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Transcript of CHA.P1'ER2 MA 1rJEJRJIAL§ ANJI}...
Chapter 2 Materials and Methods
CHA.P1'ER2
MA 1rJEJRJIAL§ ANJI} MJE1rH0]])§
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Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
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Chapter 2 Materials and Methods
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
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Chapter 2 Materials and Methods
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.
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Chapter 2 Materials and Methods
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
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Chapter 2 Materials and Methods
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
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Chapter 2 Materials and Methods
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
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Chapter 2 Materials and Methods
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.
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Chapter 2 Materials and Methods
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.
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Chapter 2 Materials and Methods
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%
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Chapter 2 Materials and Methods
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).
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Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
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Chapter 2 Materials and Methods
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
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Chapter 2 Materials and Methods
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.
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Chapter 2 Materials and Methods
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'
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Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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
Chapter 2 Materials and Methods
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