

showed that they have varying charges in their N terminal and C terminal regions. The presence

of Beta sheet especially in case of ST-11 signal sequence was a prominent difference among

these signal sequences.

Figure 47.Secondary structure analysisof poGH2,3,4&5.. Secondary structure of all four signal sequences. (a) pET signal sequence (b) DsbA signal sequence (c) ST11 signal sequence (d) ovine growth hormone signal sequence.

The construct with DsbA signal sequence (poGH-3) was chosen best among all four signal

sequences used in this study as it resulted in the accurate size (22 kDa) of recombinant ovine

growth hormone as compared to rest of the rest of the constructs.

94

3.6 Effect of medium composition on expression of poGH-3

3.6.1 Effect of LB,TB & M9NG medium on the expression of poGH-3

On the basis of our results the poGH-3 construct was selected for the further studies. The first

objective was to enhance the production of Soluble recombinant oGH. For this purpose we

studied 9 different medium compositions in 2 sets as listed in table.The first one constitue effect

of LB,M9NG and TB medium while second set constitute seven mediums based on different

carbon and nitrogen source(LB, LB modified, GM-1, GM-2, UM, TB, TBC) to analyze their

effect on the bacterial growth and expression of roGH .All the experiments were proceeded in

shake flask fermentation. The composition of these mediums were as follows ( Table .4)

Table 4.composition of different mediums used

Culture Medium Composition

TB 2.4% yeast extract, 1.2% trypton, 0.4% glycerol, 2.31%

KH2PO4, 12.54% K2HPO4

M9NG

0.5% NaCl, 1% NZ-amine Type A, 0.5% glycerol, 0.05%

glucose, 25mM NH4Cl, 25mM KH2PO4, 50mM Na2HPO4,

2mM MgSO4 and trace metals mix (0.004mM CaCl2,

0.0004mM each of CuCl2, NiCl2, Na2MoO4, H3BO3, 0.002mM each of ZnSO4, MnCl2 and 0.01mM FeCl3)

LB 1% Tryptone, 0.5% Yeast extract, 0.5% NaCl, pH 7.2

LB-1 1% Tryptone, 0.5% Yeast extract, 1% NaCl, pH 6.8

GM-1 0.1% (NH4)2SO4, 1% Tryptone, 0.5% Yeast extract, 0.05%

Glucose, 0.5% Glycerol, 0.05M KH2PO4 dibasic, pH 7.6

GM-2 0.1% (NH4)2SO4, 1% Tryptone, 0.5% Yeast extract, 0.05% Glucose, 1% Glycerol, 0.07M KH2PO4 dibasic, 0.1M KH2PO4

monobasic, pH 7.0

UM 0.1% (NH4)2SO4, 1% Urea, 0.5% Yeast extract, 0.05% Glucose, 1% Glycerol, 0.1M KH2PO4 dibasic, 0.1M KH2PO4

monobasic, pH 7.0

TBC 2.4% Yeast extract, 1.2% Tryptone, 0.4% Glycerol, 2.31% KH2PO4, 12.54% K2HPO4, 0.5mM Mannitol,4% NaCl and

0.5M Glycylglycine, ZnCl2 0.5mM

We proceeded these experiments in (2 sets 1;LB,TB & M9NG and second set LB-1GM-1GM-

2,UM &TBC on roGH production.The construct poGH3 transformed in BL21 Codon Plus (DE3)

95

RIPL cells was grown in First set ( TB, LB, and M9NG media ) for enhanced production of

roGH. The post-induction cell growth was monitored for up to 14hrs in LB, TB and M9NG

media. The (250ml) fermentations were carried out at 37°C, induction by 1mM IPTG in the

logarithmic phase and aeration of 5 times in 1L Erlenmayer shake flask. All fermentations were

performed at least in triplicates and the results presented were the averages. The cells were

harvested at pre and at 2hrs post-induction for each fermentation. All the total cellular protein

samples were processed for analysis in 12% SDS-PAGE and the expression in each medium was

observed as shown in Fig. 47a, b & c. It was found that expression level of roGH was enhanced

up to 18% in TB medium while in LB and M9NG media 10% expression.The growth pattern in

each medium was observed to be different as in LB, M9NG and TB media the maximum cell

growth reached up to OD600 1.2, 2.3 and 5.6, respectively (Fig. 48 d). Moreover, it was

observed that in TB medium, 65.3mg/L of roGH was obtained as compared to 13mg/l in LB and

16mg/l in M9NG (Table 2).

Figure 48.SDS-PAGE analysis and graphical representation of effect of medium on poGH-3 SDS PAGE (12%) showing effect of different media composition (TB, LB, M9NG) on poGH3 construct. (A) SDS PAGE analysis of poGH3 expression in TB medium. Lane M, Protein marker; lane U, un-induced poGH3 construct; lane 1, induced poGH3 sample at

10hrs post induction; lane 2, induced poGH3 sample at 12hrs post induction; lane 3, induced poGH3 sample at 14hrs post induction.

(B) SDS PAGE analysis of poGH3 expression in LB medium. Lane 1, induced poGH3 sample at 12hrs post induction; lane 2, induced

poGH3 sample at 14hrs post induction. (C) SDS PAGE analysis of poGH3 expressed in M9NG medium. Lane 1, induced poGH3 sample at 12hrs post induction; lane 2, induced poGH3 sample at 14hrs post induction. (D) Effect of OD600 on cell growth (hrs) of

poGH3 construct fermented in TB, LB and M9NG media.

96

Table 5.Effect of medium composition on production of oGH

Medium Maximum

OD600

Dry cell mass

(g/L)

Total cell

proteina

(mg/L)

roGH

(%age of total

protein)

roGH

(mg/L)

TB 5.6 2.7 390 18 65.3

LB 1.2 1.3 135 10 13

M9NG 2.3 1.6 167 10 16

Protein concentration was determined by absorbance measurements at A280.

These results suggested further optimization of fermentation conditions in TB medium such as

the effect of lowering temperature, concentration of IPTG and induction time in cell growth

cycle.

3.6. 2 Effect of temperature on poGH3 construct

As periplasmic protein processing occurs better at low temperature as stated by Novagen;

variable range of temperatures i.e. 20, 25, 28, 30, 35 and 370 C in shake flask cultures were

applied. It was observed that cell growth was maximum when fermentation was carried out at

25ºC, it reached up to OD600 5.6 while at 28ºC and 20ºC final OD600 reached up to 5 and 4.8,

respectively suggesting that the optimum temperature is between these two ranges. However,

fermentation at elevated temperatures of 30ºC, 35ºC and 37ºC resulted in reduced cell growth

and recombinant protein with OD600 of 4.1, 3.6 and 2.8 respectively and almost half in the case

of 37ºC grown culture. In conclusion, the best soluble expression of roGH was obtained at 25ºC as

shown in (Fig. 48,A).

3.6.3 Effect of induction time and IPTG concentration on poGH3 construct

The expression of soluble recombinant proteins enhances with lowering amount of inducer

(Novagen) and in the current study, IPTG concentrations ranging from 10μM to 1mM were used

for expression level of roGH and analysed in 12% SDS-PAGE. The roGH was best expressed

with 18% protein in total cell protein at 20μM IPTG while at 1mM IPTG, 14% expression was

observed (Fig. 49 B)The induction time with 20μM. IPTG was also studied .It was found that

97

induction at absorbance 0.5, 1.0 and 1.5, resulted in maximum cell growth of 2.8, 3.5 and 4.2

respectively at OD600. Induction at OD600 2.0 resulted in the best maximum cell growth i.e. 5.6

as shown in Fig. 49C. The induction at later stages like 2.5, 3.0, 3.5 and 4.0 resulted in decrease

cell growth which eventually turned at OD600 1.8 (Fig. 49C). These results showed that

induction time is best in the initial log phase of the cell cycle.

Figure 49.effect of temperature,induction time and IPTG concn. on poGH-3. Graphs showing the cell growth of poGH3 construct in TB medium at variable temperatures, induction time and IPTG concentration.

(A) Effect of temperature on cell growth of poGH3 construct fermented in terrific broth medium. (B) Effect of IPTG concentration on

cell growth of poGH3 construct fermented in terrific broth medium. (C) Effect of IPTG induction time on cell growth of poGH3

construct fermented in terrific broth medium.

3.7 Enhanced production of roGH

After achieving above optimized expression we further studied more medium inorder to

enhance the soluble production of roGH.There was still need to enhance the yield of this soluble

growth hormone. For this purpose we used combination of seven mediums based on different

carbon and nitrogen source(LB, LB modified, GM-1, GM-2, UM, and, TBC) to analyze their

effect on the bacterial growth and expression of roGH .All the experiments were proceeded in

shake flask fermentation. The composition of these mediums were as follows ( Table .4)For this

98

purpose all the mediums were inoculated with roGH as explained in materials and methods. The

samples were taken after every 2 hrs from each medium flask until it reached the final OD600.

The results were analyzed on 12% SDS-PAGE (data not shown). The graphical representation of

results showed that TBC resulted in the best bacterial growth of 10.4 while rest of the mediums

reached up to maximum 5.6 (Figure 50). In TBC medium we followed the same concentrations

of all the medium components as stated by (Barth et al, 2000) From this it was decided to study

the effect of different components on TBC medium for the enhanced production of roGH and on

this basis following parameters were studied; effect of different compatible solutes on the

bacterial growth, chemical chaperon for the stability of recombinant proteins, temperature,

inducer concentration and the time of induction. In order to understand the behavior of the

compatible solute, constant concentration of NaCl (4%) was used.

Figure 50. Growth of poGH-3 in different medium.

The T.B medium supplemented with compatible solute was used for the further analysis. For this

purpose all the variables which can effect the production and expression of recombinant OaST

were analysed. For this purpose the effect of ZnCl2, temperature, induction time, effect of IPTG

or lactose as inducer and variable combinations of compatible solute.

99

3.7.1 Effect of compatible solute on the expression of poGH-3 construct

As per our results (Fig.50) we decided to use T.B with varying combinations of compatible

solute in order to further enhance the production of recombinant oGH. The compatible solute

which was previously used was namely ( glycerol, sorbitol, glycine betaine, and hydroxyectoine)

during the production phase. In this study we have used 2 different sets (Glycylglycine ,glycine

betaine) and (sorbitol and Mannitol) of compatible solutes in production phase of recombinant

ovine growth hormone construct poGH-3.

3.7.1.1 Optimization of soluble roGH expression using compatible solutes(Glycylglycine ,

glycine betaine,sorbitol and Mannitol

In the present work we used different concentrations of each compatible solutes ( sorbitol,

mannitol, glycylglycine and glycine betaine) and compared their effect on the final absorbance

of the growing culture. It was observed that all these components increase the final OD of the

growing culture. We found that glycylglycine results in higher final density of growing culture

as compared to glycine betaine and so mannitol gives the better results as compared to sorbitol as

shown in graphs. We further studied the effect of glycylglycine and mannitol on the soluble

expression of roGH. The concentration of Glycylglycine effects the cell growth as by increasing

the concentration of Glycylglycine from 10 to 50mM. The cell growth also reached at the

maximum OD600 of 13.0. Mannitol also enhanced the cell growth and found the maximum

growth at 13.5 with optimized concentration of 0.6M as shown in Table 6. The results were also

observed on 12%SDS-PAGE separately for each of them (data not shown) and graphically

summarized in (Figure 51).

100

Table 6. Effect of compatible solutes inTB medium on the growth of poGH-3

The optimized concentration of Glycylglycine (50mM) and Mannitol (0.6M) enhanced the

percentage expression of roGH. The subcellular fractionation [periplasmic (P), cytoplasmic (C),

membrane fraction (MF)] of recombinant cells grown in above optimized conditions of TB were

proceeded as explained in materials and methods and were analyzed on 12% SDS-PAGE (Figure

52 a, b, c ). Figure 51 (a) shows the expression of roGH in TB medium supplemented with no

glycylglycine and Mannitol .we observed a good expression of roGH in total cell protein but we

could nt get the good yield of soluble roGH from membrane fraction . As we found some part of

the recombinant protein in periplasmic fraction and very diminished in the membrane fraction

while didn't receive any protein in the cytoplasmic fraction.

Figure 51.Graphical representation of the effect of 2 sets of compatible solutes on the growth of poGH-3 in TB medium

Glycylglycine Glycine betaine Mannitol Sorbitol

Conc(mM) OD600 14hrs post-

induction

Conc.(mM) OD600 14hrs post-

induction

Conc. M OD600 14hrs post-

induction

Conc. M OD600 14hrs post-

induction

10 8.2 10 6.8 0.1 7.1 0.1 7.2 20 9.3 20 7.4 0.2 8.4 0.2 8.2 30 10 30 8.7 0.3 10.1 0.3 8.9 40 11.5 40 9.2 0.4 11.9 0.4 9.8 50 13 50 9.4 0.5 12.6 0.5 10.7 60 12.6 60 9.8 0.6 13.5 0.6 11.5 70 11.8 70 10.2 0.7 13.1 0.7 12.4 80 11 80 9.8 0.8 12.7 0.8 12.6

101

However, Figure 52 (b) shows TB medium supplemented with Mannitol but no glycylglycine

and the total cell expression of roGH was observed to be good and at least half of the

recombinant protein in membrane fraction was found which was then easily solubilized by use of

40% acetonitrile. Moreover, some protein was also observed in periplasmic and cytoplasmic

fractions. While Figure 52 (c) shows TB medium supplemented with the optimized concentration

of glycylglycine and a very good expression of roGH as total cell protein was observed with

almost all of it transferred to the membrane fraction and no visible fraction was observed in

periplasmic or cytoplasmic fractions..On the basis of above observation we found that

glycylglycine and mannitol both effects the soluble expression of recombinant

( a ) ( b ) ( c )

Figure 52.effect of compatible solutes on the solublr expression of poGH-3 in TB medium . (a) shows the expression of roGH in TB medium supplemented with no glycylglycine and Mannitol.( b ) shows TB medium supplemented with Mannitol but no glycylglycine.(c) shows TB medium supplemented with the optimized concentration of

glycylglycine

3.7.2 Production of soluble roGH in TBC optimized medium

From the above observations we selected the optimized concentrations of Glycylglycine (50mM),

Mannitol (0.5M) and used it with optimized conditions of temperature (25ºC), IPTG (20μM),

induction at early logarithmic phase i.e. OD600 ~3.0 with 0.5mM of ZnCl2 and 4% NaCl in TB

medium for the batch of shake flask fermentation. Continued growth of bacterial cells was observed

up to 20 hrs post-induction. For expression studies 1ml sample was collected from the culture after

every 2hrs as explained in the materials and methods and samples were analyzed on 12% SDS-

PAGE. The optimized compatible solutes in TB medium enhanced the expression and solubility of

roGH with ~32% expression at 18hrs post-induction as shown in (Figure 53). The analysis on 15%

SDS-PAGE shows 32% expression of recombinant oGH.

102

Figure 53.SDS-PAGE analysis of optimized compatible solte in TB medium on expression of poGH-3. SDS PAGE analysis of poGH-3 expressed in compatible solute supported terrific broth medium. Lane M, prestained protein

marker;lane 2,un induced poGH-3;lane 3,induced poGH-3 after 4 hrs of induction ; lane 3,induced poGH-3 after 4 hrs of induction;

lane 4,induced poGH-3 after 8 hrs of induction; lane 5 ,induced poGH-3 after 10 hrs of induction; lane 6,induced poGH-3 after 12 hrs of induction ; lane 7,induced poGH-3 after 14hrs of induction ; lane 8,induced poGH-3 after 16 hrs of induction ; lane 9,induced

poGH-3 after 18 hrs of induction lane 10,induced poGH-3 after 20 hrs of induction .lane,11,western blot of recombinant oGH

3.7.2.1 Effect of temperature

As periplasmic protein process better at low temperature as stated by Novagen. The range

of temperatures 20̊C,25̊C,28̊C,30 ̊C and 37̊C in shake flask culture were applied .It was observed

that at low temperature conditions 20̊C, 25̊C and 28̊C the cell growth was very slow in the

beginnig till it reached upto 3.0 at O.D600 at which medium is supplemented with compatible

solute and 0.1mMIPTG ,the cell growth speeds up and continued growing after 16-20 hours of

induction. while by increasing the temperature from 28 to 30 and 37̊C the cell growth takes 5 hrs

to reach upto 3 at O.D600 and induction was given, the cell growth enhances quickly but it lasts

upto 8-9hours after induction. At lower temperature 25̊C the O.D600 of cell growth reached upto

17.2 while increasing the temperature 37̊C it reached upto 10.2 as shown in the graph (figure 54).

103

Figure 54.Effect of temperature.. Effect of temperature on cell growth of poGH-3 construct fermented in terrific broth medium supplemented with compatible solute

.

3.7.2.2 Effect of IPTG and Lactose as an inducer

The lactose and IPTG as an inducer were used seperately in the shake flask fermentation

of poGH-3 construct with above optimized conditions. a study of the effect of IPTG

concentration (10µm to 0.1mM) showed that beyond 20µM, there was no significance increase

in the expression levels (Fig. 55). A progressive decrease in expression levels was observed

following addition of IPTG till a final concentration of 0.5mM. Further increase in inducer

concentration up to 0.1 mM, however, did not result in any improvement in poGH-3 production.

20µM IPTG, therefore, was found optimal for maximal expression of poGH-3. Lactose-based

auto-induction strategy was employed to poGH-3 construct in terrific broth medium

supplemented with compatible solute. In this methodology, inducer (lactose) is added right at the

beginning of inoculation but the induction is completely repressed due to the presence of glucose

in the cultivation medium. Upon glucose depletion, induction and hence the production of

recombinant protein starts, automatically. This is advantageous, as unlike IPTG induction,

culture growth is not required to be monitored prior to induction.

104

Figure 55.effect of IPTG and lactose.. (a)SDS-PAGE analysis of poGH-3 construct in terrific broth medium with different concentrations of IPTG (b) SDS-PAGE analysis

of poGH-3 construct in terrific broth medium with lactose as an inducer

3.7.2.3 Effect of induction time

When induced at OD600 0.5-1.0,the cell growth enhanced quickly but it dropped after

reaching 4.3 at O.D600 . While by increasing the induction stage 1.5 ,2,2.5 and 3 the cell growth

enhances quickly after induction and it continued growing after 14-18 hrs of induction .As

compatible solutes has 4% NaCl2 which gives stress to the medium so its better if induction is

given in early logarithmic phase. The best induction of recombinant E. coli by IPTG, therefore,

was obtained at logarithmic phase, i.e., between OD600 of 2.5 to 3.

3.7.3 Subcellular fractionation of poGH-3 construct

The fate of the expressed was analysed by subcellular fractionation of the fermented

cells.The scheme of subcellular fractionation was as explained in materials and method .To

devise an appropriate strategy for downstream processing, the relative distribution of the

expressed oGH was examined in soluble and insoluble fractions. Cells expressing oGH were

lysed and then centrifuged as described under materials and methods. The supernatant and pellet

fractions thus obtained were analyzed by 15 % SDS-PAGE. When the cells were lysed under

native conditions, virtually all the expressed oGH was found in the supernatant representing the

105

soluble fraction while no or very little traces were found associated with the pellet .The total

cells were analyzed for the periplasmic, cytoplasmic and membrane fractions of the cell as

explained in materials and method. The subcellular fractionation was proceeded as shown in the

flow chart.The above strategy was used for the analysis of poGH-3 construct expressed under

optimized fermentation conditions of T.B medium supplemented with compatible solute. The

oGH was found in the membrane bounded form which was than solubilized with 40% of

acetonitrile. The recombianant oGH was also confirmed by western blot as shown in Fig.56.

Figure 56.Subcellular fractionation of poGH-3. SDS PAGE analysis of sub cellular fractionation of pOaST-3 construct expressed in T.B medium supplememnted with compatible

solute. lane 1, western blot of OaST. lane 2, soluble fraction . lane 3, membrane fraction..lane 4, periplasmic expression 5, cytoplasmic protein. lane, lane 6, total cell protein.

We studied the effect of sorbitol plus glycine betaine as compatible solute in the terrific broth on the

expression and final yield of recombinant oGH.we found that TB with above combination of

compatible solute produces a final yield of 189mg/L which is 3 times more than the yield obtained

from simple terrific broth medium.The results were extraordinarily high when we used Glycylglycine

plus mannitol in terrific broth medium.The glycylglycine and mannitol proved to be the better option

than sorbitol and glycine betaine as they resulted in final yield of 443mg/L which is about 9 times

higher when using TB simply without compatible solute.as shown in table

106

Table 7.Effect of compatible solute in TB medium on yield of soluble oGH

Medium Maximum

OD600

Dry cell

mass

(g/L)

Total cell

proteina

(mg/L)

roGH

(%age of

total

protein)

roGH

(mg/L)

TB

5.6

2.7

389

18

65.3

TB with

(Sorbitol and

Glycine

betaine)

10.4

4.1

1020

18

189

TB with (glycylglycine

and mannitol

17

6.4

1380

32

443

107

3.8 Effect of amino acid alterations in DsbA signal sequence on poGH

expression and secretion

The role of amino acid substitution in the tripartite structure of DsbA signal sequence in

targeting recombinant ovine growth hormone to the inner membrane of Escherichia coli cell was

investigated. Construct’s were designed by altering amino acids in the H, C and N domain of

DsbA signal sequence (DsbA ss) and for this purpose alanine, serine and lysine were replaced by

isoleucine, cysteine and arginine residues respectively.

3.8.1 PCR amplification and Cloning of pOaST varying constructs

We designed basically three types of mutant DsbA constructs

1. Mutant construct of DsbA with varying hydrophobic region

2. Mutant construct of DsbA with varying N terminal region

3. Mutant constructs of DsbA with varying C terminal region

The primer were designed for each construct as explained in materials and method. These

forward primers with one reverse primer PBGH3 (5`TAG GAT CCG CAA CTA GAA GGC

AGC 3`) were used for the PCR amplification. The wild-type growth hormone sequence in the

construct pTZoGH-1 was amplified using each of the forward and the reverse primers (Fp1-

Fp8)as shown in the (Table 7). The oGH gene was PCR amplified and analysed on 1% agarose

gel as shown in figure. All the primers resulted in 573bp oGH amplified product.

108

.Figure 57.PCR amplification of poGH-3-I-VIII. lane M; marker, lane 1-7 poGH-3 a-g constructs

These PCR products were then T/A cloned into pTZ57RT vector. The recombinant

colonies were chosen and confirmed by colony PCR analysis.

( a ) ( b )

Figure 58.colony PCR and double digestion of poGH-3-I-VIII.

(a) colony PCR of poGH-3a-g constructs.Lane 1-7 colony PCR of pOaST3a-g constructs.(b)Agarose gel analysis of double digested

T/A clone recombinant pTZ-oGH-3a-g

These (pTZoGH-3-I-VIII) series of recombinant plasmids were digested with NdeI and

BamHI (figure )and ligated at the NdeI/ BamHI site of pET22b thus generating a series of

recombinant plasmids ( poGH-3-I-VIII).

109

3.8.2 Construction of Expression plasmid poGH3-I-VIII

Figure 59.Construction of recombinant pET for poGH-3-I-VIII constructs .

3.8.3 Expression of poGH-3-I-VIII

oGH expression analysis. The recombinant plasmids of poGH-3 were transferred into E. coli

BL21 CodonPlus (DE3) RIPL strain. The transformants were grown in LB medium at 25 C and

induced with 20µM IPTG when OD600 reached 0.6. Lysates of E. coli cells of the various

transformants, after IPTG induction for 6 hrs, cells were treated with lysis buffer, and the total

cell protein was analyzed by SDS-PAGE. Equal amounts of cells (based on OD600 values) were

lysed from each culture and the protein expression was analyzed by SDS-PAGE. The expected

molecular mass of oGH is ~22 kDa, variations were observed both in mass and expression levels

pEToGH

NdeI

lacI

f1 origin

BamH1

pT7-lac

CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCG

CATATGAAAAAGATTTGGCTGATTCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCG

CATATGAAAAAGATTTGGCTGATTCTGATTGGTTTAGTTTTAGCGTTTAGCGCATCG

CATATGAAAAAGATTTGGCTGATTCTGATTGGTTTAGTTTTAATTTTTAGCATTTCG

CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAATTTTTAGCATTTCG

CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTTGTGCATGT

CATATGAGAAGGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCG

CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAATTTTTAGCGCATCG

oGH-3-I-VIII

oGH

110

when the total protein in lysates of the E. coli, transformed with different poGH constructs, were

compared (Fig. 60).

Figure 60.SDS-PAGE analysis of poGH-3-I-VIII constructs in LB medium. lane 1, oGH -3I; lane 2 , oGH-3II; lane3, oGH -3-IV; lane 4, OaST -3VI; lane 5, OaST -3VII, lane 6, OaST-3V; lane 7, OaST-3III; lane 8, OaST-3VIII; Pre-stained protein molecular markers, lane 9.

The new constructs with variations in DsbA signal peptide gave variable results. The

poGH-3 constructs III, V and VIII resulted in 25kDa of ovine growth hormone and rest of the

constructs resulted in 22kDa of band on 15% SDS-PAGE as shown in figure 60. Since, the

approximate size of 18 amino acids long DsbAss is ~2 kDa, the higher molecular mass of oGH

in these constructs is likely to be the result of incomplete processing of DsbAss.

In the case of poGH-3-IV construct, size of expressed oGH was 22 kDa but the expression level

was barely detectable on SDS-gel and therefore it was confirmed by western blot analysis (data

not shown).

3.8.4 The expression of DsbA ss constructs with substitution of alanine with

isoleucine in the H domain

The DsbAss has four Ala residues in its H- and near H-domain region, which are present at

position I, IV, IX and XI with respect to the signal peptidase cleavage site. In the present study

these alanine residues were replaced by Isoleucine in poGH-3-II to -VI constructs and the impact

of substitutions was observed on oGH expression and export in E. coli. When analysed by SDS-

1 2 3 4 5 6 7 8 9

25kDa 22kDa

kDa

250

130 100

70

55

35 25

15

111

PAGE, expression levels of oGH in these constructs ranged from undetectable (poGH-3-IV) to

up to 25 % (poGH-3-V) of the total E. coli cellular proteins. Variations in molecular mass were

also observed in the case of poGH-3-III and -V (~25 kDa) and poGH-3-II, -IV and -VI proteins

(~22 kDa).

In order to understand the behavior of expressed oGHs, the hydropathy index of each

DsbAss mutant construct was analyzed using the Swiss ExPASy Protparam tool (Table 2).

poGH-3-II and -V having the same hydropathy indices but variable molecular weight of

expressed oGH were the most interesting constructs. The subcellular fractionation of proteins

from the cells transformed with these constructs showed that in case of poGH-3-II, DsbAss

directs oGH-II into the inner-membrane of E. coli (Fig. 61). While in the case of poGH-3-V,

more than 75 % of the expressed protein remains in the cytoplasmic fraction with no traces in the

membrane fraction. Thus, Ala13 of DsbAss when substituted with Ile13 somehow hampered the

export and processing of oGH in E. coli. This suggests that it is not the hydropathy but the nature

of amino acid substitution at specific position, which influences the mechanism of protein

translocation.

In the present study, oGH-3-II, the one which is highly expressed (up to 20 % of the total E.

coli cellular proteins) with complete processing of DsbAss, was purified by employing FPLC

chromatography for further confirmation of size using the MALDI-TOF mass spectrometry. The

mass of purified oGH-II-2 was almost the same as the theoretically calculated mass of mature

oGH (data not shown), reflecting the complete processing of DsbAss.

112

(a) (b)

(c)

Figure 61.SDS-PAGE analysis of poGH-3-II-VI&I SDS PAGE analysis of subcellular fractions of construct poGH-3-I,II,VI ,in terrific broth medium.lane1,(a) SDS PAGE analysis of

subcellular fractions of construct poST-3-I;lane,M, marker;lane 2,total cell protein; lane 3,periplasmic fraction;lane 4, membrane fraction ;lane 5, cytoplasmic fraction. ,(b) SDS PAGE analysis of subcellular fractions of construct poST-3-ii;lane,1 ,membrane

fraction; lane 2, cytoplasmic fraction; lane 3,periplasmic fraction;lane 4, total cell protein; lane,M, marker. ,(c) SDS PAGE analysis of

subcellular fractions of construct poST-3-Vi;lane,1 , total cell protein; lane 2, cytoplasmic fraction; lane 3,periplasmic fraction;lane 4,

membrane fraction;lane,5,soluble fraction; lane,M, marker.

While the fractions poGH-3,III and V resulted in 25kDa protein found in both cytoplasmic and

periplasmic spaces as shown in( Fig 62).

113

(a) (b)

.Figure 62.SDS-PAGE analysis of poGH-3-III&V. SDS-PAGE analysis of sub cellular fractionation of pOST-3,III and V constructs. (a)subcellular fractions of pOaST-3-d;Lane 1 membrane fraction,M ,marker,lane-3 cytoplasmic fraction,lane 4,periplasmic fraction.,lane 5, total cell protein of pOST-3,b

construct,.(b)subcellular fraction of pOaST-3c; lane 1,membrane fraction , lane 2,periplasmic , lane 3,total cell protein ,lane

4,cytoplasmic fraction, lane 4 ,SDS marker.

The plasmid constructs poGH-3, III and V showed variation in the sub cellular localization

of the recombinant growth hormone. These constructs showed extra 3kDa size in the expected

band and it was very slightly translocated into cytoplasmic and periplasmic space while most of

it is being lost.

3.8.5 DsbA ss constructs with substitution of serine with cysteine in the C domain

In this construct two serine residues at C domain of DsbA ss were substituted with two

cysteine residues. The replacement of serine by cysteine residue in clone ( pOaST-3VIII)

affected the translocation process and the expressed recombinant ovine growth hormone was

found in the cytoplasmic fraction with 25kDa molecular weight when analyzed by SDS-PAGE

and Western blot analysis. This result is in accordance with the already reported importance of

polar C terminus which is essential for recognition of signal peptidase. The change of serine to

cysteine residue impaired the signal peptidase activity( Fig.63).

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Figure 63,SDS-PAGE analyis of poGH-3 VIII.

SDS_PAGE analysis of poGH-3, VIII constructs. Lane 1 SDS marker, lane 2 total cell

protein,lane3,cytoplasmicfraction.lane 4,periplasmicfraction.lane 5,membrane fraction

.

3.8.6 DsbA ss constructs with substitution of lysine with arginine in the N domain

The two lysine residues were substituted by arginine in the N domain in (poGH-3VII) and

the expression and subcellular localization of recombinant ovine growth hormone was analyzed

by SDS-PAGE and Western blot as described for the rest of the constructs. The ovine growth

hormone of 22kDa was found in the membrane fraction and no traces were found in cytoplasmic

fraction and periplasmic fraction as shown in fig 64.

Figure 64.SDS-PAGE analysis of poGH-3-VII. SDS-PAGE analysis of pOST-3-Vii construct.lane 1 SDS marker,lane 2 ,total cell protein,lane 3,periplasmic fraction,lane 4 cytoplasmic fraction,lane 5,membrane fraction

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3.8.7 Purification of oGH from poGH-3-II construct

Ovine growth hormone was purified by simple procedure of subcellular fractionation and

it was found in membrane bounded form. This membrane bounded growth hormone was

solubilzed by 40% acetonitrile and was observed on SDS gel. Western blot analysis of

membrane bounded growth hormone confirmed it as growth hormone (Fig.65)and MALDI TOF

analysis proved its molecular weight(Fig.66).

Figure 65.Subcellular fractionation of poGH-3II and western blot analysis. SDS PAGE analysis of sub cellular fractionation of pOaST-3-II construct expressed in T.B medium supplememnted with compatible

solute. lane 1,uninduced pOaST-3 construct.lane 2,total cell protein,lane 3,periplasmic fraction,lane 4,cytoplasmic fraction,lane

5,membrane fraction.lane 6, soluble fraction.lane 7 western blot of OaST.

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The purification of OaST from membrane bounded fraction is just single step of solubilization as

shown in Table.9

3.8.8 MALDI TOF analysis of purified ovine growth hormone

The purified product from FPLC was then applied on MALDITOF analysis and for that

0.1microlitre of the purified product was used .The MLDITOF analysis showed that purified

ovine growth hormone is of 21,059kDa of mass which is the approximately very near to the

actual 21759kDa mass of ovine growth hormone. A single sharp peak was observed at 0.5M

concentration. The identity of the purified oGH was further confirmed by MALDI-TOF analysis

as a single peak with a mass of 21,059.

Figure 66.MALADI-TOF analysis of purified ovine growth hormone

.

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3.8.9 Biological activity assessment assay

Cell-based proliferation assay was used to assess the biological activity of the purified

recombinant ovine growth hormone from oGH-3-II construct. The HeLa cells incubated with

BSA and purified recombinant ovine growth hormone were counted after 24 hrs by using

hemocytometer and it was found that cell growth in BSA was 40,000 +/- cells and growth was up

to 90,000 +/- cells in the presence of purified recombinant ovine GH. The biological activity of

oGH was checked in duplicate and the average no. of cells was determined by applying the

formula given in materials and methods. It was observed that in the presence of oGH, the growth

of the cells was found to be three-fold higher than the control

( a ) ( b )

Figure 67.Biological activity of oGH in the presence og Hela cell lines..

(a). HeLa cells in the presence of BSA (10 ug).(b), HeLa cells in the presence of ovine GH (10 ug)

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3.8.10 Computational analysis of pOaST-3-I-VIII constructs

In order to understand the behavior of these construct hydropathies and the secondary

structure of these construct was studied. On the basis of hydrophobicity all constructs were with

higher hydrophobicity than original DsbA signal sequence when calculated by Swiss Expasy

Protparam.

Table 8.Hydropathy indices of modified DsbA ss in poGH-3-I-VIII constructs

PoGH-

3

Construct

DsbA signal sequence* Description

Hydr

opath

y index

Approx.MW of oGH

PoGH-

3-I

N-terminal C-terminal

KKIWLALAGLVLAFSASA

H-domain Signalpeptidase

18 amino acids long native

DsbA signal sequence having

N-terminal, H- and C-terminal

domains incorporated at the N-

terminus of oGH sequence

1.389 22

poGH-

3-II KKIWLALIGLVLAFSASA

Modified DsbA with 1 Ala

changed to Ile 1.539 22

poGH-

3 -III KKIWLILIGLVLIFSISA

Modified DsbA with 4 Ala

changed to Ile 1.989 25

poGH-

3-IV KKIWLILIGLVLAFSASA

Modified DsbA with 2 Ala

changed to Ile 1.689 22

poGH-

3-V KKIWLILAGLVLAFSASA

Modified DsbA with 1 Ala

changed to Ile 1.539 25

poGH-

3-VI KKIWLALAGLVLIFSISA

Modified DsbA with 2 Ala

changed to Ile 1.689 22

poGH-

3-VII RRIWLALAGLVLAFSASA

Modified DsbA with 2 Lys

changed to Arg 1.321 22

poGH-3-VIII

KKIWLALAGLVLIFCICA Modified DsbA with 2 Ser changed to Cys

1.531 25

*The amino acid residues modified in native DsbA signal sequence are highlighted in grey.

The varying hydrophobicity didn’t gave the answer as the constructs poGH-3-II and

poGH-3-V with same hydropathies gave two different results .The construct (poGH-3-II)

appeared with the exact molecular weight of ovine growth hormone of 22kDa and construct

( poGH-3-V )with the additional 3 kDa. Both these constructs with the difference of alnine at

position 9 and Isoleucine at position 11 resulted in two different kind of expression levels(Table

8).One with the exact 22 kDa of oGH and the other resulted in 25kDa as shown in the figures52

and 53 respectively.

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We hypothesized that the secondary structure of the mutant DsbA signal sequence effect the

proper translocation of recombinant protein attached with it. By analysis of the secondary

structure of these mutant signal sequences it was observed that the mutation at position 11 in

DsbA signal sequence brings breakage in the alpha helix structure of hydrophobic

region(Fig.68).The homology model of DsbA signal sequence shows the alpha helix structure in

the first part of hydrophobic portion of DsbA signal sequence as shown in (Fig.69) .It is already

established fact that helix breaker in cleavable signal sequences prevents recognition by SRP,

and it appears that besides hydrophobicity the α-helix propensity of the hydrophobic core of the

signal sequence helps to determine the targeting pathway (Adams et al., 2002 ).

Figure 68.Minnou Server prediction results.

is α and other helices, is coil and is β-strand or bridge. is hydrophobicity,yellow is

hydrophobic, pale green is amphipathic, pink is polar and dark brown is charged

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Figure 69.Homology model of DsbA ss with altered alanine.

THe homology modeling and molecular docking of the altered amino acids in DsbA signal

sequence can answer all the questions of varying behaviour of DsbA by changing amino acid

alinaine at position 11.This can give good base for further work in this direction for new research.

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Discussion

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4.1 Characterization of oGH gene

The gene encoding oGH was isolated from the ovine pituitary tissue by RT-PCR methodology

and its sequence was determined. Like most other mammalian GHs (Vize and Wells, 1987;

Wallis and Wallis, 1989) oGH cDNA was found to contain 573 nucleotides and an intact ORF

coding for 191 amino acids with an in-frame stop codon (Fig.7 ). The percentage of G+C and

A+T nucleotides was 59.34 and 40.66 % respectively, which differs from 42 % GC contents of

vertebrate DNAs in general .The sequence of GH of local ovine breed (Lohi) of Pakistan was

submitted to the Gene bank with Accession numbers GQ45268 and AB24479 for genomic and

coding sequence of ovine growth hormone respectively. The genomic sequence details about the

exons, introns and regulatory sequence of the GH deduced that it constitutes 5 exons and 4

introns (Fig.7 )

While analyzing GHs of other vertebrate species, it was observed that a single residue (V130)

makes the ovine GH different from the bGH. The change i.e., valine (V130) versus glycine (G130)

in the latter, is interesting because G130 residue is widely conserved in the GHs of different

vertebrate (Fig.10 ) (Mukhopadhyay and Sahni, 2002b).It was reported that caprine GH also

differs from bGH by a single residue, i.e., glycine (G9) in bGH while serine (S9) in caprine GH.

Similarly, (Castro and Barrera, 1995) observed a single amino acid variation at position 155,

making the feline GH different from the canine and porcine GHs. The change (P155) like G130 in

ovine GH, was again in the highly conserved region.

Homology analysis revealed that the mature oGH exhibits only 67% similarity with human GH.

However, high homologies (98.5-99.5 %) were being observed between oGH and the reported

bovine, ovine, caprine and giraffe GHs (Mukhopadhay and Sahni, 2002). Due to high degree of

sequence identity between vertebrate GHs, similarities in three-dimensional structure were also

anticipated. Analysis of 3-D structure revealed that oGH exhibits a typical topology of cytokine

superfamily (Fig.16). The structure has all the salient features including four antiparallel α-

helices forming four-helix bundle structure, as described (deVos et al., 1992). In oGH like

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human GH more than 50% amino acid residues were involved in the helix formation which are

largely hydrophobic.

The sequence analysis of GH isolated from local ovine breed (Lohi) revealed the difference of

one amino acid when it was compared with the Indian and Australian ovine breeds. It showed

variation of one amino acid at position 147 where threonine is replaced with arginine as shown

in Fig.11 . The coding sequence comparison with local isolated caprine GH showed 100%

homology at amino acid level The sequence of oGH isolated from (Lohi) showed no variation at

amino acid level with the sequence of caprine growth hormone .The only variation which it has

are silent mutations (Fig 13). while its comparison with other species of same family Bovidae

showed 95-99% homology. It was also recognized that this difference increases when compared

with other families of class Mammalia (Hominidae, Canidae, Felidae, Equidae, Hippopotamidae,

Camelidae, Didelphidae) as shown in (Fig 12 & 13). The difference enhanced at inter class level

and the comparison showed that the N terminal of GH is highly variable, central region is less

conserved while C terminal is highly conserved. The phylogram of oGH showed that members of

Bovidae species are evolutionary more allied than other animals. The percentage homology of lohi

was 99, 98, 97, 97, 93, 91, 91, 90, 89, 76, 74, 69, 67, 50, 50, 49, 43 and 45 % with cow, deer, goat,

sheep, rabbit, panda, dog, pig, guinea pig, human, monkey, rat, mouse, domestic pigeon, chicken,

frog, goldfish and catfish respectively.As shown in fig below.

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Figure 70.Phylogenetic tree of ovine growth hormone

4.2 periplasmic Expression of roGH

The confirmed clones of recombinant ovine GH tested by sequencing were used for the

expression studies. For this purpose T7 promoter based expression vector pET22b was used and

E. coli strain BL21 codon plus . The oGH gene in the first construct poGH-1 was expressed with

NdeI/BamHII sites of pET22b and found negligible expression of native oGH in E. coli . As

shown ( Fig.26) the oGH expression with the clone poGH-1, carrying native gene was less than

0.5 % of the total E. coli cellular proteins. The observation was in good agreement with the

previous reports where bovine (Schoner et al., 1984), ovine (Puri et al., 1999), caprine

(Mukhopadhyay and Sahni, 2002a,c; Khan et al., 2007) and porcine GHs (Vize and Wells, 1987)

with native N-termini expressed poorly in E. coli systems. The high-level expression of the

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cloned gene in E. coli generally requires a strong promoter, a properly spaced Shine-Delgarno

(SD) sequence and an effective ribosome binding site for efficient translation of the mRNA (Das,

1990). The GHs of several vertebrate species however expressed poorly in E. coli regardless of

the promoter strength, the SD-sequence, host strains and culture conditions (Schoner et al., 1984;

George et al., 1985; Hsiung and MacKellar, 1987). Very low levels of expression resulted when

bubaline and caprine ST cDNAs were directly placed under the E. coli (trc) or phage (T7)

promoters (Mukhopadhyay and Sahni, 2002). The approximate expression levels were less than

0.1 % of the intracellular E. coli proteins, respectively. also had trouble while expressing ovine

GH in E. coli under the regulation of phage T5 promoter had a trouble too (Puri et al., 1999).

The gene encoding bGH was also found to be expressed poorly in E. coli (Tomich et al., 1989).

According to (Paik et al., 2006) GH coding sequence has some inherent properties that inhibit its

expression in E. coli. The suggested explanations for this behavior includes: two putative

secondary structures at the beginning of the coding region (Tomich et al., 1989) a basic pI value

of the bGH as described (Saito et al., 1987) and the existence of number of non-preferred

codons present in the bGH gene (Seeburg et al., 1983; George et al., 1985). The results of

(Schoner et al., 1984) however showed that the native bGH codons are not a barrier to efficient

translation.

Several different strategies were adopted to improve the expression levels of animals GHs in E.

coli. Expression levels of animal GHs could also be improved by making use of a two-cistronic

expression system. A synthetic two-cistronic expression system was constructed for high-level

expression of bGH and human GH in E. coli (Schoner et al., 1986). Puri et al., (1999) while

expressing the ovine GH in E. coli observed that the expression levels of GH are greatly affected

by nature of codon following the ATG start site. Presence of GCC (Ala) codon at position next

to ATG completely blocked the expression of ovine GH, whereas its removal or replacement

with Fusion of heterologous proteins with a short stretch of histidine residues was also found to

improve the expression levels of cloned genes in E. coli. The cDNAs encoding human and ovine

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STs were cloned with the His6 tag and expressed in E. coli under the control of T5 promoter

(Mukhija et al., 1995; Appa Rao et al., 1997). The GGC (Gly) codon restored the GH

expression, though, with different levels. High-level expression (representing ~ 20 % of the

soluble E. coli proteins) of buffalo and goat-GHs was also achieved as a fusion with glutathione-

s-transferase (gGH) partner, under the control of Trc promoter (Mukhopadhyay and Sahni,

2002). High-level expression could also be obtained by using procedures known to optimize both

gene transcription and mRNA translation (Hsiung and MacKellar, 1987). we tried to solve this

issue by utilizing PelB leader sequence of pET22b in construct poGH-2. The N terminal of ovine

GH gene was linked to PelB leader sequence of pET22b. As a result we analyzed good level

expression of about 18% of oGH but with an additional 3 kDa in its molecular weight on SDS-

PAGE (Fig 28). We further confirmed the expressed protein to be as GH by Western blot

analysis(data not shown). GH consists of 190 or 191 amino acids with two disulfide bridges. Of

particular interest for the expression of disulfide bonded proteins is a family of pET vectors

containing the N-terminal pelB secretion signal, which directs synthesized polypeptides to the E.

coli periplasm(Yoon et al., 2010) . Disulfide oxidoreductases and isomerases located in the E.

coli periplasm catalyze the formation of disulfide bonds enabling the accumulation of properly

folded, soluble protein making the periplasm an ideal compartment for expression of certain

therapeutic proteins .

Here we studied different factors effecting the release of oGH in periplasmic space by using

PelB leader sequence of PET vectore at the N terminus of oGH gene. We studied the effects of

IPTG concentration ,chemical chaperon (ZnCl2) and glycerol in the LB medium and also studied

the effect of ZnCl2 and Glycerol in the osmotic shock procedure.Glycerol is the cheapest carbon

source and is used widely to enhance the expression of recombinant protein in E.coli.We

replaced the carbon source of LB medium with glycerol with the same percentage and found that

it enhanced the expression of roGH from 18% to 22% as analysed on SDS-PAGE (fig,34)

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It has earlier been reported that a reduction of the IPTG concentration has a positive effect on

periplasmic yield of soluble protein expressed from a lac promoter( Kipriyanov et al., 1997).

The lac promoter is highly inducible and overexpression of the recombinant oGH is evident

even at IPTG concentrations as low as 20 µM(Fig 30 ). Similarly high levels of expression are

obtained at final IPTG concentrations of 60,80, 100, 1000 µM. Un-induced cultures typically

display some evidence of background (“leaky”) expression, which is well characterized for T7

promoter-based vector systems (Pushkar Malakar, 2015). IPTG is a costly chemical and here we

showed that IPTG concentrations as low as 20 µM are sufficient for high levels of roGH protein

expression. Following induction, the recombinant oGH can be recovered from the periplasm.

E. coli cells are easily lysed by several methods and for most laboratory set-ups sonication and

freeze/thaw cycles are the method of choice (Berrow et al., 2006). Mechanical and physical cell

disruption methods have been assessed for the release of periplasmic proteins. Mechanical

methods such as high pressure homogenizer (Balasundaram et al., 2008) hydrodynamic

cavitation (Balasundaram et al., 2006), bead mill (Bakir, 1997) are not selective in releasing the

individual periplasmic proteins. On the other hand, physical methods such as osmotic shock

could be considered as a mild treatment with low operation cost which also has the ability to

release periplasmic proteins with very high selectivity. we compared different methods of

somotic shocks including freeze thaw method.We found that molar concentration of Tris

buffer,sucrose concentration and time of incubations along with concentration of chelating agent

i.e EDTA effects the release of recombinant oGH into periplasmic space.We found 20% release

of oGH by using freeze thaw method for the recovery of oGH as compared to 12 and 10 % yield

by other (Koshland, 1980; Ramakrishnan et al., 2010) osmotic shock procedures( Fig.32&33

).We optimized the freeze thaw method for the better yield of roGH from shock fluid.For this

purpose different factors were studied i.e. glycerol concentration,use of chemical chaperon

(ZnCl2 )concentration and incubation time.It was observed that by increasing glycerol

concentration from 10% to 25% in the shock procedure enhanced the released of oGH when

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analysed by Bradford method and SDS-PAGE( Fig, 35& 36a) The oGH was further

aunthenticated by western blot analysis ( Fig 35 b).Optimization of osmotic shock procedure for

the specific study has been conducted by several researchers ( Chen et al., 2004; Rastgar et al.,

2007) .They claimed that the pre treatment of cells with divalent cations of calcium and

magnesium prior to the introduction of hypertonic solution would have chelated the

lipopolysaccharide and increased the recovery of creatinase from 60% to 75 on the other hand,

the addition of magnesium chloride in hypotonic solution reduced the cytoplasmic contaminants

in the medium by reducing the chelation of plasma membrane .These findings helped us to study

the effect of ZnCl2 in the medium and also in shock fluid which enhanced the release of oGH in

periplasmic space as shown in graph(Fig 29). Chaperones are known to enhance expression

yields as they facilitate folding, prevent aggregation, reactivate aggregates and reduce protein

degradation (Ying et al., 2004).The roGH thus released was 80% pure and for further

purification FPLC chromatoghraphy resulted in 95% pure roGH (Fig 37 ) .

4.3 Secretion of oGH into the inner membrane of E.Coli.

Secretory production of recombinant proteins in E. coli has been particularly useful for the

production of pharmaceutical proteins as compared to cytoplasmic production. Targeting a

protein of interest to the periplasmic space or the culture medium enables downstream

processing at a reduced process cost. Isolation and purification of the over-expressed products

can be simplified and rapid due to reduced contamination of various cellular components and

hence reduce proteolytic degradation by intracellular proteases. Correct folding of eukaryotic

proteins containing multiple disulfide bonds is also likely to occur in the reducing environment

of the periplasmic space. Secretory process allows removal of the amino-terminal signal

sequence from the recombinant on reaching the destination and appearance of mature protein and

naturally occurring sequences contain no N-terminal methionine residue.

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we utilized PelB leader sequence of pET22b in construct poGH-2. The N terminal of ovine GH

gene was linked to PelB leader sequence of pET22b. As a result we analyzed good level

expression of about 18% of oGH but with an additional 3 kDa in its molecular weight on SDS-

PAGE (Fig 28). We optimized the conditions for poGH-2 construct got enhanced yield in the

periplasmic fraction too(36 a& b ) but still the yield was very low as periplasmic space of E.coli

constitute just 6% of the cell.

In order to get the correct size (22kD) roGH with high yield. We expressed roGH in extra

cytoplasmic space and for this purpose three roGH constructs were designed with different signal

sequences; oGH, DsbA and STII. T7 promoter based expression vector pET22b(+) and E. coli

strain BL21 codon plus were utilized. Among the constructs, constructs poGH4 and 5 with signal

sequences (oGH and ST-II) respectively showed roGH expression at 25kDa while in construct

poGH3 with DsbA signal sequence, roGH was found to be 22kDa ( Fig 43 ). In order to

understand the reason of varying behaviour of these all signal sequences( including pelB) they

were analyzed based on probability of signal sequence by signal p3.0 server

(http://www.cbs.dtu.dk/services/signalP/), hydropathy plot by kyte-doolittle method and

secondary structure by PolyView prediction server (http://polyview.cchmc.org) (Porollo et al.,

2004). The hydropathies of all the signal sequences gave variable results with an optimal range

of hydropathy value above or below at which the signal sequence does not function properly

(Table 3). The secondary structure of these signal sequence were also analyzed (Fig. 47 A, B, C

& D). The hydropathy value for these signal sequences varied from least value of 0.986 for ST-II

signal sequence up to highest value of 1.840 for oGH signal sequence. The hydropathy profile of

oGH showed that it is more than 60% hydrophobic (data not shown). The amino acid sequence

of DsbA, STII, pelB and oGH signal sequence showed that they have varying charges in their N

and C terminal regions. The N terminal of pelB leader sequence constitutes KYL in which K

(lysine) was basic, Y (tyrosine) and L (leucine) were hydrophobic neutral. Whereas, C terminal

consisted of AMA, in which A (alanine) was neutral hydrophobic and M (methionine) was polar

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hydrophobic amino acid. However, N terminal of STII signal sequence had KKNI sequence,

where K was basic hydrophilic with positive charge, N (asparagine) hydrophilic neutral, I

(isoleucine) hydrophobic neutral and at C terminal A (alanine) and tyrosine were hydrophobic

neutral. However, DsbA signal sequence constituted of KKI at N terminal and ASA at C

terminal where S (serine) was polar hydrophilic. Based on the above stated facts we can explain

the reason behind inefficient translocation of native oGH, STII and pelB signal sequences. Thus,

a very high hydrophobicity (1.8) of oGH signal sequence in construct poGH2 blocked export, as

oGH itself is very hydrophobic protein. The secondary structure of STII (Fig. 47,C) showed that

it constitutes beta sheets and had aromatic tyrosine residue at cleavage region which blocked

export. The hydrophobicity of the signal sequence has been a dominant structure for proper

functioning of the signal sequence (Goder and Spiess, 2003). A wide survey performed by

(Beckwith and coworkers, 2005) identified a strong correlation between hydrophobicity of the

leader peptide and export mechanism (Huber et al., 2005). The DsbA signal sequence when

compared with npr, ST-II, PhoA signal sequences gave best periplasmic expression in E. coli

(Soares et al. 2003). In bacteria the naturally occurring cleavable DsbA signal sequence

promotes SRP-based protein export, which is attributed to its apparent hydrophobicity that is

greater than that of two other signal sequences (pelB and STII) which do not promote SRP-

dependent export. DsbA signal sequence is thought to direct the fused thioredoxin protein to the

co-translational SRP pathway by acting as an inhibitor of folding, thus allowing effective

posttranslational export (Schierle et al., 2003). Similarly, the secretion of the human GH to the

periplasm has been reported earlier (Soares et al. 2003; Becker and Hsiung 1986) using DsbA

signal sequence. Moreover, the theoretical analysis of 22 different signal peptides by using

bioinformatics tools also proved DsbA as the best signal sequence for the soluble expression of

human GH (Zamani et al., 2015). In this study we found complete translocation of roGH to the

inner membrane of E. coli (E.coli constitutes three biological boundaries: the inner membrane,

the cell wall and the outer membrane). The inner membrane separates the cell into two

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compartments; cytoplasmic space contained within the inner membrane and periplasmic space

between the inner and outer membranes. In E. coli, proteins are synthesized in the cytoplasm and

are targeted to different destinations within the cell. A number of systems have been studied for

translocation of recombinant proteins. The information about primary and secondary structure of

target protein also affects its translocation (Zamani et al., 2015) as in GH because it is highly

hydrophobic possesses alpha helical structures that resemble helical bundle class of inner

membrane proteins (IMPs). It is known that DsbA is an SRP signal sequence (Schierle et al.,

2003) and SRP pathway is primarily used by E. coli for targeting the IMPs. The combination of

SRP based DsbA signal sequence and GH structure explains its translocation into the inner

membrane.

4.4 Effect of medium composition on the expression and secretion of oGH in

E.coli

When the expression of the recombinant protein is low and cannot be increased by the proposed

mechanisms, then the volumetric yield of desired protein can be augmented by growing the

culture to higher densities. This can be achieved by changing a few parameters, like medium

composition and providing better aeration by vigorous agitation (Cui et al., 2006; Blommel et al.,

2007).

Different mediums have been applied to enhance the expression of recombinant proteins. The

poGH-3 was selected as the best construct for the expression of recombinant ovine GH as it

gave a protein of accurate size at 22kDa but with an expression level of 14% (Fig 43). Therefore,

different medium compositions were used in order to enhance the expression level of poGH-3.

We demonstrated that by using different media compositions, simple additives and changing

temperatures could easily enhance production of recombinant protein roGH. We compared seven

different mediums and found a remarkable variation in the final density of growth culture

inoculated with roGH( Fig 50). We found that cell growth stops at a relatively low density by

using LB(as shown in( fig 48). This happens because LB constitute very less amounts of carbon

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source and divalent cations (Sezonov et al.,2007). Not surprisingly increasing the amount of

peptone or yeast extract leads to little bit higher cell densities. Also divalent cation

supplementation (MgSO4 in the millimolar range) results in higher cell growth( as shown in

fig.48 ). Terrific Broth and SB (Super Broth) media recipes have been shown to be superior to

LB for reaching higher cell densities(studier, 2005) .TB constitute mainly Glycerol that aids in

forming a solvent shell around a protein molecule as a protein stabilizer, and increases the

viscosity of a solution for prevention of protein association (Swartz, 2001). It was observed that

expression level was much enhanced up to 22% in terrific broth medium supplemented with

compatible solute whereas it was up to 12% in LB and M9NG medium. A positive effect of low

molecular weight additives (chemical chaperones) supplemented in the culture medium were

being observed in various studies in terms of yields of periplasmic expressed proteins. Sorbitol

addition to the culture medium resulted in higher accumulation of a functional scFv , glycine

betaine and sucrose were beneficial for the folding of immunotoxin and cytochrome c550(

Swartz, 2001; Barth et al., 2000) . The problem of production of sufficient amounts of pure and

fully active recombinant immunotoxins, however, still remains an obstacle for clinical

application.

Thre strategy was seen and explained ( Barth et al., 2000) as a new approach for the production

of sufficient amount of pure and fully active recombinant immunotoxins in an optimized

periplasmic expression under osmotic stress. Barth and his coworkers used protein-stabilizing

compatible solutes ( glycerol, sorbitol, glycine betaine, and hydroxyectoine) during the

production phase and in the course of purification and storage to optimize the functionality and

stability of the proteins.

In this study we compared 2 set of osmolytes .1; glycylglycine and glycine betaine (both are

glycine with different side chains) and 2;Sorbitol and mannitol ( isomers) on the production

enhancement of roGH ( Fig 51 a & b). The general mechanism for stabilization with osmolytes is

believed to be through changing the protein hydration by exclusion from the hydration layer of

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the protein The resulting change in the protein hydration increases the energy needed to denature

proteins. Only compatible osmolytes, i.e., molecules which could interact favorably with protein

side chains and stabilize them against inactivation and which could potentially contribute to ATP

generation by the cell, proved to be effective solubilizers of the overexpressed proteins and

inhibited formation of inclusion bodies (Jain et al., 2008).It has been previously reported that

when sorbitol with glycyl betaine ,it enhanced the expression and solubl purification e (Barth et

al., 2000). The mechanism by which Sorbitol can be transported across E. coli via the sorbitol-

specific phosphoenol-pyruvate phospho-transferase system in the form of sorbitol-6-phosphate

(Sussman et al., 1971). Sorbitol-6-phosphate can enter glycolysis by the action of sorbitol-6-

phosphate dehydrogenase, which converts it to fructose-6-phosphate, a key intermediate of

glycolysis. However ,the mechanism of glycylglycine-mediated enhanced solubilization remains

to be understood. E. coli is known to possess specific transporters for dipetides and

oligopeptides. These in turn are of particular advantage to the bacteria, which thrive in the

peptide-rich gut lumen environment (Lengeler, 1975). Another possibility is the direct

interaction of glycylglycine with the expressed protein by acting as a chemical chaperone (Ou,

2002). Glycylglycine transport behaves similar to other shock-sensitive transport systems

requiring ATP for its transport (Cowell, 1974). In the presence of higher concentrations of

glycylglycine in the media, the bacteria probably ends up spending considerable energy in active

glycylglycine transport, thus slowing down the overall metabolic rate including the rate of

translation. This probably allows more time for the expressed proteins to be folded correctly.It is

also reported that higher concentration of glycine added in medium also enhance the periplasmic

yield of recombinant protein( kaderbhai et al., 1997).

We observed that mannitol can much enhance the production of roGH as compared to sorbitol as

shown in graph and it was also observed that mannitol also enhanced the soluble recovery of

roGH as clearly shown in fig our result is in complete accordance with (Leibly et al., 2012)

which states that. naturally occurring osmolyte mannitol can effectively aid in the stability and

134

solubility of recombinant proteins.The second set of comparative study was between

glycylglycine and glycine betaine both are derivatives of glycine.We found glycylglycine as

much better additive in the TB medium for enhance soluble production of recombinant roGH as

compared to glycine betaine as shown in (fig 51 ).

There are other factors like temperature ,concentration of inducer and induction time which

were also optimized. studies showed that rate of expression and culture temperature can affect

the proper folding of recombinant proteins and inclusion body formation( Kaushik et al.,

2003).Reducing culture temperature usually leads to slower growth of bacteria, slower rate of

protein production and lower aggregation of target protein(Clark et al., 2004). For recombinant

oGH, as shown in ( fig 54 ), culture at 25°C resulted in more expressed protein than 37°C. This

finding has shown less aggregation of recombinant protein in lower temperatures (M.rezaei et

al., 2013). It has been shown that the level of recombinant protein expression is affected by

inducer concentration. Generally most of recombinant proteins tend to be aggregated at high

concentrations of IPTG, while some other proteins are less sensitive to aggregation due to their

inherent higher solubility ( Shivcharan et al., 2013).

Based upon our findings, 20µM IPTG resulted in the highest amount of recombinant protein.

After optimizing all the above factors we recommended 0.6M mannitol,50mM

glycylglycine.4%Nacl2 in terrific broth medium with 20µM IPTG ( inducer) concentration at

25C ,at 150rpm shaking rate for the best yield of soluble recombinant oGH ( Fig 57 ).we

compared the yield by using sorbitol and glycyl-betaine as a known compatible solute used for

the soluble protein production(Barth et al., 2000) and our designed mannitol plus glycylglycine

and we found that it results in more than double concentration of soluble roGH as shown in(

table 6 .

135

4.5 Effect of mutation in DsbA signal sequence on the expression and secretion

of OaST

In order to enhance the expression and secretion level of oGH we thought to design new

constructs based on the mutation in the tripartite structure of DsbA signal sequence. In bacteria,

the naturally occurring cleavable DsbA signal sequence promotes SRP-based protein export and

is attributed to its apparent hydrophobicity that is greater than that of two other signal sequences

(PelB and ST11) which do not promote SRP-dependent export. DsbA signal sequence is thought

to direct the fused thioredoxin protein to the co-translational SRP pathway by acting as an

inhibitor of folding, thus allowing effective posttranslational export; or (ii) DsbA signal sequence

is able to direct more rapid engagement of the protein with the secretory machinery (Schierle,

2003). Similarly, the secretion of the human growth hormone to the periplasm was reported

earlier (soares et al., 2003) using DsbA signal sequence. The effect of signal peptide changes on

the expression and secretion of bovine growth hormone was also investigated by (Klein et al.,

1992) but they failed to find any significant influence of the signal sequence. However, most of

the work on recombinant growth hormone is still being carried out for cytoplasmic expression to

study refolding procedures and effect of different media or hosts systems (Patra et al., 2000).

In the present study we studied the effect of change of amino acid in all three parts of DsbA

signal sequence on the expression and translocation of recombinant oGH protein. The change of

amino acid in N terminal (poGH-3VIII construct) and C terminal (poGH-3-VII construct) did not

show any deviance from already reported facts. As in case of poGH-3g construct the substitution

of arginine did not enhance the expression of OaST while the substitution of cystein in poGH-3f

changed the polar nature of C terminal and affected the cleavage of signal peptide (Tsumoto et

al., 2010).

The substitution of each alanine with Isoleucine in the hydrophobic domain of DsbA signal

sequence showed the importance of specific position of alanine in the H domain of DsbA signal

sequence.The DsbAsignal sequence directs export via the SRP mechanism (Gerstein et al., 2005;

136

Nobuyuki et al., 2005) and recognizes its substrate by the presence of a hydrophobic signal

sequence which interacts with Ffh and 4.5sRNA ( Herskovits et al., 2000; Gerstein et al., 2005).

The N and G region of bacterial SRP binds to the hydrophobic part of the signal sequence while

M binds to SRP and 4.5sRNA (Robert et al., 2002).

In the present study, we show that the amino acid substitution in the hydrophobic (H) part of

DsbAss determines the translocation of the precursor protein. It has been proven that Gly

residues in the H region of GspB signal sequence affect the routing of a recombinant protein by

the sec pathway (Barbara et al., 2007). We investigated Ala in the H domain of DsbA signal

sequence and observed that Ala at position 11 with reference to signal peptidase site is necessary

for SRP routing of recombinant OaST to the inner membrane of E. coli.

The poGH-3I,II,IV and 3VII showed 22kDa while pOaST-3III and 3V and VIII resulted in 25

kDa band of recombinant oGH on SDS-PAGE (Fig.51) while constuct poGH-3VII resulted in

negligible expression. The hydropathy analysis of these substitution in H domain (Table 8)

showed same hydropathy of 1.539 for constructs pOaST-3II, -3V and 1.689 for constructs

pOaST-IV and VI. The hydropathy did not affect the translocation but the substitution at

specific position changed the translocation. The DsbA signal sequence has 4 alanine in its H

domain which are at position -1,-4,-9 and -11 with respect to signal peptidase site (Table 7). It

was observed that substitution of alanine with Isoleucine at position -11 changes the whole

mechanism of translocation.

On the basis of our findings we suggest a model for SRP routing of the recombinant OaST

protein with an appended DsbA signal sequence. The Ffh part of SRP constitutes N, G and M

domain of Ffh induces the conformational change in the nascent hydrophobic site of the signal

sequence ( Robert et al., 2002). This means that a specific protein conformation in the

hydrophobic part of the signal sequence affects binding with the N and G domain of the Ffh (

Manuvera et al., 2010). The Ala at position -11 in the H domain with respect to signal peptidase

site of DsbA signal sequence is important for the binding of N, G domain of Ffh in SRP

137

mechanism as replacement by Ile at this position resulted in the localization of recombinant oGH

majorly in the cytoplasmic fraction without cleavage of signal peptide as explained with the

poGH-3V construct . We suggest that there is amino acid specificity in the H domain of DsbA

signal sequence which is essential for its binding with the SRP as described in the model (Figure

71).The model explains the substitution of Isoleucine instead of alanine at position 11 with

respect to signal peptidase site effects the translocation of oGH protein through SRP mechanism.

(a)

(b)

Figure 71.Model representing the mechanism of DsbA signal sequence with altered amino acid with SRP mechanism.

SRP with Ffh , 4 . 5 s RNA

OaSt Ffh

OaSt

Ribosome DsbA with Ile at -11 position

Inner membrane

DsbA ss

4 . 5 sRNA

R i b o s o m e

Inner membrane

SRP

OaSt Ribosome

DsbA ss with Ile at - 9 position

R i b o s o m e SRP

Ffh

SRP with Ffh , 4 . 5 s RNA

4 . 5 sRNA

posi

tio

138

4.6 Purification and Biological activity Assessment

The poGH-3 construct with the best expression of 32% by compatible solute medium was used

further for the purification and biological activity assessment. In order to solubilize the

membrane bounded oGH a simple procedure of adding acetonitrile v/v was used and it was

observed that 40% acetonitrile was considered to be the best concentration for the solubility of

oGH or extraction of oGH from membrane. For this purpose ultra-centrifugation was used and

protein was recovered from the inner membrane of E. coli( kaderbhai et.al.2008) To our

knowledge this was the first study describing the oGH extraction from the inner membrane of E.

coli while all the previous studies have shown different techniques like freeze thaw, osmotic

shock etc. to obtain a protein in soluble form . The soluble oGH obtain was 90% pure.

The mass was determined by MALDI TOF analysis which showed oGH of 21059 which was a

little bit less than calculated value i.e. 21086 of ovine ST . This little variation in the molecular

weight didn’t affect the biological activity of the ovine GH.There are different cell lines that are

being used to assess the biological activity of the GH, while in the current study we used HeLa

cell lines. An enhancement or proliferation of HeLa cells was being observed in the presence of

recombinant oGH though in the presence of BSA (control) no cell proliferation was detected.

Hence, this result proved that recombinant ovine GH was biologically active.

4.7 Conclusion

In this study we have described the GH sequence of locally isolated ovine breed Lohi and its

comparison with the other Bovidae species. we report a simple system for production of

recombinant ovine growth hormone directed to the E. coli periplasm via the pET based

expression platform to yield soluble, properly folded oGH . The amount of glycerol and ZnCl2 in

the medium and shock fluid enhanced the yield of oGH from periplasmic space. The oGH

isolated by optimised shock methods and FPLC binds the GH receptor with high affinity. This

139

system will be useful for the average research laboratory wishing to produce material to study

oGH biology.

Further we studied the high yield of expression and purification of recombinant oGH from the

inner membrane of E.coli by using DsbA signal sequence.We found that recombinant protein

yields can be increased significantly by supplementing the TB medium with 0.6M mannitol and

50mMglycylglycine in the presence of 4% NaCl .

Further the effect of amino acid substitution in the tripartite structure of DsbAss emphasized the

alanine at position 11 is of most importance in the translocation mechanism of DsbA signal

sequence.

We suggest for further studies

Application of the compatible solute conditions in fermentor in order to get higher yields

of soluble ovine growth homone

To apply poGH-3 construct in mammalian host cells can also enhance the production of

oGH in soluble form .

Homology modeling of all the DsbA based constructs should be done.

140

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