CONSTRUCTION OF A STREP-TAG II MUTANT MALTOSE BINDING

PROTEIN FOR REAGENTLESS FLUORESCENCE SENSING

SITI HALIMAH BINTI HASMONI

UNIVERSITI TEKNOLOGI MALAYSIA

CONSTRUCTION OF A STREP-TAG II MUTANT MALTOSE BINDING

PROTEIN FOR REAGENTLESS FLUORESCENCE SENSING

SITI HALIMAH BINTI HASMONI

A thesis submitted in fulfillment of the requirement for the award of the degree of

Master of Science (Bioscience)

Faculty of Bioscience and Bioengineering

Universiti Teknologi Malaysia

NOVEMBER 2012

iii

To my beloved parents, my family and my soulmate, my best friend; my husband

and not forgetting to our newborn bub, Muhammad Rafiqi.

iv

ACKNOWLEDGEMENT

First and foremost, I would like thank Dr Shafinaz, my supervisor, who had been

dedicated and committed to guide me all this while. Her patience, encouragement and

trust is truly appreciated and deeply cherished. Also, thanks to Prof Dr Anthony Cass,

for his advice and insights on this research. Next, I would like to express my gratitude to

Dr. Saiful Karsani and Dr. Goh Kian Mau is members of the research group. Thank you

for the opportunity and the wealth of information shared during discussions. Also, my

greatest gratitude goes to Prof Dr. Peter Klappa, who helps me solve the missing puzzle

of my research with his knowledge and wisdom.

Furthermore, I am thankful to Ministry of Higher Education for funding my

masters study. I would like to thank the Ministry of Higher Education again for

funding this project under Fundamental Research Grant Scheme (FRGS) Vote

Number 78309. A great appreciation for FBB,UTM for the research facilities they

provided and also thank you to PSZ library for easy access of many literatures

online.

Thank you to the technical supports from most of the companies that I

contacted with for their advices and help. Not forgetting my fellow postgraduates’

friends in this faculty for giving me their encouragements, helping hand and

motivation. Thank you for being there for me. Special thanks to the best

labmates/friends a researcher could for; Edot, Yok, Maya, Kak Su and Tiqah.

I would like to thank my supportive father and mother, my beloved sisters

and brothers for supporting me no matter what. I would like to extend my

appreciation to my husband who is always there for me through the ups and downs of

this journey. And lastly, I am grateful to Allah.

v

ABSTRACT

Maltose binding protein (MBP) changes its conformational structure upon its ligand

binding.This molecular recognition element that transduces a ligand-binding event

into a physical one make MBP an ideal candidate for reagentless fluorescence

sensing. MBP gene, (malE) was amplified from a pMaL-C4x plasmid vector and was

fused to a Strep-Tag II pET-51b(+) vector. Strep-Tag II is a tag that will enable the

MBP to be unidirectionally immobilized on solid supports. A cysteine mutant of the

MBP was constructed by inverse PCR and the recombinant protein fusion was then

purified by affinity purification using Strep-Tactin resin. To sense maltose binding,

an environmentally sensitive fluorophore (IANBD amide) was covalently attached to

the introduced thiol group. The tagged mutant MBP (D95C) was successfully

generated and the protein was successfully purified with the expected molecular size

of ~42 kDa observed on the SDS PAGE. The fluorescence measurements of the

IANBD labeled of tagged mutant MBP (Strep-Tag II D95C) in the solution phase,

showed an appreciable change in fluorescence intensity with dissociation constant,

(Kd) of 7.6 ± 1.75 µM. Nonetheless, it could retain its ligand binding activity towards

maltose. However, immobilization of Strep-Tag II D95C on solid surface suffered

some limitation with the Strep-Tactin coated microwell plates because it did not give

any dependable results to support the ligand binding activity of the site directed

immobilized protein. Thus, this engineered mutant MBP (Strep-Tag II fused D95C)

could be potentially developed for biosensor application with further improvement in

protein immobilization method.

vi

ABSTRAK

Protein pengikat maltosa (MBP) mengalami perubahan struktur konformasi

semasa mengikat pada ligan. Molekul pengenalpastian yang menyebabkan transduksi

pengikatan ligan kepada bentuk fizikal menjadikan MBP calon yang sesuai sebagai

penderia pendaflour tanpa reagen. Gen MBP (malE) diamplifikasi daripada vektor

plasmid pMaL-C4x dan kemudian digabungkan dengan Strep-Tag II yang terdapat

pada vektor pET-51b(+). Strep-Tag II merupakan tag yang membolehkan MBP

disekatgerak secara seragam kepada penyokong pepejal. Mutan MBP yang

mempunyai satu residu sisteina telah dihasilkan melalui PCR berbalik dan gabungan

protein rekombinan ini ditulen melalui penulenan afiniti yang menggunakan resin

Strep-Tactin. Untuk mengesan pengikatan maltosa, flourofor yang sensitif pada

persekitaran (IANBD amida) telah diikat secara kovalen kepada kumpulan tiol yang

telah diperkenalkan pada protein itu. Mutan MBP yang bertag telah berjaya dijana

dan melalui pemerhatian SDS-PAGE protein ini telah berjaya ditulen dengan saiz

molekul ~42 kDa seperti yang dijangkakan. Ukuran pendaflour di dalam fasa larutan

bagi mutan MBP yang bertag dan berlabel dengan IANBD menunjukkan perubahan

ketara bagi keamatan pendaflour dengan pemalar penguraian Kd 7.6 ± 1.75 µM.

Walau bagaimanapun, aktiviti pengikatan ligan terhadap maltosa boleh dikekalkan.

Pemegunan protein Strep-Tag II D95C pada permukaan pepejal, berhadapan dengan

beberapa kelemahan apabila piring mikrotelaga yang bersalut Strep-Tactin

digunakan kerana ia tidak memberi keputusan yang dapat menyokong aktiviti

pengikatan ligan oleh protein yang dipegunkan pada tapak khusus. Oleh itu,

kejuruteraan mutan MBP gabungan Strep-Tag II-D95C berpotensi dibangunkan

untuk aplikasi biopenderia dengan lebih penambahbaikan dalam kaedah pemegunan

protein.

vii

TABLE OF CONTENT

CHAPTER TITLE PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENT vii

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF APPENDICES xvi

LIST OF ABBREVIATIONS xvii

1 INTRODUCTION

1.1 Introduction 1

1.2 Research Objectives 3

1.3 Significance of Research 4

1.4 Scope of Research 4

2 LITERATURE REVIEW

2.1 Periplasmic Binding Protein (PBPs) 5

2.2 Maltose Binding Protein (MBP) 6

2.3 Reagentless Fluorescence Sensors Based on PBPs 9

2.3.1 Principles of Fluorescence 10

2.3.2 Thiol-reactive Fluorophore 12

viii

2.4 Engineering MBP for Reagentless Fluorescence Sensing 13

2.5 Tagged Proteins for Site-specific Immobilization 16

3 CONSTRUCTION OF STREP-TAG II MUTANT

MBP (D95C) FOR FLUORESCENCE SENSING

3.1 Introduction 19

3.2 Research Methodology 20

3.3 Materials 21

3.3.1 Chemicals and reagents 21

3.3.2 Bacterial strains 21

3.3.3 Plasmids 22

3.3.4 Enzymes 22

3.3.5 Culture media, buffers and stock solutions 22

3.4 Molecular Biology Methods 23

3.4.1 Competent cell preparation 23

3.4.2 Heat-shock transformation of cells 24

3.4.3 Plasmid extraction and purification 24

3.4.4 Restriction enzyme digestion of DNA 24

3.4.5 Agarose gel electrophoresis 25

3.4.6 DNA ligation 25

3.4.7 Construction of pET-51b (+)- malE 25

3.4.8 Screening for recombinant clones 27

3.4.9 Construction of pET-51b(+)-D95C 28

3.5 Results and Discussion 29

3.5.1 Construction of pET-51b (+)- malE 29

3.5.2 Construction of cysteine mutants of MBP (D95C) 34

3.5.2.1 Design of primer sequence for site-directed

mutagenesis 34

3.5.2.2 Identification of cysteine mutants by

restriction enzyme digestion 35

3.6 Conclusions 39

ix

4 EXPRESSION AND PURIFICATION OF STREP-TAG II

WILD TYPE MBP AND ITS MUTANT (D95C)

4.1 Introduction 40

4.2 Research Methodology 41

4.3 Materials 42

4.3.1 Chemicals and reagents 42

4.3.2 Culture media, buffers and stock solutions 42

4.4 Expression and purification of the recombinant protein 44

4.4.1 Recombinant protein expression of Strep-Tag II

Wild Type MBP and its mutant D95C 44

4.4.2 Spectroscopic determination of protein

concentration 45

4.4.3 Protein assay by Bradford reagent 47

4.4.4 Protein electrophoresis (SDS-PAGE) 47

4.4.5 Western blotting for detection of Strep-Tag II 48

4.4.6 Western blotting for detection of Maltose

Binding Protein 48

4.4.7 Recombinant protein purification via Strep-Tag II

affinity purification 49

4.4.8 Recombinant protein purification via

size exclusion chromatography 50

4.4.9 Ultrafiltration of the pooled fractions 51

4.5 Results and Discussions 52

4.5.1 Recombinant expression of Strep-Tag II

wild type MBP and its mutant (D95C) 52

4.5.2 Strep-Tag II affinity purification of Strep-Tag II

wild type MBP 57

4.5.3 Affinity purification of Strep-Tag II mutant

MBP (D95C) in room temperature 59

4.5.4 Size Exclusion Purification of Strep-Tag II

mutant MBP (D95C) 62

4.5.5 Affinity purification of Strep-Tag II mutant MBP

(D95C) in ice 65

x

4.6 Conclusions 71

5 INTRINSIC TRYPTOPHAN OF STREP-TAG II WILD

TYPE MBP AND EXTRINSIC FLUORESCENCE

SENSING OF STREP-TAG II MUTANT MBP (D95C)

5.1 Introduction 72

5.2 Materials 73

5.3 Methodology 74

5.3.1 Intrinsic tryptophan fluorescence of Strep-Tag II

wild type MBP 74

5.3.2 Extrinsic fluorescence of Strep-Tag II mutant

MBP (D95C) 75

5.3.2.1 Fluorophore labeling 75

5.3.2.2 Extrinsic fluorescence measurement of

Strep-Tag II mutant MBP (D95C) 76

5.3.3 Immobilization of Strep-Tag II mutant

MBP (D95C) on functionalized surface 77

5.3.3.1 Immobilization of Strep-Tag II D95C on

Strep-Tactin coated microwell plates 77

5.3.3.2 Immobilization of Strep-Tag II D95C on

Strep-Tactin agarose. 78

5.3.3.3 Immobilization of Strep-Tag II D95C on

Streptavidin coated microwell plates 78

5.4 Results and Discussions 79

5.4.1 Intrinsic tryptophan fluorescence of Strep-Tag II

wild type MBP 79

5.4.2 Extrinsic fluorescence sensing of Strep-Tag II

mutant MBP (D95C) 81

5.4.3 Fluorescence response of immobilized

Strep-Tag II D95C 85

5.4.3.1 Immobilization of Strep-Tag II D95C on

Strep-Tactin coated microwell plates 86

5.4.3.2 Immobilization of D95C on

xi

Strep-Tactin agarose 89

5.4.3.3 Immobilization of Strep-Tag II D95C

on streptavidin coated microwell plates 90

5.5 Conclusion 93

6 CONCLUSIONS AND FUTURE WORK

6.1 Conclusion 94

6.2 Future Work 95

6.2.1 Protein modeling of Strep-Tag II mutant MBP 95

6.2.2 X-ray crystallography 96

6.2.3 Characterisation of binding interactions by

isothermal titration calorimetry (ITC) 96

6.2.4 Characterisation of secondary structure and

folding protein properties by circular

dichorism (CD) 97

6.2.5 Improved methods for immobilization of

Strep-Tag II mutant MBP (D95C) 97

REFERENCES 98

APPENDINCES A-E 109

xii

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Types of thiol-reactive fluorophores 13

3.1 Primers sequence used for amplification of the

cytoplasmic malE gene 26

3.2 Primers sequence used for site-directed mutagenesis of the

MBP cysteine mutants D95C 28

4.1 The concentration (Molar) of crude lysate which

was calculated according to the Beer Lambert Law 54

4.3 The concentration (Molar) of protein sample which was

calculated according to the Beer Lambert Law 69

4.4 Purification table. The concentration and total protein

of protein samples using protein assay Bradford reagent. 70

5.1 Comparisons of the dissociation constant of D95C and wild type

MBP 83

5.3 Optimization of immobilization of D95C onto streptavidin

microwell plates. 91

xiii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 A three dimensional model of a maltodextrin binding

protein with bound maltose (1ANF) 6

2.2 A model of MBP transport system 7

2.3 Schematic model of MBP bound to Tar that induces the

chemotaxis of the bacteria 8

2.4 Jablonski Diagram 11

3.1 Workflow of construction of pET-51b(+)-malE 20

3.2 Workflow of construction of cysteine mutants of MBP (D95C)

by site-directed mutagenesis 21

3.3 pET-51b(+) Multiple Cloning Site 26

3.4 Agarose gel electrophoresis showing the size of

the amplified PCR product. 30

3.5 Agarose gel electrophoresis showing the size of the pET-51b(+)-

malE construct and the Restriction enzyme test of the construct 31

xiv

3.6 The contig assemblies of forward and reverse sequence

of pET-51b(+)-malE construct using DNA Baser v.2.75.0. 32

3.7 ORF finder for the contig assemblies of forward and reverse

sequence of pET-51b(+)-malE 33

3.8 Construction of MBP cysteine mutant (D95C) 34

3.9 Agarose gel electrophoresis showing the size of the product

of inverse PCR of pET-51b(+)-D95C 35

3.10 Agarose gel electrophoresis of screening mutant MBP

(pET-51b(+)-D95C) with restriction enzyme FspI 36

3.11 Agarose gel electrophoresis of diagnostic restriction enzyme

of BamHI and HindIII on the wild type and mutant 37

3.12 ORF finder for the contig assemblies of forward and reverse

sequence of pET-51b(+)-D95C. 38

4.1 Workflow the expression and purification of Strep-Tag II

wild type MBP and its mutant (D95C). 41

4.2 SDS- PAGE (12%) of the crude lysate of Strep-Tag II wild type

and mutant (D95C) proteins. 53

4.3 A: SDS-PAGE of crude lysate of the wild type and mutant

D95C. B: Western blot of MBP for crude lysate of the wild type

and mutant D95C. C: Western blot of Strep-Tag II for crude lysate

of the wild type and mutant D95C. 55

4.4 Chromatogram of Strep-Tag II affinity purification using

Akta-Prime Plus 57

xv

4.5 SDS- PAGE of the crude lysate and pooled of the fractions

collected during elution of Strep-Tag II purification 58

4.6 (A) Western blot of maltose binding protein for the crude lysate

and fractions collected during elution of Strep-Tag II purification.

(B) Western blot of Strep-Tag II for the crude lysate and

fractions collected during elution of Strep-Tag II purification 58

4.7 Elution peak of Strep-Tag II affinity purification using

Akta-Prime Plus 59

4.8 SDS- PAGE of the crude lysate D95C and the fractions

collected during elution of Strep-Tag II purification of D95C 60

4.9 (A) Western blot of maltose binding protein for the crude lysate

and fractions collected during elution of Strep-Tag II purification.

(B) Western blot of Strep-Tag II for the crude lysate and

fractions collected during elution of Strep-Tag II purification 61

4.10 Elution peak of Strep-Tag II affinity purification using

Akta-Prime Plus 63

4.11 SDS- PAGE of the affinity purification product and the

fractions collected during elution of Strep-Tag II purification

of D95C 63

4.12 SDS- PAGE of the crude lysate of D95C and pooled

fractions of affinity and gel filtration purification 64

4.13 SDS- PAGE of the crude lysate of D95C and observed the

presence of inclusion bodies from the pellet of lysed cell 65

4.14 SDS- PAGE of the crude lysate of D95C and pooled

fractions of affinity and gel filtration purification 66

xvi

4.15 (A) Western blot of Strep-Tag II for the crude lysate and

fractions collected during elution of Strep-Tag II purification.

(B) Western blot of maltose binding protein for the crude

lysate and fractions collected during elution of

Strep-Tag II purification 67

4.16 A: SDS-PAGE (12%) of crude lysate of the wild type

and mutant D95C. B: Western blot of MBP for crude lysate

of the wild type and mutant D95C. C : Western blot of

Strep-Tag II for crude lysate of the wild type and mutant D95C 68

5.1 Maltose binding curves of intrinsic tryptophan of wild type

MBP 80

5.2 Fluorescence emission spectra of IANBD labeled D95C 81

5.3 Maltose binding curves of labeled IANBD D95C 82

5.4 Glucose binding curve of the labeled IANBD D95C 84

5.5 Illustration of unidirectional immobilization of Strep-Tag II

fused D95C on functionalized surface 85

5.6 Maltose binding curves of Immobilized labeled IANBD of

D95C 90

xvii

LIST OF APPENDICES

APPENDIX TITLE PAGE

A Preparation reagents for SDS PAGE 109

B Standard curve for Bradford assay 111

C Molecular weight of protein standard of unstained

protein marker (Fermentas) 112

D Molecular weight of protein standard of Strep-Tag II

perfect protein markers (Merck) 113

E Raw data of fluorescence measurement for

immobilization Strep-Tactin coated microwell plate 114

xviii

LIST OF ABBREVIATIONS

ºC - Degree celcius

µg/ml - Microgram per millilitre

µl - Microliter

µM - Micromolar

bp - Basepair

BLAST - Basic local alignment search tool

CaCl2 - Calcium chloride

cm - centimeter

DMSO - Dimethyl sulphoxide

DNA - Deoxyribonucleic acid

dNTP - Deoxyribonucleotides

dsDNA - double stranded Deoxyribonucleic acid

DTT - Dithiothreitol

EDTA - Ethylenediaminetetraacetic acid

ELISA - Enzyme-linked immunosorbent assay

g - gram

HRP - Hydrogen peroxidase

IANBD - N,N’-dimethyl-N-(iodoacetyl)-N’-(7-nitrobenz-2-oxa-

1,3-diazol-4 -yl)ethylenediamine

IPTG - Isopropyl β-D-1-thiogalactopyranoside

K2HPO4 - Dipotassium hydrogen phosphate

KCl - Potassium chloride

Kd - Dissoication constant

kDa - kilodalton

KH2PO4 - Potassium dihydrogen phosphate

KOAc - Potassium acetate

xix

LB - Luria Bertani

LBA - Luria Bertani Agar

M - Molar

MgSO4.7H2O - Magnesium sulfate heptahydrate

mM - Milimolar

MnCl2 - Manganese chloride

MOPS - 3-(N-morpholino)propanesulfonic acid

NaCl - Sodium chloride

nm - Nanometer

OD - Optical density

PAGE - Polyacrylamide Gel Electrophoresis

PBS - Phosphate buffer saline

PCR - Polymerase chain reaction

PMSF - Phenylmethanesulfonyl fluoride

RbCl - Rubidium chloride

rpm – Rotation per minute

SDS - Sodium dodecyl sulfate

TAE - Tris base, acetic acid, EDTA buffer

UV - Ultraviolet

V - Voltage

v/v - Volume per volume

w/v - Weight per volume

CHAPTER 1

INTRODUCTION

1.1 Introduction

Biosensor is an analytical device that functions by coupling a biological

sensing element with a detector system using a transducer (Chauhan et al., 2004). It

involves exploiting the recognition and detection system of a biology component for

a target molecule or macromolecule with a transducer that converts the biological

recognition event into an output signal (Collings et al., 1997). The signal can be

electrical, optical or thermal and is converted by a suitable transducer into a

measurable electrical parameter such as electrical or current. Since its establishment,

biosensors have been widely used in diagnostics, pharmaceutical research,

agriculture, food safety, environment and industrial monitoring (Luong et al., 2008).

There have been some limitations in producing biosensors because each

device is unique and requires an amount of time and optimization. This is because

developments of most biosensors involve the identification of natural specificity of a

biological component for an analyte and the discovery of a suitable signal transducer

that is adapted to the macromolecule (Chauhan et al., 2004). Hence, protein

engineering techniques are being used to overcome this issue where signal

transduction properties of biological molecules are being modified to adapt the

detector instrumentation rather than adapting the detector instruments to the unique

2

requirements of each natural molecule. This is achieved by integrating a functional

group that gives simple signal-transduction mechanism such as optical or electrical,

to the protein itself (Hellinga and Marvin, 1998).

Development of sensor devices requires incorporation of sensing proteins into

a detector element by encapsulation or surface immobilization on a suitable material

for interfacing with detectors (De Lorimier et al., 2006). It is crucial for the protein

to be immobilized for reagentless biosensing and for further application in biosensor

such as protein microarray. Thus, with protein engineering techniques, enzymes and

proteins can be chemically modified to make them more stable and more specific

with more regular interface for immobilization. A protein may be modified so that its

active site points outwards from the sensor surface once the protein is immobilized

because the active sites need to be accessible to the analyte (Collings et al., 1997).

Thus, in this research, rational protein engineering techniques will be applied to

molecular engineer proteins for unidirectional immobilization on functionalized

surfaces and to investigate its protein activity. Moreover, it will assess the

applicability of immobilized protein for biosensing application for high throughput

analysis of analyte.

The protein used in this research is maltose binding protein (MBP) which is

in the superfamily of periplasmic binding protein (PBP) that is extensively studied

as receptors for sensor applications (Hellinga et al., 1998). This is because PBP

superfamily has a remarkable adaptablity for their cognate ligands and can be

designed to bind nonnatural ligands (Marvin and Hellinga, 2001). The structure of

PBPs both with and without their ligands bound, has been described as a ‘Venus

Flytrap’ where the two lobes of the protein will close upon the ligand, entrapping it

(Gilardi et al., 1994). This molecular recognition element that transduces a ligand-

binding event into a physical event makes it suited for biosensor applications such as

reageantless fluorescent biosensing. MBP will be altered genetically to construct a

regeantless biosensor so that a reporter group (fluorophore) may be covalently linked

to MBP. The fluorophore will respond to the ligand binding event of the labeled

protein and this response is measured by fluorescence intensity changes.

3

It is worth noting that, to the extent of our knowledge, all of the reported works

describing signal transduction by fluorophore labeled PBPs have been studied

without their linkage to solid supports (Brune et al., 1994; Gilardi et al., 1994;

Marvin et al., 1997; Hellinga and Marvin, 1998; Marvin and Hellinga, 2001). In this

research, protein immobilization will be done by exploiting the affinity tag by

genetic engineering. Affinity tags are widely used in biotechnology to assist

purification of recombinant protein. The MBP will be fused to an affinity tag, Strep-

Tag II to aid in purification and site-directed immobilization of the MBP onto

functionalized solid surfaces. Strep-Tag II consists of eight amino acids fusion tag

that will bind to Strep-Tactin protein (Merck Biosciences, 2007). The Strep-Tag II

will act as a handle that will enable the attachment of MBP to a Strep-Tactin surface

on microwell-plates for optimal ligand binding.

1.2 Research objectives

The ultimate aim of this work was to construct tagged fusion proteins for site

directed immobilization onto surfaces with a view to investigate their potential for

biosensor applications. To achieve this, the objectives were as follows:

1. To mutate the Maltose Binding Protein (MBP) for the development of a

reagentless fluorescence sensing system for maltose.

2. To overexpress and purify Strep-Tagged II fusion MBP and its mutant via

affinity chromatography

3. To determine the ligand binding activity of fluorophore-labeled mutant MBP

via fluorescence intensity measurements.

4

1.3 Significance of Research

The immobilization of proteins onto solid surfaces remains a critical aspect in

the development of biosensors. Furthermore, to the extent of our knowledge, all of

the reported works describing signal transduction by fluorophore labeled PBPs have

been studied without their linkage to solid supports. Thus, in this research, MBP was

first mutated to construct a reagentless biosensor for maltose sensing. The Strep-Tag

II fused to the mutated MBP enabled the attachment of the ligand binding protein

onto a functionalized solid surface. The ability of the immobilized protein to sense

ligand binding was subsequently assessed.

1.4 Scope of Research

To fulfill the objectives of this research, there were four main experimental

steps in laboratory work that needed to be done. Firstly, the construction of plasmids

containing wild type malE gene and its variant were done. This involved

amplification of malE gene, site-directed mutagenesis of malE gene and cloning. The

next step was expression and purification of wild type MBP and its mutant; which

involved optimizing protein expression to produce the optimal amount of protein and

purification of the protein by affinity purification. The third step was to analyze the

ligand binding characteristics of wild type MBP and its mutant via fluorescence.

Fluorescence measurements involved labeling the mutant MBP with fluorophore and

analyzing the ligand binding characteristic of MBP via fluorescence. The final step

was to analyze the ligand binding characteristics of the immobilized protein via

fluorescence. In this final step, the labeled mutant protein was immobilized onto a

functionalized microwell plate and its ability to sense maltose via fluorescence

intensity changes was determined.

98

References

Ames, G. F. (1986). Bacterial periplasmic transport systems: structure,

mechanism, and evolution. Annual Review of Biochemistry. 55(1): 397-425

Bell, C. E. and M. Lewis (2000). A closer view of the conformation of the Lac

repressor bound to operator. Nature Structural Biology. 7(3): 209-214.

Bollag, D. M., M. D. Rozycki and S. J. Edelstein. (1996). Protein Methods. (2nd

Edition). USA. John Wiley & Sons Inc. Publication

Bondos, S. E. and A. Bicknell. (2003). Detection and prevention of protein

aggregation before, during, and after purification. Analytical Biochemistry.

316 (2):223-231.

Brune, M., J. L. Hunter, J. E. Corrie and M. R. Webb (1994). Direct, real-time

measurement of rapid inorganic phosphate release using novel fluorescent

probe and its application to actomysin subfragment 1 ATPase.

Biochemistry. 33(27): 8262-8271

Bray, D. (1998). Signaling complexes: Biophysical Constraints on Intracellular

Communication. Annual Review of Biophysics and Biomolecular Structure.

27:59-75

Cha, T. W., A. Guo and X. Y., Zhu (2005). Enzymatic activity on a chip: The critical

role of protein orientation. Proteomics. 5: 416–419

Chauhan, S., V. Rai and H. B. Singh (2004). Biosensors. Resonance. 9 (12): 33-44

Chen, H. M. and C. W. Lin. (2007). Hydrogel-coated streptavidin piezoelectric

biosensors and applications to selective detection of Strep-Tag displaying

cells. Biotechnology Progress. 23: 741-748

99

Chiti, F., N. Taddei, F. Baroni, C. Capanni, M. Stefani, G. Ramponi and C. M.

Dobson, (2002). Kinetic partitioning of protein folding and aggregation.

Nature Structural Biology. 9(2): 137-143.

Coligan, J. E., B. M. Dunn, D. W. Speicher and P.T. Wingfield. (2003). Short

protocols in protein science: a compendium of methods from current

protocols in protein science. Canada. John Wiley & Sons Inc.

Collings, A. F. and F. Caruso (1997). Biosensors: recent advances. Report on

Progress in Physics. 60:1397-1445

Copeland, R. A (1994). Methods for protein analysis: a practical guide to

laboratory protocols. London, Great Britain. Chapman & Hall

Dattelbaum, J. D., L. L. Looger, D. E. Benson, K. M. Sali, R. B. Thompson, and H.

W. Hellinga (2004). Analysis of allosteric signal transduction mechanisms in

an engineered fluorescent maltose biosensor. Protein Science.14:284-291

Davidson, A. L. and P. C. Maloney (2007). ABC transporter: how small machines

do a big job. Trends in microbiology. 15 (10): 448-455

De Lorimier, R. M., J. J. Smith, M. A. Dwyer, L. L. Looger, K. M. Sali, C. D.

Paavola, S. S. Rizk, S. Sadigov, D. W. Conrad, L. Loew, and H. W.

Hellinga. (2002). Construction of fluorescent biosensor family. Protein

Science. 11:2655-2675

De Lorimier, R. M., Y. Tian and H. W. Hellinga (2006). Binding and signaling of

surface-immobilized reagentless fluorescent biosensors derived from

periplasmic binding proteins. Protein Science. 15: 1-9

Dippel, R and W. Boos (2005). The maltodextrin system of Escherichia coli:

metabolism and transport. Journal of Bacteriology. 187(24): 8322-8331

100

Dubendorf, J. W. and F. W. Studier (1991). Controlling basal expression in an

inducible T7 expression system by blocking the target T7 promoter with lac

repressor. Journal of Molecular Biology. 219(1): 45-59.

Duy, C. and J. Fitter. (2006). How aggregation and conformational scrambling of

unfolded states govern fluorescence emission spectra. Biophysical Journal.

90(10): 3704–3711.

Dwyer, M. A. and H. W. Hellinga. (2004). Periplasmic binding proteins: a versatile

superfamily for protein engineering. Current Opinion in Structural Biology

14:405-504

Falke, J. J. and G. L. Hazelbauer. (2001). Transmembrane signaling in bacterial

chemoreceptors. Trends in Biochemical Sciences. 26(4):257-265

Gaj, T., S. C. Meyer and I. Ghosh. (2007). The AviD-tag, a NeutrAvidin/avidin

specific peptide affinity tag for the immobilization and purification of

recombinant proteins. Protein Expression and Purification. 56(1):54-61

Garman, A. (1997). Non-radioactive labelling: a practical introduction. London,

United Kingdom. Academic Press.

GE Healthcare. (2008). Strep-Trap HP manual [Manual]. Sweeden. GE Healthcare.

Gilardi, G. (2004) Protein engineering for biosensors. J. Cooper and T. Cass.

Biosensors. (185- 240). United Kingdom. Oxford University Press

Gilardi, G., L. Q. Zhou, L. Hibber and A. E. Cass. (1994). Engineering the maltose

binding protein for reagentless fluorescence sensing. Analytical Chemistry

66(21):3840-3847

Gilardi, G., G. Mei, N. Rosato, A. F. Agro and A. E. G. Cass. (1997). Spectroscopic

properties of an engineered maltose binding protein. Protein Engineering.

10(5): 479-486

101

Giuseppe, A. D., K. Forti, F. Feliziani, G. Severi and M. Cagiola. (2010).

Purification by Strep-Tactin affinity chromatography of a delete envelope

gp51 protein of bovine leukaemia virus expressed in sf21 insect cells.

Protein Journal. 29:153–160

Glenn, K. F., M. J. Smith and D. M. Markovitz. (1997). Bacterial protease Lon is a

site-specific DNA-binding protein. Journal of Biological Chemistry.

272(1):534-538

Grossman, T. H., E. S. Kawasaki, S. R. Punreddy and M. S. Osburne. (1998).

Spontaneous cAMP-dependent derepression of gene expression in stationary

phase plays a role in recombinant expression instability. Gene. 209(1-2):95-

103.

Hamid, T. H. T. A., M. A. Eltaweel, R. N. Z. R. A. Rahman, M. Basri and A. B.

Salleh. (2009). Characterization and solvent stable features of Strep-tagged

purified recombinant lipase from thermostable and solvent tolerant Bacillus

sp. Strain 42. Annals of Microbiology. 59(1):111-118

Hellinga, H. W. and J. S. Marvin. (1998). Protein engineering and the development

of generic biosensor. Trends in Biotechnology. 6: 183-189

Huang, J., J. Villemain, R. Padilla and R. Sousa. (1999). Mechanisms by which T7

lysozyme specifically regulates T7 RNA polymerase during different

phases of transcription. Journal of Molecular Biology. 293: 457- 475

Johnson, L. and M. T. Z. Spence. (2011) Molecular probes® handbook- a guide to

fluorescent probes and labeling technologies. (11th

Edition). USA.

Invitrogen, Life Technologies Corporation.

Juda, G. A., J. A. Bollinger, and D. M. Dooley. (2001). Construction,

overexpression, and purification of Arthrobacter globiformis amine

oxidase–strep-tag ii fusion protein. Protein Expression and Purification.

22: 455–461

102

Kambampati, R. and C. T. Lauhon. (2000). Evidence for the transfer of sulfane

sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in

Escherichia coli tRNA. Journal of Biological Chemistry. 275:10727-10730

Khan, F., M. Hue and M. J. Taussig. (2006). Double-Hexahistidine tag with high

affinity binding for protein immobilization, purification and detection on Ni-

nitrilotriacetic acid surfaces. Analytical Chemistry. 78(9) 3072-3079

Koiwai, O. and H. Hayashi. (1979). Studies on bacterial chemotaxis for interaction

of maltose receptor with membrane bound chemosensing component.

Journal Biochemistry. 86 :27-34

Lakowicz, J. R. (1999). Principles of Fluorescence Spectroscopy. (Second Edition).

Spring Street, New York. Kluwer academic/ Plenum Publisher New York.

Le, T. T., C. P. Wilde, N. Grossman and A. E. G. Cass. (2011). A simple method for

controlled immobilization of proteins on modified SAMs. Physical Chemistry

Chemical Physics. 13, 5271–5278

Le Grice, S. F. J., H. Matzura, R. Marcoli, S. Lida and T. A. Bickle. (1982). The

catabolite-sensitive promoter for the chloramphenicol acetyl transferase gene

is preceded by two binding sites for the catabolite gene activator protein.

Journal of Bacteriology. 150(1):312-318

Lesaicherre, M. L., R. Y. P. Lue, G. Y. J Chen, Q. Zhu and S. Q. Yao (2002). Intern

mediated biotinylation of proteins and its application in a protein microarray.

Journal of the American Chemical Society. 124 (30): 8768-8769

Lee, W., B. K. Oh, W. H. Lee and J.W. Choi. (2004). Immobilization of antibody

fragment for immunosensor application based on surface plasmon resonance.

Colloids and Surfaces B: Biointerfaces. 40 :143–148

Ling, M. M. and B. H. Robinson. (1997). Approaches to DNA mutagenesis: An

overview. Analytical Biochemistry. 254:157-178

103

Litchy, J. L., J. L. Malecki, H. D. Agnew, D. J. Michelson-Horowitz, S. Tan. (2005).

Comparison of affinity tags for protein purification. Protein expression and

Purification. 41: 98-105

Luong, J. H. T, K. B. Male and J. D. Glennon. (2008). Biosensor technology:

technology push versus market pull. Biotechnology Advances. 26: 492-500

Lyndon, L. E., R. A. W. Salins, C. M. Ensor, and S. Daunert. (2001). A novel

reagentless sensing system for measuring glucose based on the

galactose/glucose-binding protein. Analytical Biochemistry. 294: 19–26.

Maier, T., N. Drapal, M. Thanbichler and A. Bock. (1998). Strep-Tag II affinity

purification: an approach to study intermediates of metalloenzyme

biosynthesis. Analytical Biochemistry. 259: 68–73

Marvin, J. S., E. E. Corcoran, N. A. Hattangadi, J. V. Zhang, S. A. Gere and H. W.

Hellinga. (1997). The rational design of allosteric interactions in a

monomeric protein and its applications to the construction of biosensors.

Proceedings of the National Academy of Sciences of the United States of

America. 94 (9): 4366-4371

Marvin, J. S. and H. W. Helinga. (1998). Engineering biosensors by introducing

fluorescent allosteric signal transducers: Construction of a novel glucose

sensor. Journal of The American Chemical Society. 120 (1): 7-11

Marvin, J. S and H. W. Hellinga. (2001). Manipulation of ligand binding affinity by

exploitation of conformational coupling. Nature Structural Biology .

8 (9): 795-798

McCarter, J. D., D. Stephens, K. Shoemaker, S. Rosenberg, J. F. Kirsch and G.

Georgiou. (2004). Substrate specificity of the Escherichia coli outer

membrane protease OmpT. Journal of Bacteriology. 186 (17): 5919-5925

104

Merck Biosciences. (2007). Novagen Strep-tag II, Tools for protein expression,

purification and detection in bacterial, insect and mammalian cells. [Strep-

Tag Brochure]. United States of America. Merck

Mierendorf, R., K. Yeager and R. E. Novy. (1994a). The pET System: your choice

for expression. InNOVAtions. 1(1):1-3

Mierendorf, R. C., B. B. Morris, B. Hammer and R. E. Novy. (1994b). Expression

and purification of recombinant proteins using the pET system. R. Rapley

(Ed.). The Nucleic Acid Protocols Handbook. Totowa, N.J. Humana Press

Inc.

Miller, D. M. 3rd

, J. S Olson, J. W. Pflugrath and F. A. Quiocho. (1983). Rates of

ligand binding to periplasmic proteins involved in bacterial transport and

chemotaxis. Journal Of Biological Chemistry. 258 (22): 13665-13672

Miroux, B. and J. E. Walker. (1996). Over-production of proteins in Escherichia

coli: mutant hosts that allow synthesis of some membrane proteins and

globular proteins at high levels. Journal of Molecular Biology. 260:289–298

Mocz, G. (1999). The Use of Fluorescence in Research into Protein Structures.

unpublished note, A PPS ‘99 Project University of Hawaii.

Nakane, P. K. and A. Kawaoi. (1974). Peroxidase-labeled antibody a new method

of conjugation. Journal of Histochemistry and Cytochemistry. 22 (12):

1084-1091

Nikaido, H. (1994). Maltose transport system of Escherichia coli: an ABC-type

transporter. FEBS Letters. 346(1):55-58

Novy, R. and B. Morris. (2001). Use of glucose to control basal expression in the

pET system. BioTechniques. 12 (26): 1–3

105

Ntziachristos, V. (2006). Fluorescence molecular imaging. Annual Review

Biomedical Engineering. 8:1–33

Pabrosky, L. R., K. E. Dunn, C. S. Gibbs and J. P. Dougherty. (1996). A nickel

chelate microtiter plate assay for six-histidine containing proteins. Analytical

Biochemistry. 234(1):60-65

Peluso, P., D. S. Wilson, D. Do, H. Tran, M. Venkatasubbaiah, D. Quincy, B.

Heidecker, K. Poindexter, N. Tolani, M. Phelan,K. Witte, L. S. Jung, P.

Wagner, and S. Nock. (2002). Optimizing antibody immobilization strategies

for the construction of protein microarrays. Analytical Biochemistry. 312:

113–124

Perutz, M. F., B. J. Pope, D. Owen, E. E.Wanker and E. Scherzinger. (2002).

Aggregation of proteins with expanded glutamine and alanine repeats of the

glutamine-rich and asparagine-rich domains of Sup35 and of the amyloid β-

peptide of amyloid plaques. Proceedings of the National Academy of

Sciences of the United States of America. 99 (8): 5596-5600.

Rusmini, F, Z. Zhong and J. Feijen. (2007). Protein immobilization strategies for

biochips. Biomacromolecules. 8: 1775-1789

Salins, L. L. E., R. A. Ware, C. M. Ensor and S. Daunert (2001). A novel reagentless

sensing system for measuring glucose based on the galactose/glucose-binding

protein. Analytical Biochemistry. 294: 19-26

Schmidt, T. G. M. and A. Skerra. (1994). One-step affinity purification of bacterially

produced proteins by means of the “Strep tag” and immobilized recombinant

core streptavidin. Journal of Chromatography A. 676 (2): 337-345.

Seo, M. H., J. Han, Z. Jin, D. W. Lee, H. S. Park, and H. S. Kim. (2011). Controlled

and oriented immobilization of protein by site-specific incorporation of

unnatural amino acid. Analytical Chemistry. 83: 2841-2845

106

Skerra, A. and T. G. M. Schmidt. (1999). Applications of a peptide ligand for

streptavidin: the Strep-tag. Biomolecular Engineering. 16: 79–86.

Shahir, S. (2006). Engineering the Maltose Binding Protein for Metal Ions Sensing.

Doctor Philosophy. Imperial College London, United Kingdom.

Sharff, A. J., L. E. Rodseth, J. C. Spurlino, and F. A. Quiocho. (1992).

Crystallographic Evidence of a Large Ligand-Induced Hinge-Twist Motion

between the Two Domains of the Maltodextrin Binding Protein Involved in

Active Transport and Chemotaxis. Biochemistry. 31:10657-10663

Sharff, A. J., L. E. Rodseth and F. A. Quiochol. (1993). Refined 1.8-Ǻ structure

reveals the mode of binding of p-cyclodextrin to the maltodextrin binding

protein. Biochemistry. 32:10553-10559

Shrestha, S., L. L. E. Salins, C. M. Ensor and S. Dauner. (2002). Rationally

designed fluorescently labeled sulfate-binding protein mutants: evaluation in

the development of a sensing system for sulfate. Biotechnology and

Bioengineering. 78: 517–526

Sohanpal, K., T. Watsuji, L. Q. Zhou and A. E. G. Cass. (1993). Reagentless

fluorescence sensor based upon specific binding proteins. Sensor and

Actuators. 11:547-552

Sørensen, H. P. and K. K Mortensen. (2005). Advanced strategies for recombinant

protein expression in Escherichia coli. Journal of Biotechnology. 115: 113-

128

Spurlino, J. C., G. Y. Lu and F. A. Quicho. (1991). The 2.3 Ǻ resolution structure of

the maltose or maltodextrin- binding protein, a primary receptor of bacterial

active transport and chemotaxis. The Journal of Biological Chemistry.

266(6):5202-5219

107

Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff. (1990). Use Of

T7 RNA-Polymerase to direct expression of cloned genes. Methods in

Enzymology. 185: 60-89.

Szmelcman, S. and M. Schwartz. (1976). Maltose transport in Escherichia coli K12:

a comparison of transport kinetics in wild-type and a-resistant mutants

with the dissociation constants of the maltose-binding protein as measured

by fluorescence quenching. Journal Biochemistry. 65: 13-19

Taddei, N., C. Capanni, F. Chiti, M. Stefani, C. M. Dobson and G. Ramponi (2001).

Folding and aggregation are selectively influenced by the conformational

preferences of the alpha-helices of muscle acylphosphatase. Journal of

Biological Chemistry. 276(40): 37149-37154.

Taraska, J. W., and W. N. Zagotta (2010). Fluorescence application in molecular

neurobiology. Neuron. 66(2):170-89.

Templin, M. F., D. Stoll, M. Schrenk, P. C. Traub, C. F. Vohringer and T. O. Joos

(2002). Protein microarray technology. Trends in Biotechnology. 20 (4):

160-166

Tsai, C. W., C. I. Liu and R. C. Ruaana. (2009). A novel strategy for oriented

protein immobilization. World Academy of Science, Engineering and

Technology. 53: 975-979

Vercillo, N. C., K. J. Herald, J. M. Fox, B. S. Der, and J. D. Dattelbaum. (2006).

Analysis of ligand binding to a ribose biosensor using site-directed

mutagenesis and fluorescence spectroscopy. Protein Science. 16: 362-368

Villaverde, A. and M. M. Carrio. (2003) Protein aggregation in recombinant bacteria:

biological role of inclusion bodies. Biotechnology Letter. 17: 1385-1395

108

Voss S. and A. Skerra. (1997). Mutagenesis of a flexible loop in streptavidin leads

to higher affinity for the Strep-tag II peptide and improved performance in

recombinant protein purification. Protein Engineering. 10 (8): 975-982

Walker, J. M. (2002). The Protein Protocol Handbook. (2nd

Edition).United

Kingdom. Humana Press.

Wemmer, E. M. (2003). The energetic of structural change in maltose binding

protein. Proceedings of National Academy of Sciences. 100 (22): 12529-

12530

Ye, L., C. D. M. Filipe, M. Kavoosi, C. A. Haynes, R. Pelton and M. A. Brook.

(2009). Immobilization of TiO2 nanoparticles onto paper modification

through bioconjugation. Journal of Materials Chemistry. 19: 2189–2198

Zhou, Q. L. and A. .E. G. Cass. (1991). Periplasmic binding protein based

biosensors: preliminary study of maltose binding protein as sensing element

for maltose biosensor. Biosensors and Bioelectronics. 6 (1): 445-450

Zhu, H. and M. Snyder. (2003). Protein Chip Technology. Current Opinion in

Chemical Biology. 7: 55-63