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Chapter 2 Materials and Methods

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43


2.1 MATERIALS

Cell culture media, culture flasks, enzymes, chemicals, reagents, kits,

X-ray films, membranes and radio-isotopes were obtained from the

following companies-

Ambion- RNAzap

Amresco- Ampicillin, Potassium chloride, Tris base,

BD biosciences- Thrombin

GBiosciences (Genotech) - Protease Arrest (protease inhibitor

cocktail), FAST silver™

GE Healthcare- Lysozyme, Tris-saturated phenol, proteinase K,

RNase A (DNase free), agarose, restriction enzymes, IPTG, X-gal, f3-mercaptoethanol, PMSF, urea, Sephadex G-50 (DNA grade), X-ray

films (Hyperfilm-MP), Hybond N+ membrane, Hybond ECL NC

membrane, Hybond C+ nitrocellulose membrane, ECL Advance

Western blotting detection kit, Tween-20, Amplify fluorographic

reagent, mice anti-His antibody, Superdex 200HR 10/300 column,

glutathione sepharose high performance, protein A sepharose™ CL-

4B

GIBCO-BRL (Invitrogen)- Platinum Pfx DNA polymerase, lOObp DNA

ladder, lkb DNA ladder, Trizol, DNase I, EDTA, Supercsript™ first

strand synthesis system for RT-PCR, Albumax II, Freund's adjuvant

(complete and incomplete),

Hi-media- Agar powder, Luria Bertani broth, tryptone, yeast extract,

sodium azide, Silicone grease, soluble starch, hydrolysed casein,

meat extract, soybean meal, calcium carbonate, oatmeal powder,

malt extract, mycological peptone 44


ICN- PAGE preservation system, Urea, protein markers

IDT- PCR primers

BRIT, India- [y-32P] GTP, L- 2SS-Methionine

Life technologies- Auprep DNA gel extraction kit

Lobachemie- Formic acid

Macherey-Nagel- Protino® glutathione agarose 48, nitrocellulose

membrane

MBI-Fermentas- Restriction enzymes and buffers, protein molecular

weight markers, DNase I enzyme, T4 DNA ligase, DNA markers.

Merck- Acetone, isopropanol, methanol, 1-butanol, NDSB-256,

Sodium thiocyanate, n-propanol, chloroform

Millipore- Protein concentrator (Centricon), sterile filters, glass fibre

filters

Molecular Probes- Enzchek phosphate detection kit, mant-GDP,

mant-GTP

Nestle- Non-fat dry milk

New England Biolabs- Restriction enzymes and buffers, RNA Marker,

100 bp and 1 kb DNA marker, amylose resin, Factor Xa

Novagen- Protein refolding kit, S-protein agarose

Promega- Taq DNA polymerase, T4 DNA ligase, pGEM®-T Easy

Vector Systems

45


Qiagen- Gel extraction kit, Ni-NTA superflow, Qiagen plasmid mm1

kit, Qiagen plasmid midi kit, Qiagen plasmid maxi kit

Qualigens- Acetic acid, acetone, hydrochloric acid, sulphuric acid,

g1emsa stain, glycerol, copper sulphate, EDTA, isoamyl alcohol,

sodium carbonate, sodium chloride, sodium potassium tartarate,

hydrogen peroxide, tri-sodium citrate, ammonium thiocyanate,

trichloroacetic acid

Roche- Monoclonal anti-GFP antibody, Digoxigenin luminescent

detection kit

Ranbaxy- EDTA, chloroform, formic acid, glycerol

S.D. fine chemical- Dimethyl sulphate, methanol

Sigma-Aldrich- Acrylamide, RPMI -1640 media, incomplete modified

RPMI, glucose, sodium bicarbonate, gentamycin, hypoxanthine, D

sorbitol, saponin, glycerol, sodium chloride, L-glutamine, L-cysteine,

giemsa stain, DEPC, sucrose, guanidium thiocyanate, MOPS, sodium

acetate, formamide, ethidium bromide, xylene cyanol, bromophenol

blue, ammonium acetate, ammonium bicarbonate, malachite green,

sodium cacodylate, N-laurylsarcosine, PCR primers, Tris-saturated

phenol, water saturated phenol, agarose, plasmid midiprep kit, Gene

elute plasmid miniprep kit, Potassium iodide, silicon dioxide,

coomass1e brilliant blue R, Ponceau stain, acrylamide, bis

acrylamide, Tris-saturated phenol, sodium dodecyl sulphate,

ammonium persulfate, TEMED, Tricine, glycine, triton X-100,

imadazole, calcium chloride, salmon sperm DNA, sodium

deoxycholate, glasswool, BSA, DAPI, ammonium chloride, Igepal

(Nonidet P-40), paraformaldehyde, glutaraldehyde, formaldehyde,

poly-L-lysine, sodium borohydride, citric acid, DTT, EDTA, Potassium

chloride, sodium di-hydrogen phosphate, di-sodium hydrogen

46


phosphate, iodoacetamide, DMSO, bicinconinic acid (BCA) protein

assay kit, magnesium chloride, potassium chloride, magnesium

sulphate, sodium azide, dialysis tubing, dialysis tubing closures, 0-

phenyldiamine, imidazole hydrochloride, diaminobenzidine, silver

nitrate, ampicillin, chloramphenicol, developer, fixer, potassium

thiocyanate, sheep anti-rabbit HRP conjugate, Sodium dodecyl

sulphate, pyruvate kinase/lactate dehydrogenase mix (PK/LDH),

phosphoenolpyruvate (PEP), thrombin

Spectrochem- Acetonitrile, Diethyl ether

SRL- Tris-base, sodium acetate, sodium chloride, isoamyl alcohol,

sodium hydroxide, glycine, sodium bicarbonate, boric acid, petroleum

ether, ethyl acetate

Stratagene- QuickChange XL Site-Directed Mutagenesis Kit

Tarsons- 25cm2 culture flasks, 75cm2 culture flasks, 60 mm culture

dishes, 90 mm culture dishes, disposable sterile pipettes, 50 ml and

15 ml falcon tubes, ELISA plates, round bottom polypropylene

centrifuge tubes.

47


2.2. METHODS

2.2.1. In vitro Plasmodiumfalciparum culture

2.2.1.1. Culture media

2.2.1.2. Processing of red blood cells (RBC)

2.2.1.3. Initiation, maintenance and sub-culturing of P.

falciparum cultures

2.2.1.4. Synchronization of parasite cultures

2.2.1.5. Cryopreservation and revival of parasites

2.2.1.6. Revival of D 10ACP (leader)-GFP strain

2.2.2. Total genomic DNA isolation from P. falciparum culture

2.2.3. Molecular cloning

2.2.3.1. Cloning of Elongation factor Ts gene (EF-Ts)

2.2.3.2. Isolation of total P. falciparum RNA

2.2.3.3. RT-PCR of EF-Ts transcript

2.2.3.4. Agarose gel purification of linear DNA

2.2.3.5. Cloning of PCR-amplified DNA fragments into

expression vectors

2.2.3.5.1. PjEF-Ts gene expression construct

2.2.3.5.2. PjEF-Tsexonl construct

2.2.3.5.3. Cloning of PjEF-Ts signal and transit peptide

[ PjEF-TS(s/t)

2.2.3.5.4. PjEF-Tu as GST fusion construct

2.2.3.5.5. Cloning of PjEF-Ts & PjEF-Tu in pETDuet-1

vector

2.2.3.5.6. GST-PjEF-G(I-IVa) expression construct

2. 2. 3. 5. 7. 6xHis-PjEF -G(I-IIIl expression construct

2.2.3.5.8. EF-Tu mutagenesis

48


2.2.4. Recombinant protein expression and purification

2.2.4.1. Expression and purification of PJEF-Ts

2.2.4.2. Expression of PJEF-Tsexonl

2.2.4.3. Refolding and purification of PJEF-Tu

2.2.4.4. Expression and purification of PjGST-EFTu

2.2.4.5. Expression and purification of PjEF-Ts & PjEF-Tu

cloned in pETDuet-1 vector

2.2.4.6. Expression and purification of P.fEF-G(I-IVa)

2.2.4.7. Expression and purification of P.fEF-G(I-III)

2.2.5. Generation of polyclonal antisera

2.2.5.1. Preparation of PJEF-Ts protein

2.2.5.2. Preparation of PJEF-Tu protein

2.2.5.3. Preparation of P.fEF-G(I-III) protein

2.2.5.4. Raising antisera in rabbit and mice

2.2.5.5. Purification of antibodies by immobilisation on

nitrocellulose membrane

2.2.6. Western blotting and immunoprecipitation

2.2.6.1. Western blotting

2.2.6.2. Detection of PjEF-Ts in P. falciparum lysate

2.2.6.3. Immunoprecipitation of PJEF-Tu from parasite lysate

2.2.6.4. EF-Ts pull down assay

2.2. 7. Production and purification of antibiotics

2. 2. 7. 1. Culture of actinomycetes strains

2.2.7.2. Production and purification of GE2270A

2. 2. 7. 3. Production and purification of pulvomycin

49


2.2.8. Nucleotide binding, hydrolysis and release

2.2.8.1. Nucleotide binding by PjEF-Tu

2.2.8.2. Nucleotide release by PjEF-Ts

2.2.8.3. Intrinsic GTPase activity of PjEF-Tu and effect of

antibiotics

2.2.8.4. GTPase activity of PjEF-G(I-IVa)

2.2.8.5. Effect of kirromycin on GDP dissociation from PjEF

Tu.GDP

2.2.8.6. Kinetics of GDP release from PJEF-Tu by PjEF-Ts

2.2.9. Assay of insulin disulfide reduction

2.2.10 Structural modeling and molecular dynamics

50


2.2. METHODS

2.2.1. In vitro Plasmodiumfalciparum culture

2.2.1.1. Culture media

Complete RPMI -1640 medium (Rosewell ,eark Memorial Institute) was

used to maintain RBCs infected with P. falciparum (strains 3D7 and

D10 ACP (leader) -GFP). To make 1L of RPMI culture medium, 16.4g of

RPMI -1640 (HE PES modified), 1 Og glucose and 2 g sodium

bicarbonate were dissolved in 1L of water. This is the incomplete

medium and is used for processing of erythrocytes. To make complete

culture medium, 10% vjv human serum or 0.5% wjv Albumax II

(Invitrogen) was added to incomplete medium and pH was adjusted to

7.4. Thus, the complete medium contains 25mM HEPES, 1% glucose,

0.2% sodium bicarbonate and 10% vjv human serum or 0.5% wjv

Albumax II. To avoid bacterial contamination, gentamycin sulfate was

added at a final concentration of 25)lg/ ml and the medium was filter

sterilized. Hypoxanthine was added at a final concentration of 92 J.LM.

The P. falciparum cell line, D10ACP (leader)-GFP (ATCC number: MRA-

568) was maintained under selection pressure of 10nM

pyrimethamine (Waller et al., 2000).

2.2.1.2. Processing of red blood cells (RBC)

Processed RBCs were used for parasite culture. For RBC processing,

20ml of human blood was collected in a sterile 50ml tube containing

7.5ml anticoagulant (ACD; For 100ml, 0.0375M citric acid, 0.075M

tri-sodium citrate and 0.15M dextrose). The cells were pelleted at

2,000 rpm for 10 min at 4°C, supernatant was discarded and the

pellet was washed twice with incomplete RPMI medium. Equal

volume of complete RPMI medium was added to the pellet to make a

RBC stock of 50% hematocrit. The RBC stock was stored at 4°C and

used for around 15 days.

51


2.2.1.3. Initiation, maintenance and subculturing of P.

falciparum cultures

In vitro cultivation of erythrocytic stages of P. falcipantm was carried

out in human erythrocytes at 37°C under low 02 (5-8%) and 2-5%

C02 with a balance of N2 gas. The candle jar method of Jensen and

Trager (Jensen and Trager, 1978) is the simplest way to achieve this

gas phase. The culture medium was changed every 24h. A thin smear

was made from the culture, stained with Giemsa stain (Schichtherle

et al., 2000) and percent parasitemia was determined by counting

infected and uninfected cells under the microscope. After 4-5 days,

when the parasites were predominantly at trophozoite stage and

percent parasitemia was about 6-8%, sub-culturing of the cells was

performed. For sub-culturing of parasites, fresh RBC stock (50%

hematocrit) was diluted to 5% hematocrit in complete medium and

added to an appropriate volume of culture such that the final

parasitemia was 1-2%. The culture was maintained in 60 mm dishes

as well as in 25 cm2 and 75 cm2 culture flasks (6 ml and 12 ml of

parasite culture, respectively).

2.2.1.4. Synchronization of parasite culture

Synchronization of parasites was carried out at high parasitemia

when the parasites were predominantly in the ring stage. 5% D

sorbitol solution was used to synchronize the mixed stages in vitro

continuous culture of P. falcipantm (Lambros and Vanderberg, 1979).

Normally erythrocytes are impermeable to sorbitol but the developing

parasites change the erythrocyte membrane permeability to this

sugar so that washing the erythrocytes in an aqueous sorbitol

solution lyses all trophozoite and schizont infected erythrocytes,

leaving only uninfected and ring-infected erythrocytes.

52


For synchronization, three volumes of 5% D-sorbitol was added to the

parasite pellet and incubated at 37°C for 10 min with intermittent

shaking. The cells were centrifuged and washed twice with

incomplete medium. Erythrocytes (at 5% hematocrit) and complete

medium were added to the pellet and the culture was maintained in a

25-cm2 culture flask. The cultures containing ring stage parasites

were allowed to develop synchronously.

2.2.1.5. Cryopreservation and revival of parasites

Frozen stock of P. falciparum culture was prepared by the Stockholm

Sorbitol method (Schichtherle et al., 2000). The freezing

solution/ cryoprotectant consisted of 28% glycerol, 3% sorbitol and

0.65% NaCl. To make 250 ml of freezing media, 180 ml of 4.2%

sorbitol in 0.9% NaCl was mixed with 70 ml glycerol and filter

sterilized. The parasite culture at 2 to 3% parasitemia, predominantly

at ring stage, was pelleted by centrifugation at 1,500 rpm for 5 min at

4°C. To the 0.2 ml parasite pellet, 0.3 ml of human serum of

complimentary blood group was added. Equal volume of the

cryoprotectant was then added drop by drop, while shaking gently.

The culture was transferred to a sterile cryovials and stored in liquid

nitrogen until use.

To revive the frozen culture, cryopreserved cells were taken out from

liquid nitrogen and thawed at 37°C for 1-2 min. The culture was

transferred to a 50 ml tube, 0.1X volume of 12% NaCl was added

drop wise, while shaking the tube gently. The tube was left for 5 min

at room temperature followed by addition of 1 OX volume of 1.6% NaCl

slowly, drop wise. Cells were centrifuged at 20°C for 5 min at 1,500

rpm, supernatant was removed and lOX volume of complete medium

was added drop wise. The cells were centrifuged again at 1500 rpm

for 5 min at 20°C and the supernatant was removed. RBCs (at 5%

53


hematocrit) and complete medium were added to the pellet and the

culture was maintained in a culture flask (Schichtherle et al., 2000).

2.2.1.6. Revival of DlO ACP (leader)·GFP Strain.

To revive the frozen culture, cryopreserved cells were taken out from

liquid nitrogen and thawed at 37°C for 1-2 min. The culture was

transferred to a 50 ml tube; 1ml of 3.5% NaCl was added drop wise,

while shaking the tube gently. The tube was left for 5 min at room

temperature followed by centrifugation at 1,300 rpm for 5min at room

temperature, supernatant was removed and washing with 3.5% NaCl

was repeated till clear supernatant was obtained. Complete medium

was added drop wise. The cells were centrifuged again at 1300 rpm

for 5 min at room temperature and the supernatant was removed.

RBCs (at 5% hematocrit) and complete medium were added to the

pellet and the culture was maintained in a culture flask.

2.2.2. Total genomic DNA isolation from P. falciparum culture

Total genomic DNA was isolated from P. falciparum culture according

to the method used by Qari et al. (1998). Briefly, parasites were

released from infected RBCs by 0.05% saponin lysis and washed

extensively with ice cold 1X PBS. The DNA was released by adding

450 pl of lysis buffer (50mM Tris-Cl pH 8.0, 5mM EDTA, 100mM

NaCl and 1% SDS). 200 pg of proteinase K was added and mixed by

swirling. The reaction mixture was incubated at 42°C for 45 min.

RNase A (2 pg) was added, mixed and incubated at 37°C for 15 min

to degrade RNA. DNA was extracted with phenol:chloroform mix (1:1).

To the aqueous phase, 0.04 M NaCl and twice the volume of ethanol

was added for precipitation of DNA. The DNA pellet was washed with

70% ethanol, dried and suspended in TE buffer ( 10 mM Tris-Cl pH

8.0 and 1mM EDTA).

54


2.2.3. Molecular Cloning

All the PCR amplifications, restriction digestions, ligations and

transformations were carried out by standard procedures (Sambrook

J, 1989).

2.2.3.1. Cloning of Elongation factor Ts gene (EF-Ts).

The putative Elongation factor Ts (EF-Ts) gene sequence was obtained

from Plasmodium genome database PlasmoDB (PlasmoDB accession

number PFC0225c). EF-Ts gene comprises of two exons (1276bp) and

encodes a protein of 44.8kDa.

P. falciparum EF-Ts protein (PJEF-Ts) comprises of 390 amino acids.

It contains an N-terminal signal and transit peptide required for

apicoplast targeting. ClustalW alignment with other EF-Ts analogs

showed that first 54 amino acid stretch may putatively act as signal

and transit peptide. Thus, amino acid 55 was taken as starting

residue for processed and functional EF-Ts. The predicted processed

protein ( -39kDa) was encoded by a 1011 bp DNA fragment. eDNA

prepared from total parasite RNA was used as template for PCR

amplification of the DNA sequence encoding processed EF-Ts.

2.2.3.2. Isolation of total P. falciparum RNA

Total RNA isolation by Guanidium isothiocyanate (GITC) method

P. falciparum cultures at high parasitemia ( -7-8%, at late

trophozoite/early schizonts stage) were lysed with 0.05% saponin.

Released parasites were washed with ice cold 1X PBS at 6,000 rpm

for 10min at 4°C. To the pellet, 500 J.!l of solution D (4M GITC, 25mM

sodium citrate, 0.5% sarcosyl, 0.1M PME in DEPC water) was added.

50J.!l of 2M sodium acetate (pH 4.0), 500 J.!l of water saturated phenol

and lOOJ.!l of chloroform:isoamylalcohol (49: 1) were added, mixed well 55


and the tube was incubated on ice for 15 min. The aqueous phase

was collected after centrifugation at 12,000 rpm for 20 min at 4°C

followed by the addition of 500 J.!l isopropanol to aqueous phase for

precipitation of RNA. The sample was incubated at -20°C for 3h

followed by centrifugation at 12,000 rpm. The pellet was suspended

in 300J.!l solution D, isopropanol (300J.!l) was added and left overnight

at -20°C. After centrifugation, the RNA pellet was washed with 70%

ethanol, dried and suspended in DEPC-treated deionized water.

Total RNA isolation by Trizol method

Total RNA from parasite culture was also isolated by Trizol solution

according to manufacturer's instructions. Briefly, parasite culture at

high parasitemia was lysed with 0.05% saponin and washed with ice

cold 1X PBS. Released parasites were lysed by addition of 500J.!l of

Trizol and incubation for 5 min at room temperature. This was

followed by addition of 300J.!l chloroform, 5min incubation at room

temperature and centrifugation at 12,000 rpm for 20min at 4°C.

Aqueous phase was taken out and 400J.!l of isopropanol was added to

this fraction. After 1 Omin incubation at room temperature the sample

was centrifuged at 12,000 rpm for 20 min at 4°C. The RNA pellet was

washed with 70% ethanol and finally suspended in DEPC treated

deionized water.

2.2.3.3. RT-PCR of EF-Ts transcript

For the RT-PCR of EF-Ts transcript, eDNA was synthesized from

DNase I treated total RNA isolated from parasite cultures followed by

PCR amplification of target sequences.

DNase I treatment of RNA

P. falciparum RNA (-2J.tg) was treated with 2J.!l of DNase I (stock

1 U I J.!l) to remove any DNA contamination. After incubation at room

temperature for 30min, DNase I was inactivated by addition of 25mM

56


EDTA followed by incubated at 65°C for 10 m1n. RNA was

precipitated with 1/ lOth volume of sodium acetate (3M, pH 5.2) and

two volumes of ethanol, washed with 70% ethanol, dried and

suspended in DEPC treated deionized water.

eDNA synthesis

eDNA synthesis was carried out from DNase I treated RNA using first

strand superscript™ eDNA synthesis kit (Invitrogen). The reaction

mixture, containing RNA, dNTPs and gene specific downstream

primerjhexamer primer was incubated at 65°C for 5 min. After

incubation, the reaction mixture was immediately kept on ice for 1-2

min to prevent RNA secondary structure formation. Reaction buffer,

MgCl2, DTT and RNase out (RNase inhibitor) were added and

incubated at 42°C for 2 min. lJ.!l reverse transcriptase enzyme (50

U I J.!l) was added to the reaction and incubated at 42°C for 50 min for

reverse transcription to take place. The reaction was terminated at

70°C for 15 min. The RNA strand of the eDNA was removed by

treatment with lJ.!l of RNase H at 37°C for 20min. The first-strand

eDNA obtained was amplified directly by PCR using platinium Pfx

DNA polymerase. 10% of the first-strand reaction was used as a

template in the PCR reaction.

2.2.3.4. Agarose gel purification of linear DNA

DNA Fragments were resolved on agarose gel in lX TAE buffer and

the desired DNA bands were excised from the gel and transferred to a

pre- weighed sterile micro tube. The gel slice was weighed and three

volumes of DNA binding silica suspension (1% wjv silica in 6 M KI in

TE) were added to one volume of gel ( 100111 for 1 OOmg of gel weight).

The mixture was incubated at 55oc till the agarose block was

dissolved completely. DNA bound silica was then washed once each

with 50% ethanol and acetone. Silica pellet was air dried and bound

57


DNA was eluted either in autoclaved water or TE buffer. Alternatively,

DNA was extracted from the agarose gel using DNA gel extraction kit

(Qiagen, Invitrogen) following manufacturer's instructions.

2.2.3.5. Cloning of PCR amplified DNA fragments into expression

vector

PCR amplifications were carried out using Taq DNA polymerase for

routine applications (Promega, USA). Platinum Pfx DNA polymerase

(Invitrogen) was used to amplify gene fragments for cloning into

expressiOn vectors. All the PCR reactions were carried out by

standard procedures (Sambrook J, 1989) using eDNA, genomic DNA

or plasmid DNA as template in a Gene Amp PCR System (Applied

Biosystems). The reaction conditions and primer sequences (with

restriction enzyme sites highlighted) for all amplified gene segments

are given in Table 2.1 and Table 2.2, respectively. The PCR amplified

gene fragments for PjEF-Ts and PjEF-G were cloned in expression

vectors like (pQE-30, pET23a or pGEX) (Fig. 2.1).

BamHI Sali I r+;. $%** I

~ Digestion \Vith BamHI and Sali t

~ ----------1

Fig. 2.1. Cloning strategy for inserts (PJEF-Ts, PJEF-Tsexonl, PjEF-G (I-III) and PJEF-G

(I-IVa) in pGEX, pET23a and pQE-30 vectors.

58


Table 2.1: PCR conditions for amplification of gene fragments

FRAGMENT DENATURATION ANNEALING EXTENSION POLYMERASE

TEMPERATURE TEMPERATURE (0 C) TEMPERA TURE(0 C) USED

(.C)

PJEF-Ts 94 53.5 68 Platinum Pfx

PJEF-Tsexonl 94 53.5 68 Platinum Pfx

PjEF-Gn-Hn 94 54.5 60 Taq PJEF -Gu-tval 94 54.5 60 Taq P.fF._F-Tslsttl 94 51 60 Taq PJEF-Tu<mtx) 94 48 60 Taq for pETDuet-1

Table 2.2: Primers for amplification of gene fragments

Primer name Primer sequence

TsF2 5'-CGCGGATCCGATCATCTAAAACTATTAAAATATG -3'

TsR2 5'-CGCGTCGACTTCCAT AAGAACGmTTTTCCCC --3'

TsRl 5'-CGCGTCGACCTGCTCTAGGAA TACTATG -3'

EF-GU 5'-CGCGGATCCGTTAATGGTTCAACAAAAAATG -3'

EF-GD 5'-CGCGTCGACCTCCAACAACCTTATAGACGT -3'

EF-GmnD 5'-CGCGTCGACAGATATCTGAGGTTTCCCAT AA -3'

EF-TuU 5'-CGCCAATTGATGAATAATAAGTTGTTmAAG-3'

EF-TuD 5'- CCGCTCGAGATTTTTTATTTCTGTTATAATACCTGC-3'

GSTU 5'-CATGCCATGGACTCCCCTATACTAGGTTATTGGAAAATTAAG -3'

GSTD 5'-CGGGGATCCACGCGGAACCAGATCGGATTTTGGAGG-3'

EF-Ts stF 5'-GATCTCGAGATGAAGTTGTTTTATTTTTTCTTGTTAAG -3'

EF-Ts stR 5'-CACCCCGGGATTGmGTAGAATATAATCTATTTTT -3'

59


2.2.3.5.1. Cloning of PjEF-Ts gene

Full-length PjEF-Ts gene encoding the predicted processed protein

(1011 bp) was amplified from P. falciparum eDNA using primers TSF2

(forward primer) containing a BamHI site and TSR2 (reverse primer)

containing a Sall site (Fig. 2.2A). pGEX vector and the PCR product

were digested with BamHI and Sall enzymes and the fragments were

gel purified and ligated using T4 DNA ligase. DHSa competent cells

were transformed with the ligation product and plated on LB

ampicillin plate and kept overnight at 37°C. The colonies were

screened and clones were confirmed by digestion with BamHI and

Sall enzymes. The resultant construct, pGEX-Ts, carried PjEF-Ts

fused to GST at the N-terminus.

2.2.3.5.2. Cloning of P.JEF-Tsexonl

The exon1 (876 bp) of PJEF-Ts gene was PCR amplified using the P.

falciparum genomic DNA as template and upstream TsF2 and

downstream TsR 1 primers carrying BamHI and Sall tags, respectively

(Fig. 2.2B). The amplified DNA was cloned into pET23a with a His

tag at theN-terminus generating the construct pET-Tsexonl.

2.2.3.5.3. Cloning of PjEF-Ts signal and transit peptide [P.JEF

TS(s/tl]

The sequence encoding the predicted signal-transit peptide of PjEF-Ts

(amino acids 1-54) was PCR-amplified using the P. falciparum

genomic DNA as template and the forward (EF-Ts stF) and reverse

primers (EF-Ts stR) (Fig. 2.2C). The sequence was cloned as an Xhol

Xmal insert into the pGlux.l vector (kind gift from Prof. Alan

Cowman).

2.2.3.5.4. PjEF-Tu as GST fusion construct

The full-length tufA gene had been cloned previously in the pUC18

vector and subcloned in pMALC2 vector generating the construct 60


pMAL-tufA. This construct encodes PjEF-Tu with maltose binding

protein as fusion partner (Chaubey et al., 2005). EF-Tu was further

subcloned into pGEX vector at the EcoRI and Sali restriction sites

yielding a construct pGEX tufA that carried tufA fused to GST at the

N-terminus.

2.2.3.5.5. Cloning of PJEF-Ts & PjEF-Tu in pETDuet-1 vector

Full length PjEF-Ts gene was amplified from pGEX-Ts vector as

template using Taq polymerase and cloned in MCS 1 of pETDuet-1

vector at BamHI and Sail restriction sites. N-terminal Histidine tag in

MCS1 of pETDuet-1 vector was replaced with GST tag using GSTU

(forward primer) with Ncol and GSTD (reverse primer) with BamHI

site. GST fragment was amplified from pGEX vector as template and

ligated to MCS1, upstream of the EF-Ts gene. PjEF-Tu gene (tufA) was

amplified using EF-TuU (forward primer) with Munl and EF-TuD

(reverse primer) with Xho I restriction sites (Fig. 2.2D) and ligated to

MCS2 of the pETDuetl vector containing S-tag at C-terminal.

2.2.3.5.6. Cloning of PjEF-G(I-IVa)

The putative Elongation factor G (EF-G) gene sequence was obtained

from Plasmodium genome database plasmoDB (PlasmoDB accession

number (PFF0115c). EF-G gene comprises of 4 exons, encodes a

protein of -108 kDa. P. falciparum EF-G (PjEF-G) comprises of 937

amino acids and is predicted to be apicoplast targeted. ClustalW

alignment with E. coli and T. thennophilus EF-G showed that the first

88 amino acid stretch may act as the bipartite signal and transit

sequence. Thus, amino acid 89 was taken as start residue for the

processed and functional EF-G (-97.5kDa). The first exon (2289bp) of

PjEF -G gene encodes domain I to IVa. This sequence was PCR

amplified from P. falciparum genomic DNA using primers EF-GU

(forward primer) containing a BamHI site and EF-GD (reverse primer)

containing a Sail site (Fig. 2.2E). PCR product was gel purified and 61

Chapter 2 Materials and Methods ·

cloned in pTZ57R/T vector (MBI Fermentas). PjEF-G(I-IVa) was further

subcloned in pGEX vector with GST fused at theN-terminus.

2.2.3.5. 7. Cloning of PJEF-G(I-IIIJ

PjEF-G(r-m), containing EF-G domains I to III was PCR amplified with

pGEX EF-G(r-rva) as template and EF-GU (forward primer) and EF

GomD (reverse primer) with BamHI and Sall restriction sites,

respectively (Fig. 2.2F). The 1776bp DNA fragment was cloned in

pQE-30 to generate the clone PjEF-G(r-m) carrying domains I-III of

PjEF-G fused to a 6X His tag at theN-terminus.

2.2.3.5.8. EF-Tu mutagenesis

The EF-TuT62A mutant for intrinsic GTPase activity (Krab and

Parmeggiani, 1999), EF-TuE153A mutant for interaction with EF-Ts

(Weiden et al., 2002) and EF-Tuw196G mutated for single tryptophan

present in helix F, were generated using the pMAL-tufA as template.

The QuickChange XL Site-Directed Mutagenesis Kit (Stratagene) was

used to mutate the tufA gene. The mutations were confirmed by DNA

sequencing.

62

Chapter 2

A

c

E

1600

1000

500

200

100

M EF-Ts

EF -G(I-IVa) M

2200

1011bp

B

800

600

D

1500

1000

Materials and Methods

M EF-Tsexonl

876bp

M EF-Tu

1233bp

EF-G (I-Illl M

F

2500 2000

1776 1500 2000

Fig. 2.2. PCR amplification of P. falciparum elongation factors

PCR products of(A) lOllbp PjEF-Ts, (B) 876bp PjEF-Tsexont, (C) 162 bp ~F-Tsrs/t),

(D) 1233bp PJEF-Tu, (E) 2200bp PjEF-Grt-IVa) and (F) 1776bp PjEF-Grr-111)

63


2.2.4. Recombinant protein expression and purification

2.2.4.1. Expression and purification of PjEF-Ts

E. coli XL1 Blue cells were co-transformed with the expression vector

pGEX-Ts and the RIG plasmid (kind gift from Prof. W.G.J. Hoi). RIG

provides rare tRNAs of amino acids arginine, isoleucine and glycine

(Baca and Hol, 2000). Provision of these tRNAs is essential due to the

codon bias of Plasmodium genes. A single colony was picked and

grown overnight in LB medium containing ampicillin (100pgjml) and

chloramphenicol (25pgjml) at 37°C as primary culture. One liter

medium of LB containing antibiotics as above was inoculated with a

10 ml primary culture and grown at 37°C until the A6oo reached 0.5.

The cultures were induced by the addition of 0.5mM isopropyl-1-thio

~-D-galactopyranoside (IPTG) and grown for 17h at 20°C. The

induced cells were harvested, resuspended in PBS (8.0), and

sonicated. The cell lysate was centrifuged at 12,000 rpm for 20min at

4°C to separate the soluble and insoluble fractions of the lysate. Both

fractions were loaded on to a 10% SDS-PAGE to check for expression

of the recombinant protein.

The soluble fraction of the lysate was then loaded onto the

glutathione sepharose resin (GE healthcare) pre equilibrated with

PBS (pH 8.0). The column was washed with 12 bed volumes of PBS

and the bound protein was eluted with 1 OmM reduced glutathione in

PBS. Fractions were checked on SDS PAGE. For cleavage of the GST

tag, bound GST-EFTs was subjected to thrombin cleavage for 16h at

25°C and fractions were collected by washing the column with PBS.

Cleavage was confirmed by Western blotting using mouse anti GST

EFTs antibody (section 2.5.4).

64


2.2.4.2. Expression of PjEF-Tsexonl

E. coli BL21 (DE3) cells were co-transformed with the expression vector

pET23a bearing the PJEF-Tsexonl DNA fragment and RIG plasmid. A

single colony was picked and grown overnight in LB medium

containing ampicillin (100pgjml) and chloramphenicol (25pg/ml) at

37°C as primary culture. One liter medium of LB containing

antibiotics as above was inoculated with a 10 ml primary culture and

grown at 37°C until the A6oo reached 0.5, the cultures were induced

by the addition of 0.3mM IPTG and grown for 4h at 37°C. The

induced cells were harvested, resuspended in PBS (pH 8.0), and

sonicated. The cell lysate was centrifuged at 12,000 rpm for 20min at

4°C to separate the soluble and insoluble fractions of the lysate. Both

fractions were loaded on to a 10% SDS-PAGE to check the expression

of the recombinant protein. Expression was confirmed by Western

blotting using mouse anti-His antibody (GE healthcare).

2.2.4.3. Refolding and purification of PjEF-Tu

The tufA gene encoding EF-Tu had been previously cloned as an MBP

fusion in the pMALC2 vector (Chaubey et al., 2005). E. coli TG-1 cells

were co-transformed with the pMAL-tufA construct and the RIG

plasmid. Large scale culture (-1.6L) was induced with 0.5 mM IPTG

and grown for 1 7h at 20°C. The inclusion body fraction was isolated

and washed twice with buffer containing 20mM Tris-HCl pH 7.5,

10mM EDTA and 1% Triton X-100 followed by solubilization with

buffer containing 0.3% N-lauroylsarcosine and 2mM DTT in 50mM

CAPS (pH 11.0) according to manufacturer's instructions (Protein

Refolding Kit, Novagen). After centrifugation at 10,000 x g for 10 min

at room temperature, the solubilised protein was added to refolding

buffer (50mM Tris, pH 7.6, 200mM NaCl, 1mM EDTA, 1mM DTT and

0.5M NDSB-256) in the ratio of 1:10 (v:v) with fast mixing and

incubated at 4 OC for 1 h. This was followed by dialysis in refolding

buffer lacking NDSB-256 followed by two changes of refolding buffer 65


lacking NDSB-256 and D'IT. The refolded protein was purified on

amylose resin (New England Biolabs). Purified PjEF-Tu was dialyzed

in (50mM Tris pH 7.5, 200mM NaCl, 1mM EDTA, 1mM PMSF and 1%

glycerol) and concentrated using centricon units (Millipore). Refolding

of the protein was confirmed by CD spectroscopy. The mutants

PjEFTUT62A, P.fEFTuE153A and P.fEFTuwi96G proteins were also

expressed as inclusion bodies in E. coli and were refolded and

purified as described for wild-type PjEFTu.

2.2.4.4. Expression and purification of PJGST-EFTu

E. coli XL-1B cells were co-transformed with pGEX-tufA containing

construct and the RIG plasmid. A single colony was picked and

inoculated 1n LB containing ampicillin (1001Jg/ml) and

chloramphenicol (251Jg/ml). The culture was incubated at 37°C

overnight. One litre LB containing antibiotics as above was inoculated

with the overnight grown primary culture and induced with 0.5mM

IPTG and grown at 37°C for 4hrs. The cultures were pelleted down

and resuspended in PBS (pH 8.0), sonicated and centrifuged. The

soluble and insoluble fractions were loaded on 10% SDS PAGE and

Western blotted. The blot was probed with mouse anti MBP EF-Tu

antibody (Chaubey et al., 2005) to check the expression of protein.

The soluble fraction was loaded on to glutathione sepharose affinity

resin pre-equilibrated with buffer containing (50mM HEPES-KOH pH

7.5, 150mM KCl, 10mM MgCb, 1mM PMSF, 1mM EDTA, 1% Triton X-

100, 1% Glycerol). The column was washed with 12 column volumes

of the same buffer without 1% Triton X-100 and bound protein was

eluted with 1 OmM reduced glutathione in the same buffer without 1%

Triton X-100. The eluted protein was checked on 10% SDS PAGE.

66


2.2.4.5. Expression and purification of PJEF-Ts & P.fEF-Tu from

pETDuet-1 vector

E. coli BL21 (DE3) cells were co-transformed with the pETDuet-1

construct bearing the EF-Ts and EF-Tu genes and the RIG plasmid.

The secondary culture grown in LB containing 100pgjml ampicillin

and 25pg/ml chloramphenicol was induced by the addition of 0.5mM

IPTG and grown for 17h at 20°C. After sonication, the cell lysate was

centrifuged at 12,000 rpm for 20min at 4°C to separate the soluble

and insoluble fractions. Expression was checked by Western blotting

using mouse anti-GST antibody (Sigma) and rabbit anti EF-Tu

antibody (section 2.5.4). The blot was stripped and probed with rabbit

anti EF-Ts antibody (section 2.5.4).

The soluble fraction of the lysate was loaded onto the glutathione

sepharose resm (GE healthcare) pre-equilibrated with buffer

containing 50mM Tris-Cl pH 8.0, 200mM NaCl, 1mM EDTA, 1mM

PMSF, 1mM DTT. The column was washed with 12 column volumes

of the same buffer and the bound protein was eluted with 1 OmM

reduced glutathione. The soluble fraction of the lysate was also

loaded onto the S-protein agarose (Novagen) resin pre-equilibrated

with lysis buffer containing 20mM Tris-Cl pH 7.5, 150mM NaCl and

1% Triton X-100. The column was washed with 12 column volumes

of the same buffer and the bound protein was eluted with 3M MgCb

in lysis buffer.

2.2.4.6. Expression and purification of P.fEF-G(I-IVa)

E. coli XL1B cells were co-transformed with pGEX EF-G(exoniJ and RIG

plasmid. A single colony was picked and grown overnight. The

secondary culture (LB containing 1 OOpg/ ml ampicillin and 25pg/ ml

chloramphenicol) was grown at 37°C until the A6oo reached 0.5. The

culture was induced by the addition of 0.5mM IPTG and grown for

1 7h at 20°C. The induced cells were harvested, resuspended in 67


50mM Tris-HCl (pH 7.5), 50mM KCl, 2mM MgC12, and 5% glycerol

and sonicated. The cell lysate was centrifuged at 12,000 rpm for

20min at 4°C to separate the soluble and insoluble fractions of the

lysate. Both fractions were loaded on to an 8% SDS-PAGE to check

the expression of the recombinant protein. Expression was confirmed

by Western blotting using mouse anti-GST antibodies (Sigma). The

soluble fraction of the lysate was then loaded onto glutathione

sepharose resin (GE healthcare) pre-equilibrated with the buffer

(mentioned above). The column was washed with 12 bed volumes of

the same buffer and the bound protein was eluted with 1 OmM

reduced glutathione.

2.2.4. 7. Expression and purification of PjEF-G(I-III)

E. coli XL1B cells were co-transformed with pQE-30EF-G(I-III) and RIG

plasmid. The secondary culture (LB containing 100pg/ml ampicillin

and 25pg/ml chloramphenicol) was grown at 37°C until the A6oo

reached 0.5. The culture was induced by the addition of 0.8mM IPTG

and grown for 1 7h at 20°C. The induced cells were harvested,

resuspended in 50mM Tris-HCl (pH 7.5), 50mM KCl, 2mM MgCb, 5%

glycerol and sonicated. The cell lysate was centrifuged at 12,000 rpm

for 20min at 4°C to separate the soluble and insoluble fractions of the

lysate. Both fractions were loaded on to an 8% SDS-PAGE to check

the expression of the recombinant protein. Expression was confirmed

by Western blotting using mouse anti-His antibodies (GE healthcare).

Purification of PJEF-G(I-III) was performed with Ni-NTA affinity

chromatography. The soluble fraction was loaded onto Ni-NTA

column pre-equilibrated with lysis buffer (50mM Tris-HCl pH8.0,

200mM NaCl, 1 mM PMSF, and 10mM immidazole) at 4°C. The

column was washed with five bed volumes of lysis buffer followed by

12 bed volumes of wash buffer (SOmM Tris-HCl pH 8.0, 200mM NaCl,

1mM PMSF, and 60mM imidazole). Bound P.fEF-G(I-III) protein was 68


eluted with the elution buffer (50mM Tris-HCl, pH 8.0, 200mM NaCl,

1mM PMSF, 250mM imidazole). Fractions were collected and loaded

onto an 8% SDS PAGE to check for the homogeneity of the purified

protein.

2.2.5. Generation of polyclonal antisera

SDS-PAGE purification of recombinant proteins

P. falciparum EF-Ts, EF-Tu and EF-G proteins were purified using

preparatory SDS polyacrylamide gels (Ohhashi et al., 1991)

2.2.5.1. Preparation of P.fEF-Ts protein

Inclusion bodies containing His tagged EF-Ts protein were obtained

from the insoluble fraction of the induced PjEF-TS(exonl) culture.

Induced cell culture was grown at 37°C, pelleted and suspended in

the lysis buffer (50mM Tris-HCl pH 8.0, 200mM NaCl). This

suspension was sonicated at 20% amplitude and 20 pulses of 10

seconds each. Soluble and insoluble fractions were separated by

centrifugation at 12,000 rpm for 20 min. The inclusion bodies were

extensively washed with PBS containing 1% TritonX-100 in order to

remove other cellular contaminants. Semi-purified inclusion bodies

were suspended in a minimal volume of lysis buffer and

electrophoresed on a preparatory 10% SDS PAGE. The preparatory

gel was negatively stained with 4M sodium acetate for 30 min and the

band corresponding to the His EF-Ts protein was excised from the

gel. The gel band was chopped into small pieces and filled in a 12kDa

cut off dialysis bag along with 10mM EDTA. Protein was electro

eluted at 4°C for 6h with an intermediary EDTA change and collection

at 3h. 10mM EDTA was also used as tank buffer. The eluted protein

was checked by SDS-PAGE and its identity was confirmed by Western

blotting using anti-His antibody (Qiagen). The protein was

concentrated on a centricon and estimated using the bichinchonic

69


assay kit (Sigma). This protein was used to raise antiserum against

PjEF-Ts in rabbit.

GST-EFTs was purified as mentioned above (section 2.4.1). The

protein was concentrated on a centricon and estimated using the

bichinchonic assay kit (Sigma). This protein was used to raise

antiserum against PjEF-Ts in mice.

2.2.5.2. Preparation of PjEF-Tu protein

PjEF-Tu expressed as fusion protein with GST was found to be

autocleaved in the bacterial lysate. Presence of 47kDa PjEF-Tu was

also detected using mouse anti MBP EF-Tu antibody (Chaubey et al.,

2005) by Western blot in bacterial lysate expressing GST-EFTu. The

soluble fraction containing cleaved PjEF-Tu was electrophoresed on a

preparatory 10% SDS PAGE and the desired band was excised from

the gel after negative staining with 4M sodium acetate for 30 min.

The protein was electroeluted in 10mM EDTA (as described above),

concentrated using centricon and confirmed by Western using mouse

anti MBP EF-Tu antibody.

2.2.5.3. Preparation of PjEF-G(I-IIIJ protein

PjEF-G(r-III) protein was purified on Ni-NTA column, concentrated

using centricon and used for raising antibody.

2.2.5.4. Raising antisera in rabbit and mice

Antibodies against PjEF-Ts and PjEF-Tu were raised in rabbit as well

as in mice using the purified recombinant proteins. Antibody against

PjEF-G was raised in rabbit. Ethical clearance was obtained from the

Institutional Animal Ethics Committee. Approximately 150!-lg and

50!-lg of SDS-PAGE purified proteins, emulsified in Freund's complete

adjuvant (GibcoBRL/Sigma/Santacruz) were injected subcutaneously

in rabbit (New Zealand Red) and mice (Swiss strain) respectively. Pre-70


immune serum was collected before administering the antigen. Two

booster injections in Freund's incomplete adjuvant in rabbit (-801-lg

protein/booster) were given after 28 days at the interval of 15 days

and a single booster dose (-301-lg protein) in Freund's incomplete

adjuvant was administered to mice after 28 days. Rabbit and mice

were bled after 10 days of last booster dose. Blood was stored at 37°C

for 30 min and then transferred to 4°C overnight for separation of

serum. Serum was collected by centrifugation at 2,000 rpm for 10

min at 4°C, aliquoted and stored at -80°C. Western blotting of the

bacterial lysates expressing the corresponding recombinant protein

was done to check the specificity of the raised polyclonal sera. ELISA

was performed to determine the titre of the antisera raised in rabbit

and mice.

2.2.5.5. Purification of antibodies by immobilisation on

nitrocellulose membrane

Antibodies were affinity purified using recombinant Plasmodium EF

Ts, EF-Tu and EF-G(I-III) proteins immobilized on nitrocellulose

membrane (Smith and Fisher, 1984). Purified recombinant protein

was electrophoresed on a 10% SDS PAGE and transferred onto

nitrocellulose membrane. After visualization by Ponceau S, the band

of interest was excised from the blot and incubated overnight at 4°C

in blocking solution (5% skimmed milk in lX PBS). The blot was

washed thoroughly with PBS-T (0.05% vjv Tween-20 in lX PBS) and

incubated for 2h at room temperature in corresponding antiserum

(dilution: 1 ml antiserum in 10 ml of 2% BSA in PBS). The blot was

washed five times with PBS-T, cut into small pieces and put in a

micro-centrifuge tube. Antibody bound to the protein on

nitrocellulose membrane was eluted by addition of 5001-ll glycine

buffer (0.2M glycine-HCl pH 2.3, 500mM NaCl); the eluate was

immediately neutralized by the addition of 125 111 1M Tris (pH 8.0)

71


such that the pH of the resulting solution reached 7.4. This elution

step was repeated three times. After elution at pH 2.3, the blot was

washed three times with PBS-T, followed by three additional elution

steps with potassium thiocyanate buffer (50mM Tris pH 8.0, 3M

K4SCN, 150mM KCl). Individual aliquots of each elution step were

checked on 10% SDS PAGE and stored separately at 4°C. Specificity

of the eluted antibody was checked by Western blotting of bacterial

lysates expressing the respective proteins.

2.2.6. Western blotting and immunoprecipitation

2.2.6.1. Western blotting

For Western blotting, protein samples were resolved on SDS

polyacrylamide gels and electroblotted onto nitrocellulose membranes

in Tris-glycine buffer (15.6mM Tris pH 8.2, 120mM glycine, 15%

methanol) at 50V for 2 h. Blots were blocked with 5% milk in PBS

overnight at 4°C and then incubated with required dilution of primary

antibodies for 4-5 hrs at 16°C. After five washes with PBS-T (PBS

containing 0.05% (v jv) Tween-20), blots were incubated with HRP

conjugated secondary antibody for another 90min followed by

washing with PBS-T. Blots were developed either by chromogenic

substrate diaminobenzidine (DAB) or by chemiluminescence

substrate (GE Healthcare).

2.2.6.2. Detection of PJEF-Ts in P. falciparum lysate

Total parasite lysate was prepared from parasite cultures at 6 to 8%

parasitaemia, predominantly at the trophozoite stage. Parasites were

released from infected RBC by 0.05% saponin lysis, washed with

PBS, and suspended in 1X SDS loading buffer containing protease

inhibitors (Protease Arrest, GBiosciences). After 30 min incubation on

ice followed by brief vortexing, the cell lysate was separated on a 10%

SDS-PA gel. Western blotting was carried out and the blot was probed 72


with purified rabbit anti EF-Ts and developed using a

chemiluminescent system (GE Healthcare).

2.2.6.3. Immunoprecipitation of PjEF-Tu from parasite lysate

For immunoprecipitation, parasite cultures at 6 to 8% parasitemia

were harvested when cells were predominantly at the late trophozoite

stage. Cells were washed with PBS and parasites were released by

0.05% saponin lysis. The parasite pellet was washed with PBS and

lysed in chilled immunoprecipitation (IP) buffer containing 50mM

Tris-HCl pH 7.5, 200mM NaCl, 5mM EDTA, 1mM PMSF, and

protease inhibitor cocktail on ice for 30 min. After brief sonication,

th~ lysate was centrifuged at 12,500 rpm for 10 min. The

supernatant was precleared for lh by addition of 3mg Protein A

sepharose CL-4B beads (GE Healthcare). The precleared supernatant

was incubated with rabbit anti EF-Tu for 2h on ice with concomitant

mixing. After centrifugation at 10,000 rpm for 2 min at 4°C, the

supernatant was incubated overnight with 5 mg Protein A sepharose

beads at 4°C with continuous mixing. Sepharose beads were pelleted

at 12,000 rpm for 2 min at 4°C and washed five times with chilled IP

buffer followed by two PBS washes. Immunoprecipitated proteins

were obtained by treating the beads with non-reducing SDS lysis

buffer. The sample was electrophoresed on a 10% SDS PAGE and

transferred onto a nitrocellulose membrane. The membrane was

probed with mouse anti EF-Tu as primary antibody and anti-mouse

HRP conjugate as secondary antibody followed by development of the

blot using a chemiluminescent detection system (GE Healthcare).

2.2.6.4. EF-Ts pull down assay

For the PjEF-Ts pull down assay, 1!-!g of purified PjEF-Ts (GST tag

cleaved out) was incubated with 3!-!g of total lysate from E. coli cells

over-expressing GST-EFTu in binding buffer containing 50mM

HEPES-KOH pH 8.0, 60mM KCl, 5 mM ~-ME, 1mM PMSF and 1% 73


glycerol at 4°C for 30 min. The lysate containing GST-EFTu as well as

autocleaved EF-Tu was prepared by suspending E. coli cells in lysis

buffer containing 50mM HEPES-KOH pH 8.0, 60mM KCl, 5mM ~-ME,

1mM PMSF, 10% glycerol, 5mM MgCb and 10 J.tM GDP. 3mg Protein

A sepharose CL-4B beads were incubated in the binding buffer for 30

min at 4°C followed by centrifugation at 12,000 rpm for 3 min. Beads

suspended in 880 J.tl of binding buffer were incubated with 100 J.tl of

the lysate. EF-Ts mix and 20 J.tl of rabbit anti-MBP-EFTu serum

overnight at 4 OC with gentle mixing. Sepharose beads were

centrifuged at 12,000 rpm for 3 min at 4°C and washed five times

with chilled 1X PBS. Bound proteins were obtained by treating the

beads with SDS loading buffer. The second wash and the eluted

protein were electrophoresed on two 10% SDS PAGE and transferred

onto nitrocellulose membranes. The membranes were probed with

mouse anti MBP-EFTu serum or mouse anti GST-EFTs serum

followed by development of the blot using a chemiluminescent

detection system (GE Healthcare).

2.2. 7. Production and purification of antibiotics

2.2. 7 .1. Culturing of Actinomycetes strains

GE2270 is produced by fermentation of Planobispora rosea ATCC

53773™ (Selva et al., 1991), a strain belonging to one of the rarest

genera of actinomycetes, as a complex of 10 structurally related

compounds (Selva et al., 1995). Pulvomycin is produced by

fermentation of Streptoverticillium netropsis ATCC 23940™. P. rosea

and S. netropsis spores were streaked on ISP4 media plates and kept

at 28oC for 15 days and 4 days, respectively. ISP4 media (g/L) was

prepared by adding agar, 20.0; soluble starch, 10; CaC03, 2.0;

(NH4)2S04, 2.0; K2HP04, 1.0; MgS04.7H20, 1.0; NaCl, 1.0;

FeS04.7H20, l.Omg/L ; MnCb.7H20, l.Omg/L; ZnS04.7H20,

l.Omg/L in TDW and pH was adjusted to 7.2 at 25°C. The media was 74


gently heated and brought to boiling with frequent agitation for

dissolving the components. Autoclaving of media for 15min at 15 psi

was done.

2.2. 7 .2. Production and purification of GE2270A

A single colony of P. rosea was inoculated in vegetative medium/Seed

media and incubated at 28°C for 48hrs with gentle shaking.

Vegetative medium/seed media having the following composition

(g/L): soluble starch, 20; polypeptone, 5; yeast extract, 3; meat

extract, 2; soybean meal, 2; calcium carbonate, 1 was prepared in

distilled water and the pH was adjusted to 7.0 before sterilization at

121 oc for 20 min. One ml of vegetative medium/seed media was

transferred to flasks containing fermentation medium and

fermentation was carried out for 7 days at 28°C with gentle shaking.

Fermentation medium had the following composition (g/L): glucose,

10; soluble starch, 25; hydrolysed casein, 5; yeast extract, 8; meat

extract, 3.5; soybean meal, 3.5; calcium carbonate, 2. Medium was

prepared in distilled water and the pH was adjusted to 7.2 before

sterilization at 121 oc for 30 min.

Cultures were pelleted down at 1,200 g for 10 min and biomass was

calculated as packed mycelium volume. One volume of cells was

mixed with two volumes of acetonitrile and shaken at room

temperature for 20 min. After centrifugation at 15,000x g for 10 min,

the supernatant was collected and injected in preparatory HPLC

(LC8A, Shimadzu) equipped with UV detector at 254 nm. GE2270A

was eluted with a mobile phase composed of 20 mM

NaH2P04/ acetonitrile (3:7) as eluent. HPLC Peaks were collected and

submitted for fast atom bombardment analysis (FAB). GE2270A

purified by prep HPLC was pooled and concentrated under reduced

pressure to remove acetonitrile, thus obtaining separated residual

solution containing antibiotic GE2270A. This solution was extracted 75


twice with an equal volume of ethyl acetate in separating funnel and

the antibiotic product was obtained by precipitation from the

concentrated organic phase by the addition of petroleum ether.

GE2270A in petroleum ether was dried under reduced pressure by

passing N2 gas.

2.2. 7 .3. Production and purification of pulvomycin

A single colony of S. netropsis was inoculated in S. netropsis media

and incubated at 28°C for 48hrs with gentle shaking. S. netropsis

medium composed of dextrose 20 g/L, yeast extract 2 g/L, soybean

meal 8 g/L, NaCl 1 g/L and CaC03, 4 g/L in deionized water before

sterilization at 121 oc for 20 min. One ml of culture was transferred to

flasks containing S. netropsis medium and fermentation was carried

out for 5 days at 28°C with gentle shaking. After 5 days, the broth

was harvested at 1,200 rpm for 10 min and the mycelium cake was

suspended in five volumes of acetone. After stirring of the sample, the

mycelium was removed by filtration and the filtrate was concentrated

under vacuum. The residue was extracted with an equal volume of

ethyl acetate. The organic phase dissolved in methanol was

fractionated on silica TLC chromatography using chloroform

methanol (10: 1) as the solvent. The major portion of the antibiotic

displayed an Rr of 0.4, the bands were marked by giving UV 254

exposure and scratched from the TLC plates and antibiotic was

eluted by addition of absolute alcohol. Sample was given for ESI-MS

analysis.

2.2.8. Nucleotide binding, hydrolysis and release

2.2.8.1. Nucleotide binding by PjEF-Tu

The affinity of EF-Tu for GDP and a non-hydrolyzable analog of GTP

(GMPPCP) were determined by fluorescence titration using a LS50B

spectrofluorimeter (Perkin Elmer) with a slit band-width of 8nm for 76


excitation and 6nm for emission. 1JJM nucleotide-free PjEFTu

(purified in the presence of 1mM EDTA to chelate Mg2+) was titrated

with increasing concentrations of the nucleotides. Masking of the

Trp-196 residue present in the vicinity of the nucleotide-binding

pocket in the G domain of EF-Tu was measured by excitation at 280

nm and recording the emission maxima at 340 nm. All spectra were

corrected for buffer containing the corresponding nucleotide

concentration. Kd values were determined by plotting ~F /Fmax where

F max is Trp fluorescence in the absence of nucleotide and ~F is the

difference between F max and emission maxima at each nucleotide

concentration (Shukla et al., 2006). Kd was calculated by curve fitting

using non-linear regression in GraphPad PRISM software.

2.2.8.2. Nucleotide release by P.fEF-Ts

The interaction of GDP with PjEFTu as well as GDP release mediated

by EF-Ts was assayed usmg Mant-GDP [2'/3'-0-(N-methyl

anthniloyl-GDP)] (Molecular Probes). For the nucleotide-binding (NB)

assay, PjEFTu was incubated with 1.6JJM mant-GDP at 3TC for 15

min in NB buffer containing 60mM Tris-HCl pH 7.6, 30mM KCl,

30mM NH4Cl, 1 OmM MgCb and 2mM DTT. Tryptophan was excited

at 280 nm. Mant-GDP bound to PjEF-Tu was excited by fluorescence

resonance energy transfer (FRET) from tryptophan (mant-GDP

excitation at 366 nm) and the emission of mant-GDP (emission

wavelength of 450 nm) was recorded in scan spectra. Background

emission spectra of 1.6JJM Mant-GDP in NB buffer was also recorded.

In order to assay EF-Ts mediated GDP release from EF-Tu, the

PjEFTu.mant-GDP complex was prepared by adding 3-fold molar

excess (1.6JJM) of mant-GDP to PjEFTu (0.5pM) in NB buffer as above.

Increasing molar ratios of PjEF-Ts were added to the complex along

with 50JJM GDP to prevent re-binding of mant-GDP to PjEFTu.

Change in mant-GDP emission as a result of FRET from tryptophan 77


was recorded. Nucleotide release by PJEF-Ts from E. coli EF-Tu.mant

GDP complex was also studied in the same manner.

2.2.8.3. Intrinsic GTPase activity of PjEF-Tu and effect of

antibiotics

Intrinsic GTPase activity of refolded and purified P.fEF-Tu was

assayed in buffer (50mM Tris pH 7.5, 50mM KCl, 2mM MgCh, 5%

glycerol) that contained [y-32P] GTP (3200cifmmol). Varying

concentration (0- 15nmoles) of PjEF-Tu was taken in a 20JJ1 reaction

and incubated at 37° C for 30min. The reactions were stopped by

adding 1% SDS and 1pl of each reaction was spotted on 3MM

Whatman paper and resolved in a solution containing n-butanol, n

propanol, acetone, 80% formic acid and 30% trichloroacetic acid at a

ratio of 40:20:25:25:15 (v fv). The chromatogram was dried and

autoradiographed.

GTPase assay of PjEF-Tu was also done by malachite green.

Malachite green was prepared in concentrated H2S04. Briefly, 60ml of

18N H2S04 was supplemented with 0.44g of malachite green and

volume was made up to 300m! with TDW. The solution was cooled to

room temperature. Resulting orange solution is stable at 4°C for 1

month. On the day of use, 2.5ml of 7.5% ammonium molybdate was

added and left on a rocker for 4-5 hrs. Before use, the solution was

centrifuged at 8,000 rpm for 5 min. and supernatant was taken for

phosphate estimation.

Refolded and purified PJEF-Tu was taken in varying concentrations

(50nmoles-400nmoles) and incubated with 200JJM GTP in 100JJ1

buffer containing 50mM Tris-Cl pH 7.5, 200mM NaCl, 30mM NH4Cl,

10mM MgCb, and 2mM DTI. The reaction was incubated for 10 min

and stopped by adding 25JJ1 of malachite green. Absorbance was

78


taken at 650nm in an ELISA plate reader (PowerWave XS, Biotek).

The amount of enzymatically released Pi by the hydrolysis of GTP was

quantitated by comparing with a standard curve. Standard curve was

prepared with dilutions of a 500 pM KH2P04 solution in the above

buffer, over a range of 0.1 to 1 pM. 1.5 ml tubes used in the assay

were pre-treated with 'Pi-mop' (1U/ml nucleotide phosphorylase,

750J.!M 7-methyl guanosine).

The effect of kirromycin on the intrinsic GTPase activity of PJEF-Tu

was assayed using y32P-GTP (BRIT, India) as described by Mesters et

al. (1994). Briefly, 2 J.!M PjEF-Tu was incubated with 0.2 JIM y32P-GTP

(3200Ci/mmol}, 2J.!M GDP, 64mM Tris-HCl pH 7.6, 10mM MgCb,

80mM NH4Cl, 10mM ~-mercaptoethanol, 83J.!M phosphoenol

pyruvate, 40J.!g/ml pyruvate kinase/lactate dehydrogenase at 3TC for

15 min followed by addition of kirromycin (0-50pM) to the final 30J.!l

reaction volume and further incubation at 37·c for 15 min. The

reactions were stopped by addition of 15J.!l of 25% (v fv) formic acid.

One pl of each reaction was spotted on 3MM Whatman paper and

chromatographed (Schwemmle and Staeheli, 1994). Phosphorimager

signals were quantitated by densitometric analysis (ImageMaster 1D

Elite V3.0 1).

The concentration of antibiotics GE2270A and pulvomycin were

calculated using molar extinction coefficients of GE2270A (30,613 at

310 nm) and pulvomycin (74,582 at 320 nm) in methanol,

respectively. The effect of these antibiotics on intrinsic GTPase

activity of PjEF-Tu was studied using malachite green protocol for

inorganic phosphate estimation. Briefly, refolded and purified PJEF

Tu was pre-incubated with GE2270A (0.005 to 0.1pM) or pulvomycin

(5 to 60pM) in GTPase buffer containing 50mM Tris-Cl (pH 7.5),

200mM NaCl, 30mM NH4Cl, 10mM MgCb, 2mM D'IT for 15 min at

room temperature in independent sets of experiments. One mM GTP 79


was added to initiate the reaction and incubated for 10 min at room

temperature. The reaction was stopped by adding malachite green

solution and absorbance at 650 nm was recorded.

2.2.8.4. GTPase activity of PjEF-G(I-IVa)

GTPase assay was carried out in buffer (50mM Tris pH 7.5, 50mM

KCl, 2mM MgCb, 5%glycerol) that contained [y-32P] GTP

(3200Ci/mmol). Varying concentration of P.fEF-G(I-IVaJ (50pmoles-

500pmoles) was added in a final reaction vaolume of 20pl and

incubated at 37° C for 30min. The reactions were stopped by addition

of 1% SDS and 1pl of each reaction was spotted on 3MM Whatman

paper and resolved in a solution containing n-butanol, n-propanol,

acetone, 80% formic acid, 30% trichloroacetic acid at a ratio of

40:20:25:25:15 (v fv). The chromatogram was dried and

autoradiographed (Schwemmle and Staeheli, 1994).

2.2.8.5. Effect of kirromycin on GDP dissociation from PJEF

Tu.GDP

The effect of kirromycin on PjEFTu.GDP dissociation was estimated

by measuring the dissociation of the P.fEFTu.mant-GDP complex with

increasing kirromycin concentrations. The P.fEFTu.mant-GDP

complex was prepared by incubation of 111M PJEF-Tu and 151-lM

mant-GDP for 15min at 3TC. 251-lM GDP and increasing

concentration of kirromycin (0.1pM-200pM) were added followed by

incubation at OOC for 40 min. Mant-GDP emission as a result of FRET

was recorded at each kirromycin concentration. Background

correction was made by subtraction of emission spectra of 151-lM

mant-GDP in NB buffer mentioned above.

80


2.2.8.6. Kinetics of GDP release from PjEF-Tu by PjEF-Ts

The kinetics of GDP release from PjEF-Tu mediated by PjEF-Ts was

studied by fluorescence stopped-flow measurements on a JASCO J-

810 spectro-polarimeter with stopped-flow attachment (SFM-300/S,

BioLogic Science Instruments, France) in a buffer containing 50mM

Tris-HCl pH 7.5, 70mM NH4Cl, 30mM KCl, 7mM MgCb, and 1mM

DTT at 20°C. The change in fluorescence of the PjEF-Tu.mant-GDP

complex in the presence of PjEF-Ts was recorded. The fluorescence of

mant-GDP bound to PjEF-Tu was excited via FRET from tryptophan

excited at 280 nm and measured after passing 400nm filter

(BioLogic). Experiments were performed by rapid mixing of the EF

Tu.mant-GDP complex (0.5!lM PjEFTu) and 4!lM of PjEF-Ts (in buffer

containing 25!lM GDP) and fluorescence was monitored over time.

Time course profiles were obtained by averaging three individual

transients. The rate constant (ki) was determined by fitting the data

to an exponential function of the form y(t)= at+ b + ~Ai e-ki.t where y

is the fluorescence at time t and the slope (a) and offset (b)

correspond to the linear drift after the reaction; the best-fitting

amplitude (Ai) and apparent rate constant (Ki) were determined with

the Bio-Kine software (BioLogic)(Huecas et al., 2007).

2.2. 9. Assay of insulin disulfide reduction

The reduction of insulin by a disulfide oxidoreductase was assessed

spectrophotometrically by recording absorbance at 650 nm (Bardwell

et al., 1991). Insulin contains two polypeptide chains A and B that

are linked by two interchain disulfide bonds. When these bonds are

broken upon reduction (mechanism shown below), the insoluble free

B chain precipitates, leading to an increase in absorbance at 650nm.

SH S __.. -:----? __.. I

PjEF-Tu-S2 +DTT--.SH ~ PjEF-Tu-(SH,h+DTT--....8

PjEF-Tu-(SH)2 + insulin-S2 < > PjEF-Tu-S2 + insulin-(SH)2 81


For the assay reaction, the incubation mixture contained the

following in a final volume of 100 Ill: 0.1M N2-equilibrated potassium

phosphate pH 6.6, 0.3mM EDTA, 0.13mM porcine pancreas insulin

and 0.3mM DTT in the presence or absence of 2!lM PJEF-Tu. Insulin

stock solutions of 10 mgjml (1.67mM) were prepared by suspending

SOmg of insulin in 4ml of O.OSM Tris-Cl, pH 8.0 and the pH was

adjusted to pH 2 to 3 by addition of 1.0M HCl. This was followed by

rapid titration of the solution back to pH8.0 with 1.0M NaOH. Finally,

the volume was adjusted to Sml with water. The solution of insulin

was perfectly clear and was stored at -20°C.

2.2.10 Structural modelling and molecular dynamics

P.fEF-Ts structure model:

The structure of PjEF-Ts was modeled on the crystal structure of

chain B of E.coli EF-Ts (PDB code: 1EFU). The sequence identity

between the two is 23.4%. The model was constructed using the

program MODELLER interfaced with lnsightll. In order to assess the

overall stereochemical quality of the modeled protein, Ramachandran

plot analysis was performed using the program PROCHECK v3.4.4.

The Ramachandran plot showed normal distribution of points with

phi (cp) values and psi ("4-!) values clustered in a few distinct regions

with 86.4% and 11.9% of residues occupying favored and allowed

regions, respectively. Although the sequence identity between the

template and target was not high, a highly reliable model of the target

structure was constructed owing to considerably similar three

dimensional fold of the two molecules.

P.fEF-Tu.P.fEF-Ts complex:

The three dimensional model of the PjEF-Tu.PjEF-Ts complex was

constructed using E. coli EF-Tu.EF-Ts as template (PDB: lEFU chain

A&B) (Kawashima et al., 1996). The alignment of PjEF-Ts and PjEF-82


Tu was performed using ClustalW, as implemented at the EBI server

(http:/ fwww.ebi.ac.uk/Toolsfclustalw2/ index.html). The resulting

model was subsequently energetically minimised with 500 steps of

steepest descent minimisation, followed by 2000 steps of conjugate

gradient minimisation to remove the geometrical strain. GDP and

Mg2+ were manually docked to the PJEF-Tu binding site based on

previous structural information of the template (Song et al., 1999).

The model was constructed using the program MODELLER (Sali and

Blundell, 1993) interfaced with Insightii. MODELLER is an

implementation of an automated approach to comparative modeling

by satisfaction of spatial restraints (Sali and Overington, 1994; Sali et

al., 1995). In order to assess the overall stereochemical quality of the

modeled protein Ramachandran plot analysis was performed using

the program PROCHECKv3.4.4 (Laskowski et al., 1993; Morris et al.,

1992).

Molecular Dynamics:

The PjEF-Tu.Mg.GDP.PjEF-Ts ternary complex was further solvated

by using Explicit Spherical Boundary with harmonic restraints

(sphere of 50 radius). This solvated complex was subjected to energy

minimisation first by 500 steps of steepest descent followed by 2000

steps of conjugate gradient method. The system was heated from 50

K to 300 K over a period of 50 psec with a time step of 1 fsec and the

velocities being reassigned in the system every 0.05 psec. The system

was further equilibrated with a 1 fsec time step, for 100 psec so that

the energy of the system achieves complete stability. Production runs

were performed at 300 K and carried out under a constant number of

particles, volume and temperature conditions for 1 nsec with a 1 fsec

time step. All the bonds involving hydrogen atoms were constrained

using the SHAKE algorithm in all simulations (Ryckaert et al., 1977).

The molecular trajectory for the systems generated by the molecular

dynamics simulations were analyzed using the VMD (Humphrey et 83


al., 1996) software and CHARMM program (Brooks et al., 1983). All

MD simulations were carried out with CHARMM program using

CHARMM force field (Brooks et al., 1983).

84

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