materials and methods - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/29931/7/07_chapter...

Materials and Methods

MATERIALS AND METHODS

35


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-

Amersham-Pharmacia- Lysozyme, Tris-saturated phenol, proteinase K,

RNase A (DNase free), T4 polynuc1eotidyl kinase, agarose, ATP, Poly (dA

dT), RNase out (RNase inhibitor), restriction enzymes, IPTG, X-gal, 13-mercaptoethanol, PMSF, Urea, Poly vinyl pyrrolidon K-90, dextran

sulphate, Ficoll-type 400, mineral oil, Phast gel blue R-350, coomassie

tablets, Mega Prime DNA labeling system, Multi Prime DNA labeling

system, Sephadex G-50 (DNA grade), X-ray films (Hyperfilm-MP), Hybond

ECL NC membrane, Hybond C+ Nitrocellulose membrane, hyperfilm ECL,

ECL Advance western blotting detection kit, Tween-20, Amplify

Fluorographic Reagent, high range Rainbow molecular weight marker,

Superdex 200HR 10/300 column

Clontech- Smart RACE cDNA amplification kit, RACE kit, Gel elution kit

G biosciences (Genotech)- Leupeptin, pepstatin A, MSG-Trypsin (Mass

spectroscopy grade)

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

DNA polymerase, 100bp DNA ladder, 1kb DNA ladder, Trizolt, DNase I,

EDTA, dATP, dGTP, dTTP, dCTP, SupercsriptTM 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 thio-sulphate

ICN- PAGE preservation system, Urea

Jonaki, India- [a-32PJdATP, [a-32PjdCTP, L- 35S-Methionine

Life technologies- AupreP DNA gel extraction kit

MBI-Fermentas- Restriction enzymes and buffers, protein molecular

weight markers, DNase I enzyme, T4 DNA ligase


Merck- acetone, isopropanol, methanol, I-butanol

Molecular Probes- MitoTracker deep Red 633, Alexaflour-tagged goat

anti-rabbit IgG (H+L), Cy5-tagged goat anti-rabbit IgG (H+L) and Oregon

green-tagged goat anti-mice IgG (H+L)

Nestle- non-fat dry milk

New England Biolabs- Restriction enzymes and buffers, RNA Marker, 100

bp and 1 kb DNA marker, calf intestinal alkaline phosphatase, T4

polynucleotidyl kinase, T4 DNA ligase

Oncogene- Fluorescent mounting media

Promega- Taq DNA polymerase, restriction enzymes, T4 DNA ligase,

Klenow enzyme, T/A cloning kit (pGEM-T easy vector).

Qiagen- Gel extraction kit, Ni-NTA fastflow, Qiagen plasmid mini kit,

Qiagen plasmid midi kit, Qiagen plasmid maxi kit

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

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

carbonate, sodium chloride, sodium potassium tartarate, hydrogen

peroxide, tri-sodium citrate, ammonium thiocyanate

Ranbaxy- EDTA, chloroform, formic acid, glycerol

Roche- Monoclonal anti-GFP antibody

Sartorius- Vivaspin concentrator

S.D. fine chemical- Dimethyl SUlphate, methanol

Sigma- 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 cyan 01 , bromophenol blue, ammonium acetate,

ammonium bicarbonate, potassium ferricyanide, sodium cacodylate, N

laurylsarcosine, PCR primers, Tris-saturated phenol, agarose, plasmid

midiprep kit, Gene elute plasmid miniprep kit, Potassium iodide, silicon

dioxide, coomassie brilliant blue R, Ponceau stain, acrylamide, bis

acrylamide, sodium dodecyl SUlphate, ammonium persulfate, TEMED,

Trizma Hel, Trizma base, glycine, triton X-lOO, imadazole, calcium

chloride, Nickel(II) sulfate hexahydrate, actinomycin D, methyl green,

37


distamycin A, salmon sperm DNA, sodium deoxycholate, glasswool, BSA,

DAPI, ammonium chloride, Igepal (Nonidet P-40), paraformaldehyde,

glutaraldehyde, formaldehyde, poly-L-Iysine, sodium borohydride, citric

acid, DTT, EDTA, Potassium chloride, sodium di-hydrogen phosphate, di

sodium hydrogen phosphate, sigmacote, iodoacetamide, DMSO, protease

inhibitor cocktail, bicinconinic acid (BeA) protein assay kit, magnesium

chloride, potassium chloride, magnesium sulphate, sodium azide, dialysis

tubing, dialysis tubing closures, OPD, imidazole hydrochloride, DAB,

silver nitrate, ampicillin, chloramphenicol, developer, ftxer, potassium

thiocyanate, sheep anti-rabbit HRP conjugate

Spectrochem- Piperadine, Trifluoro acetic acid (TFA), acetonitrile

SRL- Sodium dodecyl sulphate, Tris-base, sodium acetate, sodium

chloride, isoamyl alcohol, sodium hydroxide, acrylamide, glycine, sodium

bicarbonate, boric acid

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.

38


2.2 METHODS

2.2.1. In vitro Plasmodium/alciparum culture 2.2.1.1. Culture media

2.2.1.2. Processing of red blood cells

2.2.1.3. Initiation, maintenance and subculturing of P.falciparum , cultures

2.2.1.4. Synchronization of parasite cultures

2.2.1.5. Cryopreservation and revival of parasites

2.2.2. Transcriptional analysis of apicoplast genome

2.2.2.1. Total genomic DNA isolation from P.falciparum culture

2.2.2.2. Isolation of total P.falciparum RNA

2.2.2.2a. Total RNA isolation by GITC method

2.2.2.2b. Total RNA isolation by Trizol method

2.2.2.3. RT-PCR of apicoplast transcripts

2.2.2.3a. DNase I treatment of RNA

2.2.2.3b. cDNA synthesis of the transcript

2.2.2.3c. PCR-amplification of cDNA

2.2.2.4. Northern blotting of LSU and ORF470 transcripts

2.2.2.5. 5'-end mapping of Large subunit (LSU) transcript by RACE

(rapid amplification of cDNA ends)

2.2.3. Parasite protein extract preparation and analyses of DNA

protein interactions at replication ori of P./alciparum

apicoplast genome

2.2.3.1. Preparation of parasite protein lysate

2.2.3.2. Radiolabeling of DNA probe

2.2.3.3. Electrophoretic mobility shift assay (EMSA)

2.2.3.3a. With parasite proteins

2.2.3.3b. With recombinant PfDnaJint and PfDnaJlm

2.2.3.4. Analyses of major /minor groove binding

2.2.3.5. DNase I footprinting

2.2.3.5a. Preparation of Gladder

39


2.2.3.5b. Preparation of G+A ladder

2.2.3.5c. DNase I cleavage and electrophoresis

2.2.3.6. Southwestern blotting

2.2.3.7. MALDI-TOF analysis of trypsin-digested proteins eluted from

EMSA gel

2.2.3.7a. Protein elution after EMSA

2.2.3.7b. Silver-staining of eluted proteins in SDS-PA gel

2.2.3.7c. Destaining and in-gel digestion of the protein bands (peptide

mass fingerprinting)

2.2.4. Expression and characterization of mature PIDnaJ and internal

PIDnaJ protein 2.2.4.1. RT-PCR of PfDnaJ transcript

2.2.4.2. PfDnaJ gene amplification by polymerase chain reaction

2.2.4.3. Cloning of fragments encoding PfDnaJint and PfDnaJrn in pQE-

30 expression vector

2.2.4.4. Expression and purification of recombinant PfDnaJint and

PfDnaJrn proteins

2.2.4.5. Generation of anti-PtDnaJ antiserum and purification of

antibody

2.2.4.5a. Purification of PfDnaJint by preparatory gel

2.2.4.5b. Immunization of rabbit and mice and determination of

antibody titre

2.2.4.5c. Purification of PtDnaJint antibodies by immobilization on

nitrocellulose membrane

2.2.4.6. Specificity of raised antibodies

2.2.4.6a. Preparation of P.jalciparum, RBC and E.coli cell lysate

2.2.4.6b. Western blotting

2.2.4.7. Oligomeric status ofPtDnaJint protein

2.2.4.7a. Chemical crosslinking by glutaraldehyde

2.2.4.7b. Size-exclusion chromatography

2.2.4.8. Immuno-fluorescent labeling and localization of PfDnaJ

40

2.2 METHODS

2.2.1. In vitro Plasmodiumfalciparum culture 2.2.1.1. Culture media


Complete RPMI-1640 medium (Rosewell Park Memorial Institute) was

used to maintain RBCs infected with P.falciparum (strains 3D7 and NF54).

To make 1 L of RPMI culture medium, 16.4 g of RPMI-1640 (HEPES

modified), 10 g glucose and 2 g sodium bicarbonate were dissolved in 1 L

of water. This is the incomplete medium that is used for washing the

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 25 mM

HEPES, 1% glucose, 0.2% sodium bicarbonate and 10% vjv human

serum or 0.5% w jv Albumax II. To avoid bacterial contamination,

gentamycin sulfate was added at a final concentration of 25 /-Igjml and

the medium was filter-sterilized. Hypoxanthine was added at a final

concentration of 92 11M when Albumax II was used to make complete

medium.

2.2.1.2. Processing of red blood cells

Processed RBCs were used for parasite culture. For RBC processing, 20

ml of human blood was collected in a sterile 50 ml tube containing 7.5 ml

anticoagulant (ACD; For 100 ml, 0.0375 M citric acid, 0.075 M tri-sodium

citrate and 0.15 M 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 use,d for around 15 days.

2.2.1.3. Initiation, maintenance and subculturing of P.falciparum

cultures

In vitro cultivation of erythrocytic stages of P.falciparum was carried out

in human erythrocytes at 37°C under a low 02 (5-8%) and 2-5% C02 with

41


a balance of N2 gas. The candle jar method of Jensen and Trager (Jensen

pud Trager, 1977; Trager and Jensen, 1978) is the simplest way to achieve

this gas phase. The culture medium was changed every 24 h. 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) .

P.falciparum cell line, D 10 ACP(leader)-GFP (ATCC number: MRA-568) was

maintained under the pyrimethamine drug pressure (10nM) (Waller et aI.,

2000). The cell line was used in immunoflorescence localization for

PfDnaJ protein.

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.

jalciparum (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.

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

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

42


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 solutionfcryo

protectant 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 filte~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 cryovial and stored iIi

liquid nitrogen until use.

To thaw 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,O.lX volume of 12% NaCI was added drop wise, while shaking

the tube gently. The tube was left for 5 min at room temperature followed

by addition of lOX volume of 1.6% NaCI 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 1,500 rpm for 5 min at 20°C, the supernatant was

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

the pellet and the culture was maintained in a culture flask (Schichtherle

et ai., 2000).

43


2.2.2. Transcriptional analysis of apicoplast genome 2.2.2.1. 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 IX PBS. The DNA was released by adding 450 111 of lysis buffer

(50 mM Tris-Cl, pH 8.0, 5 mM EDTA, 100 mM NaCl and 1% SDS). 200 11g

of proteinase K was added and mixed by swirling. The reaction mixture

was incubated at 42°C for 45 min. RNase A (2 11g) was added, mixed and

incubated at 37°C for 15 min to degrade RNA. DNA was extracted with

phenol-chloroform. 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 1 mM EDTA).

2.2.2.2. Isolation of total P.falciparum RNA o

2.2.2.2a. Total RNA isolation by Guanidium iso-thi .. cyanate (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 IX PBS at 6,000 rpm for 10 min at 4°C. To the

pellet, 500 I-ll of solution D (4 M GITC, 25 mM sodium citrate, 0.5%

sarcosyl, 0.1 M PME in DEPC water) was added. 50 I-ll of 2 M sodium

acetate (pH 4.0), 500 I-ll of water saturated phenol and 100 I-ll chloroform

isoamylalcohol (49: 1) were added, mixed well 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 I-ll

isopropanol to aqueous phase for precipitation of RNA. The sample was

incubated at -20°C for 3 h followed by centrifugation at 12,000 rpm. The

pellet was suspended in 300 I-ll solution D, isopropanol (300 I-ll) 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.

44


2.2.2.2b. 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 IX

PBS. Released parasites were lysed by addition of 500 III of Trizol and

incubation for 5 min at room temperature. This was followed by addition

of 300 III chloroform, 5 min incubation at room temperature and

centrifugation at 12,000 rpm for 20 min at 4°C. Aqueous phase was takeri

ou t and 400 III of isopropanol was added to this fraction. After 10 min'

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.

2.2.2.3. RT-PCR of apicoplast transcripts

For the RT-PCR of apicoplast transcripts, cDNA was synthesized from

DNase I-treated total RNA isolated from parasite cultures followed by PCR

amplification of target sequences.

2.2.2.3a. DNase I-treatment of RNA

P.falciparum RNA (-2 Ilg) was treated with 2 III of DNase I (stock 1U/IlI)

to remove any DNA contamination. After incubation at room temperature

for 15 min, DNase I was inactivated by addion of 25 mM EDTA followed by

incubated at 65°C for 10 min. RNA was precipitated with 1/ 10th volume of

sodium acetate (3 M, pH 5.2) and two volumes of ethanol, washed with

70% ethanol, dried and suspended in DEPC- treated deionized water.

2.2.2.3b. eDNA synthesis of the transcript

cDNA synthesis was carried out from DNase I-treated RNA using first

strand superscriptTM cDNA synthesis kit (Invitrogen). The reaction

mixture, containing RNA, dNTPs and gene-specific downstream primer

/hexamer 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 the

RNA secondary structure formation. Reaction buffer, MgCb, DTT and

RNase-out (RNase inhibitor) were added and incubated at 42°C for 2 min.

1 III reverse transcriptase enzyme (50 U / Ill) was added to the reaction and.

45


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 cDNA

was removed by treatment with 1 III of RNase H at 37°C for 20 min. The

reaction was kept at -20°C till further use.

2.2.2.3c. peR amplification of cDNA

cDNA was PCR amplified by primer pairs designed for specific target

sequences.

For the determination of the mono- /poly-cistronic nature of ORF470,

primer pairs: OINT (U)-OINT (D), LSU (2)-OINT (D), and OINT (U)-rpoB (D)

were used.

Table I shows the sequences of primers used for amplification of cDNA.

Table II shows PCR conditions for cDNA amplification.

Table I. Primers used for amplification of gel10mic DNA and cDNAs.

Primer Primer sequence

U: 5'-CGC GAA 'ITC ATG GCT AAT A'IT AAT TCA ATC

GINT ATC TTI AGT G-3'

D: 5'-CGC GGA TCC 'ITA AGG AAT AGT TAC TGT TAA

AGA A'IT-3'

TufA U: 5'-TAG ATG GTA TAA TGC CTC AM C'IT-3'

D: 5'-CTC CGG A'IT GTG C'IT GTG T-3'

ClpC U: 5'-AM CCT 'ITA ATA 'ITA CCT ACA ACT CCA T-3'

D: 5'-TCC 'ITC TGA ATA ACC AAC ATA ACC-3'

rpoB(D) 5'-CTG AAT AGT ATC AAT AGA A'IT TAT A'IT TAT A'IT

AG-3'

LSU-2 U: 5'-AGT ACG AGA GGA TCA AM AM 'ITA TAC C-3'

Table II. PCR Conditions:

Pre-denaturation:

Post-extension:

Reaction volume:

94°C, 2 min

72°C, 5 min

50 III PCR cycle conditions standardized for ThermostarIII thermal cycler

46


Gene Enzyme Fragment Cycle conditions name size

(bpI Denaturation Annealing Extension

OINT Platinum 690 94°e, 50oe, 1 min 72°e, Taq 30 sec 35 cycles 1 min

Platinum 528 94°e, 45°e, 1 min 72°e, tufA Taq 30 sec 35 cycles 1 min

Platinum 545 94°e, 45°e, 1 min 72°e, clpC Taq 30 sec 35 cycles 1 min

LSU(2)- Platinum 1542 94°e, 40oe, 1 min 72°e, OINT(D) Taq 30 sec 5 cycles 2 min

4Te, 1 min 30 cycles

OINT (U)- Platinum 1580 94°e, 40oe, 1 min 72°e, rpoB (D) Taq 30 sec 5 cycles 2 min

50oe, 1 min 35 cycles

2.2.2.4. Northern blotting of LSU and ORF470 I suJB transcripts

Total P.falciparum RNA (-10 ~g) was electrophoresed on 1.2%

formaldehyde agarose gel at 75 volt in IX formaldehyde gel running buffer

(0.1 M MOPS, 40 mM sodium acetate, 5 mM EDTA) and capillary

transferred to nylon membrane (Hybond N+, Amersham) in 20X sse

buffer (3 M sodium chloride and 0.3 M trisodium citrate, pH 7.0)

overnight. After transfer, the membrane was baked at 800 e for 2 h. The

membrane was prehybridized in a solution (5X sse, 5X Oenhardt

solution, ,0.5% SOS and 10 ~g/ml salmon sperm DNA) at 55°e for 5 h.

Rediolabelling of DNA fragment was carried out with [a-32p]deTP using

Megaprime radiolabeling kit (Amersham) as per manufacturer's

instructions. The probe was denatured at 1000 e for 10 min and added to

the prehybridization solution. Hybridization was carried out at 55°e for

overnight. The membrane was sequentially washed twice with 2X sse for

5 min each at room temperature, 0.2X sse for 15 min at 55°e, and 0.1%

SDS for 15 min at 55°e and autoradiographed.

47


Northern blotting was also performed by the method as described by Kyes

et al. (2000). Briefly, Total P.falciparum RNA (-10 ).!g) was electrophorsed

on 1.2% agarose gel containing 5 mM guanidine thiocyanate in IX TBE

(0.089 M Tris, 0.089 M boric acid and 2 mM EDTA) and electrophoresed

in IX TBE gel running buffer. After staining with EtBr, the gel was soaked

in 7.5 mM NaOH twice for 10- min and capillary transferred to nylon

membrane (Hybond N+, Amersham) in 7.5 mM NaOH overnight. The

membrane was neutralized with 2X SSC buffer for 5 min and baked at

80°C for 2 h. The membrane was prehybridized for 5 h at 55°C in a

solution containing 7% SDS, 0.5 M sodium phosphate (NaH2P04), pH 7.2,

and 2% dextran sulphate, and then hybridized overnight at 55°C in the

same solution in the presence of radi~_olabeled probe. The membrane was

washed twice with 2X SSC at room temperature for 5 min each, once with

0.2X SSC at 55°C for 10 min and then autoradiographed.

2.2.2.5. 5'-end mapping of LSU transcript by RACE

5'-end of the LSU transcript was determined by using RACE kit

(Clontech) according to manufacturer's instructions (Fig. 2.1). Briefly, -2

).!g of DNase I-treated total P.falciparum RNA was used for 1st strand cDNA

synthsis. The reaction mixture containing RNA, downstream primer (Lsu-

2) and Smart II oligo, was incubated at 70°C for 2 min and chilled on ice

for 1 min. To this, first strand reaction buffer, DTT, dNTPs and

Powerscript reverse transcriptase was added and incubated at 42°C for

1.5 h. Tricine-EDTA buffer was added, incubated for 7 min at 72°C and

stored at -20°C. The synthesized cDNA was PCR amplified using universal

primer as upstream primer and Lsu-2 as downstream primer pair. The

PCR conditions were:

Predenaturation- 94°C, 2 min;

Denaturation- 94°C, 30 sec;

Annealing- 4 TC, 1 min;

Extension- 72°C, 3 min for 35 cycles;

Final extension- 72°C, 5 min.

48


RACE PCR products were resolved on 1.2% agarose gel along with DNA

ladder. DNA fragments of different sizes were eluted from the gel, ligated

into T / A cloning vector (pG EM -T easy vector, Promega) and transformed

into E.coli DHSa cells using standard protocol. Positive clones were

selected on X-gal LB-agar plate containing ampicillin antibiotic by

blue/white colony screening. DNA was isolated from insert containing

clones and sequenced in an automated sequencer.

49


RNA 5'-cl =========================i~~'-3' C 1 GSP

SMART II oligo 5'-1 ··'GGG'

CCC r· . '.'."-"

:::::::::::J-3' 1-5'

GSP

5'-cl =. :::::::=:J1GGG[1 ===============::J1-3'

Universal Primer 5'-1 I' I

3'-r I·

Nested Universal Primer

3'-cl =I::=~'~'~···§···-~·lht-===::::::::=====:r:=::::J1-5'

5'-·cl ~§'§~~§~§E~' §S,-3' 3'-1:; . II 1-5'

Cloning in T / A cloning vector (pGEM-T easy vector, Prom ega)

+

. ·'-5'

Miniprep and sequencing of insert containing plasmid DNA.

First strand cDNA synthesis

RT template switching

5'-RT PCR

Second round of PCR

Fig. 2.1. Diagrmatic scheme for steps involved in 5'··end of the transcript by 5'-RACE method.

50


2.2.3 Parasite protein extract preparation and analyses of DNA

protein interactions at replication on of P./alciparum apicoplast

genome

2.2.3.1. Preparation of parasite protein extract

To analyze the DNA-protein interaction(s) at apicoplast replication ori, first we standardized a protocol for isolation of proteins from apicoplast

enriched fraction of P.falciparum infected erythrocyte culture by

differential centrifugation (Fig. 2.2, A).

Parasite culture (6 to 8% parasitemia at late trophozoite-early schizont

stage) was centrifuged at 2,500 rpm for 5 min at 4°C. Cells were washed

twice with ice-cold IX PBS and parasites were released by treatment with

0.05% saponin followed by centrifugation at 6,000 rpm for 5 min at 4°C.

The pellet was again washed twice with ice-cold IX PBS and the cells were

suspended in a buffer containing 50 mM Tris-CI, pH 7.6, 25 mM KCI, 10

mM MgCb and 380 mM sucrose and homogenized (Ultra-Turrax T25

Basic, Janke-Kunkel GmBH, Germany). The cells were lysed using 15

strokes of stirrer rotating at 13,000 rpm for 10 sec each. Intact nuclei

were pelleted by centrifugation at 2,000 rpm for 3 min and processed. for

preparation of nuclear extract using a method modified from Voss et al. (2002) while the supernatant was processed. for preparation of extracts

enriched in cytoplasmic or organellar proteins.

The nuclear pellet was washed twice with a ice-cold buffer containing 20

mM HEPES, pH 7.8, 10 mM KCI, 1 mM EDTA, 1 mM DTI and 1 mM

PMSF and resuspended in one pellet volume of extraction buffer (20 mM

HEPES, pH 7.8, 800 mM KCI, 1 mM EDTA, 1 mM DTI and 1 mM PMSF, 3

J.lM pepstatin A and 10 J.lM leupeptin). After vigorous shaking at 4°C for 30

min, the extract was cleared by centrifugation at 12,000 rpm for 30 min.

The supernatant (containing nuclear proteins) was diluted with one

volume of dilution buffer (20 mM HEPES, pH 7.8, 1 mM EDTA, 1 mM DTI

and 30% glycerol) and stored at -70°C.

51


The supernatant obtained after separation of nuclei was centrifuged at

18,000 rpm for 35 min at 4°C to collect the organelle-enriched pellet using

a protocol modified from Fernandez-Silva et al. (1996). The supernatant

from this step was stored in 10% glycerol as the cytoplasmic fraction. The

pellet was washed twice in a buffer containing 10 mM Tris-CI, pH 6.7, 380

mM sucrose and 0.15 mM MgCb followed by centrifugation at 18,000 rpm

for 2 min. The pellet was suspended in lysis buffer (25 mM HEPES-KOH,

pH 7.6, 5 mM MgCb, 0.5 M KCI, 0.5% Tween-20, 0.5 mM EOTA, 1 mM

OTT, 1 mM PMSF, 3 11M pepstatin A, 10 11M leupeptin and 10% glycerol)

and kept on icc for 20 min with intermittent vortexing every 5 min. The

supernatant obtained after centrifugation at 12,000 rpm for 45 min at 4°C

was collected and stored at -70°C as the organellar protein extract. BCA

kit (Sigma) was used to determine protein concentration in all extracts.

The presence of apicoplast proteins in above extracts was confirmed by

immunoprecipitation of apicoplast encoded EF -Tu followed by western

blotting (Fig. 2.2, B) (Chaubey et al., 2005). For immunoprecipitation,

nuclear, cytoplasmic and organellar parasite protein extracts were

precleared by the addition of 3 mg of Protein A sepharose CL-4B. The

cleared supernatant was incubated with primary antibody (mouse anti

EFTu serum) for 2 h on ice with concomitant mixing. After centrifugation

at 12,500 rpm for 10 min, the supernatant was incubated overnight with

5 mg of Protein A sepharose CL-4B at 4°C with continuous mixing.

Sepharose beads were pelleted at 12,000 rpm for 3 min at 4°C and

washed six times with chilled buffer (buffer in which proteins were

suspended) followed by a PBS wash. Immunoprecipitated proteins were

obtained by treating the beads with SOS lysis buffer. The samples were

electrophoresed on a 10% SOS-polyacrylamide gel, transferred onto

nitrocellulose membrane and proceed for western blotting. After transfer,

the membrane was blocked overnight with IX PBS containing 5% dry

skimmed milk at 4°C. The blot was then incubated with mouse anti-EFTu

serum in 2% non-fat dry milk/PBS for 90 min at room temperature. The

52


(A) Parasite Culture ... Saponin Lysis

Parasite Pellet

Lysis by homogenization

Nuclear pellet

Pellet suspend in extraction bufer

Vigorous shaking at 4°C, 30 min

~ Centrifugation at

12,000X rpm

~ Supernatant (Nuclear fraction)

(B)

... Centrifugation at 2,000X rpm, 5 min

/~~ Supernapt (cytoplasmic and organellar enriched)

... Centrifugation at I8,000X rpm

~------,~ Supernatant Pellet (Cytoplasmic (Organellar enriched) fraction) ...

f""'< ...........,

\ .

Washing with suspension buffer

... Lysis with lysis buffer

... Incubation on ice, 45 min.

I Centrifugation at • 12,000X rpm

Supernatant (Organellar enriched protein fraction)

. J ""/'~ ... 46 kDa

1 2 3 4

Fig 2.2. (A) Flow chart for the isolation of apicoplast-enriched proteins from parasite culture by differential centrifugation method. (B) Immunoprecipitation of proteins isolated from different parasite fractions, Nuclear (lane 1), cytoplasmic (lane 2) and organellar-enriched (lane 3 and 4), with anti EF-Tu antibody. A -46 kDa band of Ef-Tu is visible below the shadow ofIgG heavy chain (-55 kDa).

53


membrane was washed with PBS-Tween followed by 90 min incubation

with secondary antibody (anti-mouse HRP conjugate, Sigma, USA). After

further washing with PBS-Tween, the blot was developed using a

chemiluminescentdetection system (Amersham Biosciences, UK).

Parasite protein extracts were also prepared by NP-40 lysis and by silicon

grinding. However, proteins isolated using these methods did not exhibit

specific binding in EMSAs.

2.2.3.2. Radiolabeling of DNA probe

Radiolabelling of 195 bp DNA fragment was carried out by end-filling

using Klenow fragment. 195 bp DNA fragment was taken out from the

1988 bp DNA fragment covering part of the inverted repeat region of the

apicoplast genome that had been cloned in pUC18. pUC18-1988 clone

was digested with HindIII-ClaI restriction enzymes generating 5'-overhang~

150 ng of 195 bp DNA was radiolabeled in the presence of a-32P [dCTP]

using 0.5 unit of Klenow fragment (Promega) by incubation at 30°C for 15

min. The probe was purified through Sephadex G-50 column (single drop

method) and counts were taken in Beckmen scintillation counter.

2.2.3.3. Electrophoretic mobility shift assay

2.2.3.3a. With parasite proteins

Binding reactions with parasite proteins extracted from nuclear,

cytoplasmic and organeller-enriched fractions were carried out by

incubation of 4 I1g extract with end-labeled 195 bp DNA fragment. The

binding reaction was carried out in a buffer containing 10 mM HEPES, pH

7.5, 10 mM MgCb, 50 mM NaCI, 0.5 mM DTT, 5% glycerol and 1 I1g (500

to 1000-fold excess) of poly(dA-dT) at 4°C for 15 min. The reaction was

loaded on a 4% poly-acrylamide gel and electrophoresed at 120 V in IX

TAE buffer at 4°C. Gel was dried at 80aC for 2 h and subjected for

autoradiography and/or phosphorimaging. In competition experiments, a

200-fold molar excess of unlabeled competitor DNA was added prior to the

addition of organellar extract.

54


2.2.3.3b. With recombinant PtDnaJint and PtDnaJm

Binding reactions with recombinant PfDnaJint were carried out by

incubation of -150 ng protein with the 195 bp probe in a buffer

containing 10 mM HEPES, pH7.5, 10 mM MgCb, 100 mM NaCl, 0.5 mM

011, 5% glycerol, 50 mM ATP and 0.02% Tween-20. Binding reactions for

PfDnaJrn were as for PfDnaJint but contained 200 mM NaCl and lacked

Tween-20. Gel running and autoradiography was performed as described

above.

2.2.3.4. Analyses of major/minor groove binding

The radiolabeled 195 bp DNA fragment was pre-incubated for 30 min at

room temperature with varying concentrations of methyl green (0.01 to 1

mM), distamycin A (0.01 to 1 mM) or actinomycin D (0.01 to 2.5 mM).

Methyl green specifically binds to major groove whereas distamycin A and

actinomycin D interact with the minor groove of the DNA double helix

(Copenhaver, et al., 1993; Kim, et al., 1993). The DNA-drug mix was then

analysed for binding with organellar protein extract in an EMSA as

described earlier.

2.2.3.5. DNase I footprinting

DNase I footprinting with both stands of 195 bp DNA fragment was

carried out as described by method of Schmitz and Galas (1978). Gladder

was prepared by the chemical cleavage method (Maxam and Gilbert, 1980)

and G+A ladder was prepared;a modified protocol (Papavassiliou, 2001). A

fragment of 973 bp (ends defined by HindIII and ClaI sites) comprising the

195 bp region at the 5'-end was labeled by end-filling followed by digestion

with ClaI. This gave the 195 bp fragment labeled at the HindIII site such

that the 3' end of the complementary strand was labeled.

2.2.3.Sa. Preparation of Gladder

G nucleotide of the radiolabeled probe was modified by di-methyl

sulfate in presence of OMS reaction buffer (50 mM sodium cacodylate, pH

8.0 and 1 mM EDTA) at 25°C for 30 min. The reaction was stopped by

addition of 50 /-II DMS stop buffer (1.5 mM sodium acetate, pH 7.0 and 1

55


M &ME). DNA was precipitated by addition of 750 ,.ll of chilled ethanol,

incubation at -80°C for 30 min and centrifugation at 13,000 rpm for 15

min. The DNA pellet was washed twice with chilled 70% ethanol and re

precipitated with 3 M sodium acetate and absolute ethanol at -80°C for 5

min. The pellet was completely dried in a Speed-Vac. The modified base

was cleaved by addition of 70 ,..1 of 10% piperadine at 90°C for 30 min~

The pellet was dried, resuspended in 30 ,..1 deionized water, dried again to

completely remove any trace of piperadine and stored at -70°C.

2.2.3.Sb. preparation of G+A ladder

A reaction mixture containing radiolabeled 195 bp fragment, 1.5 ,..g

salmon sperm DNA and l.5 ,..1 of 88% aqueous formic acid was incubated

at 37°C for 14 min in a water bath. 150 ,..1 of freshly prepared 1 M

aqueous piperadine was added and the reaction was incubated at 90°C for

30 min. After brief centrifugation, 1 ml of I-butanol was added to the

reaction and vortex vigorously until only one phase was visible. The

sample was centrifuged at 12,000 rpm for 2 min. The pellet was

suspended in 150 ,..1 of 1% SDS and vortexed. 1 ml of I-butanol was

added again and centrifugation at 12,000 rpm for 2 min was repeated.

The DNA pellet was dried in Speed-Vac and kept at -70°C till use.

2.2.3.Sc. DNase 1 cleavage and electrophoresis

The probe was incubated at 4°C for 10 min with increasing

concentrations of P.falciparum organellar lysate (2 ,..g to 10 ,..g) in the

binding buffer as used for EMSA. Cleavage was performed with 0.05U of

Dnase I for 2 min at 3TC. The reaction was stopped with 100 ,..1 of stop

buffer containing 1% SDS, 200 mM NaCI and 20 mM EDTA. DNA was

extracted with phenol-chloroform, precipitated with ethanol, dried and

stored at -70°C.

Gladder, G+A ladder and DNase I-treated DNA fragments were suspended

in 3 III of sequencing loading dye (90% v Iv deionized formamide, IX TBE,

0.025% xylene cyanol and 0.025% bromophenol blue). The samples were

heated at 90°C for 10 min and electrophoresed in 6% denaturing

polyacrylamide gel containing 7 M urea in IX TBE at 1800 V. The gel was

56


fixed in methanol and acetic acid, dried and exposed to phosphorimager

screen.

2.2.3.6. Southwestern blotting All operations were carried out at 4°C unless otherwise stated.

Organellar protein extract (-100 Jlg of protein) was fractionated on 10%

SDS-polyacrylamide gel and transferred onto' a nitrocellulose membrane

in a buffer containing 25 mM Tris and 190 mM glycine at 30 rnA for 16 h.

The membrane was incubated with blocking buffer (2% non-fat dry milk,

1% BSA, 10 mM HEPES-NaOH, pH 7.5, 0.1 mM EDTA, 200 mM NaCI, 50

mM MgCb, and 16 Jlg/ml salmon sperm DNA) for 2 h and then incubated

with binding buffer (blocking buffer with 0.2% non fat dry milk) containing

radiolabeled 195 bp DNA probe for 16 h, washed briefly and subjected to

autoradiography as described earlier (Rao et ai, 1990).

2.2.3.7. MALDI-TOF analysis of trypsin-digested proteins eluted from EMSAgel 2.2.3.7a. Protein elution after EMSA

Binding reactions with organellar protein extract were carried out as

described earlier and electrophoresed on a 4% polyacrylamide gel at 120 V

in IX TAE buffer at 4°C. The wet gel was autoradiographed at room

temperature. Both DNA-protein complex bands were cut from the gel by

aligning the X-ray film and proteins were eluted by electro-elution in

elution buffer (25 mM Tris, 200 mM glycine, 0.025% SDS, 1 mM PMSF, 3

JlM pepstatin A and 10 JlM leupeptin) at 50 V for 3 h at 4°C. Eluted

protein-DNA complexes were concentrated in -12 kDa cut-off Vivaspin

concentrator (Sartorius) and further concentrated by using sephadex G-

50.

2.2.3.7b. Silver-staining of eluted proteins in SDS-PAgel

Concentrated protein samples and free DNA probe were resolved ona

10% SDS-polyacrylamide gel along with a protein molecular weight

marker. The gel was silver stained using the method of Bloom et al.,

(1987). The gel was fixed in 50 ml of formaldehyde fixing solution (40%

57


methanol and 0.5 ml of 37% formaldehyde per liter in water) for 10 min

with slow agitation at room temperature. It was then washed twice with

distilled water fot 5 min each. The gel was sensitized by 1 min incubation

in 50 ml of 0.02% sodium thio-sulphate and rinsed twice with distilled

water for 20 sec each. After rinsing, the gel was then submerged for 10

min in 50 ml of 0.1% silver nitrate solution. The gel was again rinsed

twice with distilled water for 1 min each and then developed in thio

sulfate developing solution (3% sodium cabonate, 0.0004% sodium thio

sulfate·and 0.5 ml of 37% formaldehyde per liter in water) with intensive

shaking until band intensity was adequate. After the desired intensity of

staining was achieved, development was terminated by discarding the

reagent, followed by washing for 10 min with 5 ml of 2.3 M citric acid per

100 ml solution. The silver stained gel was washed with distilled water for

10 min, dried between cellophane paper (leN) and autoradiographed to

confirm the position of DNA bands in the gel.

2.2.3.7c. Destaining and in-gel digestion of the protein bands

(peptide mass fingerprinting)

Removal of silver ions, in-gel digestion with trypsin and peptide

extraction from gel was carried out as described by Gharahaghi et al.,

(1999) and Shevchenko et al., (1996).

The silver-stained protein bands were destained with chemical reducers

potassium ferricyanide and sodium thio-sulfate to remove the silver ions.

The chemical agents were prepared prior to digestion as two stock

solutions of 30 mM potassium ferri-cyanide and 100 mM sodium thio

sulfate. A working solution was prepared freshly by mixing a 1: 1 ratio of

the above stock solutions (final working concentration 15 mM potassium

ferri-cyanide and 50 mM sodium thio-sulfate). The silver stained protein

band was excised and soaked in 1: 1 mixing solution in a volume enough

to cover the gel and kept until the brownish colour disappeared. The gel

was rinsed a few times with water to stop the reaction followed by

treatment with 100 mM ammonium bicarbon~te for 20 min. The gel was

cut into small pieces that were rinsed with water and then dehydrated

with 100 J..II of acetonitrile until the gel pieces turned opaque. The gel

58


pieces were dried completely in a Speed-Vac. Reduction was carried out by

1 h treatment with 10 mM DTI at 5TC. After cooling to room

temperature, the sample was alkylated using 55 mM iodoacetamide for 45

min in dark. The gel pieces were washed three times for 5 min each

alternatively with 100 mM ammonium bicarbonate and 50% acetonitrile.

The liquid phase was removed and the gel pieces were dried completely in

a Speed-Vac. Enzymatic digestion was carried out by addition of three gel

volumes of 12 nglJ,l1 trypsin (Genotech) in 25 mM ammonium bicarbonate.

The reaction was incubated overnight at 3TC. The resultant peptides were

extracted sequentially (three to four times) with 5% (v Iv) TFA in 50% (v Iv)

acetonitrile. The peptide extract was dried in Speed-vac, suspended in

deionized water and subjected to MALDI-TOF analysis. The peptide mass

fingerprint was searched against the P.falciparum peptide database using

Mascot search engine (Perkins et aI., 1999).

2.2.4. Expression and characterisation of mature PIDnaJ and internal

PIDnaJ protein 2.2.4.1. RT-PCR of PIDnaJ transcript

The PIDnaJ transcript in the parasite was detected by RT-PCR analysis.

cDNA was synthesized from total P.falciparum RNA using hexamer primer

as described earlier. The cDNA was PCR amplified with upstream primer

(5'-CGAGCTCGATCCTIATACAGTITIAGGTTIATCT-31 and downstream

primer (5'-CCCAAGCTIGTTICACATIAGTTIGAGGGTIAGGTA-3l The

PCR amplified product was resolved on 1% agarose gel along with DNA

molecular weight marker.

2.2.4.2. PIDnaJ gene amplification by polymerase chain reaction

To amplify PfDnaJ gene, upstream and downstream primer pairs were

designed using DNAstar Primer select and Oligo software. The segments

encoding mature PfDnaJ (amino acids 61 to 672) as well as the conserved

DnaJ domain (amino acids 61 to 415) were amplified by PCR using

P.falciparum genomic DNA as template. Upstream (5'-CGAGCTCGATCCTI

ATACAG'IT'ITAGGTTIATCT-31 and downstream (5'-AAGCTTICAATTITTI·

59


GGCTCTAACTIATCAGATAT-31 primers carrying Sad and HindIII sites

(underlined), respectively were used to amplify a l.9 kb fragment that

encoded the entire protein but lacked the N-terminal 60 amino acids

encoding predicted apicoplast targeting elements. An internal DnaJ

segment (amino acids 61 to 415) encoding conserved DnaJ domains was

amplified using the downstream primer (5'-CCCAAGCTIGTTICACATIAGT

TIGAGGGTIAGGTA-31 carrying a HindIII tag (underlined) paired with the

upstream primer as above. Table I shows PCR conditions for the above

reactions. The reaction was carried out in Gene Amp PCR system 9700

(Applied Biosystems)

Table I. PCR conditions for PjDnaJ

Reaction volume: 50 /-11

Pre-denaturation: 94°C, 4 min

Post extension: 68°C, 10 min

Genes Enzyme Cycle conditions Denaturation Annealing

Platinum 94·C, 30 sec 46·C, 30 sec PjDna.lint Pfx 5 cycles

polymerase 55·C, 30 sec

30 cycles Platinum 94°C, 30 sec 46·C, 30 sec

PjDnaJm Pfx 5 cycles polymerase

55·C, 30 sec 30 cycles

Extension 68°C, 2 min

68°C, 2 min

2.2.4.3. Cloning of fragments encoding P/DnaJint and PfDnaJm in pQE-30 expression vector

Standard protocols (Sambrook et al., 1989) were used for cloning of

PCR fragments into appropriate vectors, plasmid isolation, restriction

enzyme digestion, DNA ligation and transformation of ligated product into

E.coli host strains. E.coli competent cells (DH5a and TG-l strains) were

prepared by CaCb method. The plasmid mini- or midi-DNA purification

kits (Qiagen and Sigma) were used for plasmid DNA isolation.

60


PCR-amplified PjDnGJint and PjDnaJm fragments digested with Sad and

HindIII restri~tion enzymes were eluted from the agarose gel (AuPreP gel

extraction kit, Life Technologies) and ligated into pQE-30 E.coli expression

vector carrying the N-terminal RGS-6xHis tag (also digested with similar

restriction enzymes) to yield the expression vectors pQE-30- PjDnGJint and

pQE-30- PjDnaJm (Fig. 2.3).

Sad HindIII

Digestion with Sad and HindIII

Ligation

pQE30-PjDllaJint

pQ E30-PjDllaJ m

Fig. 2.3. Cloning strategy for inserts (DnaJint and DnaJrn) in pQE-30 vector.

2.2.4.4. Expression and purification of recombinant PIDnaJlnt and

PIDnaJm proteins

2.2.4.4a. Expression E.coli TG-l cells were co-transformed with pQE30-PjDnaJint and pQE30-

PjDnaJm together with the RIG plasmid (kind gift from Prof. W.G.J. HoI)

and plated on LB-agar plates containing ampicillin (50 Jlg/ml) and

chloroamphanicol (34 Jlg/ml). RIG plasmid with carries tRNAs of amino

acids arginine, isoleucine and glycine for enhanced expression of

61


P.falciparum proteins. A single colony was picked and grown overnight in

3 ml LB medium containing ampicillin (100 f.l.gjml) and chloro-amphanicol

(34 f.l.gjml) antibiotics at 37°C as primary culture. 250 ml LB medium

containing both antibiotics was inoculated with 1.0 ml of primary culture

and grown at 37°C until the A600 reached 0.5-0.6. The cultures were

induced by adding 0.5 mM IPTG (isopropyl-1-thio-(3-D-galactopyranoside)

and grown for 18 h at 20°C. The IPTG induced cells were pelleted at 7,000

rpm for 10 min at 4°C. Pellet was resuspended in 15 ml of lysis buffer (50

mM HEPES, pH 7.6, 300 mM NaCI, 10 mM imidazole and 10% glycerol)

and sonicated. The sonicated cells were centrifuged at 12,000 rpm for 30

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

The uninduced, induced total cell protein, soluble and insoluble fractions

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

recombinant proteins followed by western blotting analysis using anti-His

antibodies (Amersham).

2.2.4.4b. Purification

The recombinant proteins were purified by affinity chromatography on

Ni-NTA Superflow (Qiagen). The soluble fraction of the lysate was loaded

onto the Ni-NTA column pre-equilibrated with lysis buffer followed by

column washing with four bed volumes of lysis buffer. An extra wash was

given with wash buffer containing 50 mM HEPES, pH 7.6, 500 mM NaCI,

60 mM imidazole and 10% glycerol. Protein was eluted in 10 fractions

with elution buffer (50 mM HEPES, pH 7.6, 500 mM NaCI, 250 mM

imidazole and 10% glycerol). All fractions were checked on 10% SDS

PAGE. Western blotting was carried out using anti-His antibodies

(Qiagen). The purified proteins were dialysed against dialysis buffer

containing 20 mM HEPES, pH7.6, 300 mM NaCI, 20% glycerol, 0.2 mM

EDTA, 0.5 mM DTT and 0.5 mM PMSF, concentrated and stored at -70°C.

2.2.4.5. Generation of anti-PIDnaJ antiserum and purification of antibody through nitrocellulose membrane

Polyclonal anti sera were raised against recombinant PrDnaJint in both

rabbit and mice.

62


2.2.4.Sa. Preparation of P/DnaJ,nt by preparatory gel

PfDnaJint protein was electrophoresed on 10% SOS-polyacrylamide

preparatory gel followed by gel elution (Ohhashi et al., 1991). Negative

staining was carried out with 4 M sodium acetate for 30 minutes and the

band corresponding to the PIDnaJint protein was excised from the gel. The

gel band was chopped into small pieces and placed in a dialysis bag along

with 6 ml of 10 mM EOTA. Protein was electro eluted for two hours at 4°C

at 50 V and checked on a SOS-PA gel to reconfirm the correct size. The

identity of protein as PIDnaJint was confirmed by western blotting using

anti-His antibody (Amersham). Eluted protein was concentrated in a

centricon.

2.2.4.Sb. Immunization of rabbit and mice and determination of

antibody titer

Antibodies against PIDnaJ were raised in rabbit as well as in mice

using recombinant PIDnaJint protein. -150 Ilg and -50 Ilg of SOS-PAGE

purified PfDnaJint protein, emulsified in Freund's complete adjuvant

(GIBCO, BRL) were injected subcutaneously in rabbit (New Zealand Red)

and mice (Swiss strain), respectively. Rabbit and mice were bled to collect

pre-immune sera before administering the antigen. One booster injection

in Freund's incomplete adjuvant in rabbit (-80 Ilg protein) and one

booster injection in mice (30 Ilg protein) after 28 days was given. Rabbit

and mice were bled after 10 days of booster dose. Blood was stored at

37°C for 30 min and then transferred at 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 -20°C. Western blotting of the recombinant

PIDnaJint and PfDnaJm protein was done to check the specificity of the

polyclonal sera. ELISA was performed to determine the titer of the

antisera raised in rabbit and mice.

For ELISA, 50 III of purified antigen (stock 2 Ilg/ml in carbonate buffer)

was coated per well on the ELISA plate for 6 h at 4°C (250 ml of carbonate

buffer contained 0.39 g of sodium carbonate and 0.73 g of sodium

bicarbonate in water, pH 9.6). After repeated washing with IX PBS, the

63


wells were blocked overnight at 4°C. with 100 /JI of blocking buffer

containing 5% nonfat dry milk in PBS-T (IX PBS containing 0.05% Tween-

20). Plate was washed with PBS-T for four to five times. Primary antibody

dilutions were prepared by serial dilution in the blocking buffer (2%

nonfat dry milk in PBS-T) and 50 /JI of different dilutions of anti-sera were

added per well for 2 h at 25°C followed by six washes with PBS-To Anti

rabbit HRP-conjugated secondary antibodies (Sigma) were diluted at

1:8,000 in blocking buffer, whereas anti-mice HRP-conjugated secondary

antibodies (SantaCruz) were diluted at 1:2,000 in blocking buffer. 50 /J1 of

each diluted secondary Ab was added to the corresponding antigen-coated

wells and incubated for 2 h. After six washes with PBS-T, plates were

developed for 5 min by adding ortho-phenylene diamine (OPD) solution as

the chromogenic substrate in citrate buffer with traces of H202 as catalyst

(100 ml of citrate buffer contained 0.73 g of citric acid and 0.947 g of di

sodium hydrogen phosphate, pH 5.0). For preparing the OPD substrate

solution,S mg of OPD was dissolved in 10 ml of citrate buffer with 5 JlI of

30% H202. Reaction was stopped by adding 50 /JI of 7.5% H2S04 to each

well and the reading was taken at 492 nm in an ELISA reader (/J Quant,

BIO-TEK, USA)

2.2.4.Sc. Purification of P/DnaJint antibodies by immobilization on nitrocellulose membrane

Antibodies were affinity-purified using recombinant PfDnaJint and

immobilised on nitrocellulose membrane as follows.

Purified recombinant PfDnaJint protein was electrophoresed on a 10%

SDS-PA gel and transferred onto nitrocellulose membrane. After

visualization by Ponceau stain, the band of interest was excised from the

blot and incubated overnight at 4°C in blocking solution (5% skimmed

milk in IX PBS). The blot was washed thoroughly with PBS-T (0.05% v/v

Tween-20 in IX PBS) and incubated for 2 h at room temperature in anti~

rabbit PfDnaJint serum collected after the first booster dose (1 ml serum in

10 ml IX PBS + 2% BSA). The blot was washed five times with PBS-T, cut

into small pieces and put in 2 ml micro-centrifuge tube. Antibody bound

to PfDnaJint on nitrocellulose membrane was eluted by addition of 500 /JI

64


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

was taken and immediately neutralized by the addition of 125 1-11 1 M Tris,

pH 8.0 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 thio-cyanate buffer (3 M K4SCN, 150 mM KCl, 50 mM

Tris, pH 8.0). Individual aliquots of each elution step were checked on

10% SDS-PA gel and stored separately at 4°C (Smith and Fisher, 1984).

2.2.4.6. Specificity of raised antibodies

2.2.4.6a. Preparation of P.falciparum, RBC and E.coli cell lysate

P.falciparum lysate: For preparation of total parasite lysate, parasites at

mixed stages were released by 0.05% saponin lysis, washed three times

with ice-cold IX PBS, suspended in 20 1-1-1 of 5X SDS loading buffer (25

mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 0.3% w/v bromophenol blue,

10 mM 13M E) and sonicated giving 5 pulses for 10 seconds at 10% duty

cycle. After sonication, 80 1-1-1 deionized water was added so as to make up

the final concentration to IX SDS lysis buffer.

E.coli lysate: 3 ml of E.coli TG-l cells were grown overnight and pelleted

at 12,000 rpm for 1 min. The pellet was resuspended in 5X SDS lysis

buffer, sonicated briefly and adequate amount of deionized water was

added to make final concentration to IX SDS lysis buffer.

RBC lysate: Human RBC lysate was prepared by hypotonic lysis in

deionized water. Briefly, the RBC pellet was washed three times with ice

cold physiological saline (0.9% NaCl). RBCs were lysed by the addition of 4

packed cell volume of ice-cold deionized water and kept for 10 mm.

Supernatant was taken after centrifugation at 3,000 rpm for 10 min.

2.2.4.6b. Western blotting

The P.Jalciparum, E. coli and RBC cell lysates prepared as described

above were separated on a 10% SDS-polyacrylamide gel. Proteins were

transferred onto nitrocellulose membrane and processed for western

blotting using standard protocol. The rabbit anti-PfDnaJint serum was

65


used as primary antibody and sheep anti-rabbit HRP conjugate (Sigma)

was used as secondary antibody. The blot was developed using a

chemiluminescent detection system (Amersham Biosciences).

2.2.4.7. Oligomeric status of PfDnaJint protein

The dimeric and/or oligomeric status of PfDnaJint protein was

determined by the chemical cross-linking as well as by size-exclusion

chromatography.

2.2.4.7a. Chemical crosslinking by glutaraldehyde

-150 J..lg of Ni-NTA purified PIDnaJint was treated with 100 J..lI of 25%

glutaraldehyde solution to make a final concentration of 1%

glutaraldehyde. This sample was incubated at 25°C for 1 min followed by

quenching of the cross-linking reaction by addition of 2 J..lI of f3-ME. After

20 min incubation, 3 J..lI of 10% aqueous sodium deoxycholate solution

was added. The pH of the reaction mixture was lowered to 2-2.5 by the

addition of ortho-phosphoric acid (85%), which resulted in precipitation of

the cross-linked product. The sample was centrifuged at 12,000 rpm for

30 min at 4°C. The resultant precipitate was dissolved in 0.1 M Tris-CI,

pH 8.0. The sample was heated at 100°C for 1 min and analysed on 8%

SDS-PA gel along with protein marker.

2.2.4.7b. Size-exclusion chromatography

Gel filtration experiment was carried out on a Superdex 200HR 10/300

column (manufacturer's exclusion limit 600 kDa for proteins) on AKTA

FPLC (Amersham Biosciences). The column was calibrated with various

molecular weight standard markers (Amersham Biosciences). The column

was equilibrated and run with buffer containing 10 mM HEPES, pH 8.0

and 200 mM NaCl. 600 J..lL (1 J..lg/J..lI) of purified PIDnaJint protein was

loaded on the column and run at 250C at a flow rate of 0.3 ml/min with

detection at 280 nm.

66


2.2.4.8. Immuno-fiuorescent labeling and localization of PIDnaJ Mixed stage P.Jalciparum culture was processed for immuno

fluorescence labeling and confocal microscopy according to the method of

Tonkin et al., 2004). Cells were washed with IX PBS and flxed in solution

containing 4% paraformaldehyde and 0.0075% glutaraldehyde in IX PBS

for 30 min. After one wash with PBS, fIxed cells were permeabilized with

0.1% TritonX-100/PBS for 10 min. After another PBS wash, cells were

treated with sodium borohydride (0.1 mg/ml) in PBS for 10 min. Cells

were washed once with PBS and blocked in 3% BSA/PBS for 1 h. Cells

were incubated overnight with anti-rabbit PfDnaJint serum (1 :50 dilution

in PBS containing 3% BSA) at 4°C. After three washes (10 min for each

wash) with PBS, the cells were incubated with Alexaflour-tagged goat anti

rabbit secondary antibody (Molecular Probes) for 2 h at room temperature

and allowed to settle onto coverslip coated with poly-L-Iysine (100 ~g/ml).

The coverslips were then washed three times with PBS and mounted in

anti-fade mounting media (Oncogene). For mitochondrial labeling, live

cells were incubated in 25 ng/ml MitoTracker deep Red 633 (Molecular

Probes) in PBS for 20 min at 4°C prior to fIxation. All slides were viewed in

a confocal laser-scanning microscope (Biorad Radiance 2000) under a 60X

l.4NA oil immersion lens.

For co-localization, apicoplast targeted ACP-GFP transfected P.Jalciparum

strain (D 10 ACP(leader)-GFP; ATCC number: MRA-568) was used. The

immunoflorescence labeling of the parasites was carried out by the

method as describe above except the primary and secondary antibodies

used. Primary antibodies were purifIed rabbit anti-PfDnaJint antibody

(1:50 dilution in Ix PBS) and monoclonal anti-GFP antibody, Roche (1:100

dilution in Ix PBS). The secondary antibodies were Cy5-tagged goat anti

rabbit antibody and Oregon green-tagged goat anti-mice antibodies (1:300

and 1:2000 dilution in Ix PBS, respectively). DAPI (20 llg/ml) was added

along with secondary antibodies to counterstain the nuclei. All slides were

examined in a fluorescence microscope as well as in a confocal laser

scanning microscope (Leica).

67

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