PREPARATION AND ANALYSIS OF PROTEIN

2.4.1 Preparation of protein extracts

Preparation o f T. cruzi protein. To obtain whole cell lysates, 2 x 101 T. cruzi cells were lysed with 200 pi of protein sample buffer (0.4 M Glycine, 2% (w/v) SDS, 5% (w/v) 2-mercaptoethanol, 0.1% bromphenol blue), vortexed and boiled for 5 min.

Samples were stored at -20°C. Alternatively, T. cruzi cells were lysed in protein lysis buffer N (1% (v/v) Nonidet P-40, 50 mM Tris pH 8.0, 150 mM NaCl, Roche Mini- Complete™ protease inhibitors), 30 min on ice. Lysates were centrifuged in a micro- fuge at 13,000 rpm for 5 min to separate the soluble and insoluble fractions. To assess the effect o f salt concentration on MET3 protein solubility, the NaCl concen­

tration in protein lysis buffer N was adjusted as required.

Preparation o f extracts fo r detection o f phosphorylated proteins (adapted from Mynott et al., 1999). 1.5xl08 T. cruzi cells were collected by centrifugation at 1640 g for 5 min. The cell pellet was washed once in PBS, suspended in 700 pi protein lysis buffer T, containing phosphatase inhibitors and protease inhibitors (25 mM Tris pH 7.4, 75 mM NaCl, 0.4 mM EDTA, 0.5% Triton X-100, 0.4 mM sodium ortho­

vanadate, 10 mM Na F, 10 pg ml"1 leupeptin, 740 pm PMSF, 0.7 pg ml'1 pepstatin A, 1 pg ml'1 E64, 100 pM TLCK) and incubated on ice for 30 min until lysis was complete. The suspension was centrifuged in a microfugc at 13,000 rpm for 20 min at 4°C. The resulting supernatant (soluble fraction) was rapidly frozen in liquid nitrogen and stored at -80°C. The pellets (insoluble fraction) were stored at -20°C.

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Preparation o f protein extracts fo r detection o f cruzipain activity. Parasites were collected by centrifugation at 1640 g at 4°C for 10 min. The cell pellet was washed once in PBS, solubilised at lx 108 cells ml'1 in TBS/ 1% (v/v) Nonidet P-40 and vortexed rapidly for 15 sec. The suspension was centrifuged in a microfuge at 13,000 rpm for 3 min at r/t and the resulting supernatant was rapidly frozen in liquid nitro­

gen and stored at -20°C (Greig and Ashall, 1990).

Determination o f protein concentration. The protein content of parasite extracts was estimated using the BCA protein assay system (Pierce), using bovine serum albumin (BSA) as a standard and performed according to the manufacturer's instructions.

2.4.2 Expression of recombinant MET2 and MET3 in E. coli

100 ml of an overnight culture of E. coli BL21 pTrcHis-met2 or E. coli BL21 pTrcHis-met3 in NZCYM containing 100 pg ml"1 ampicillin (NZCYM-amp) was diluted 1:10 into 1 litre NZCYM-amp, and incubated at 37°C with shaking for 1-3 hours. Expression was induced by adding IPTG to a final concentration of 1 mM and the culture was incubated for an additional 2 hours (for MET2 expression) or over­

night (for MET3 expression). The cells were collected by centrifugation in a Beck­

man J2-21 centrifuge at 4,000 rpm for 20 min. The cell pellets were frozen at -80°C for at least 30 min prior to lysis.

2.4.3 Purification of recombinant MET2 and MET3 from E. coli

For preparation of cleared lysates under native conditions, cell pellets were lysed for 30 min on ice in lysis buffer (50 mM NaHjP04 pH 8.0, 300 mM NaCl, 10 mM imi­

dazole, 1 pg ml'1 lysozyme, 1% Triton X-100, Roche Mini Complete™ protease inhibitors, EDTA-free), before sonication on ice for six 10-second bursts, with a 10- second cooling period between each burst. The lysate was centrifuged in a Beckman J2-21 centrifuge at 12,000 rpm for 20 min at 4°C. 2 ml of 50% Ni-NTA slurry (Qiagen) was added to the cleared lysate and mixed by shaking at 4°C for at least one hour. The lysatc-Ni-NTA mixture was loaded into a column and the flow­

through collected. The column was washed with 4x 5 ml wash buffer (50 mM

Purified 6xHIS-MET3 or 6xHIS-MET2 protein was excised from an SDS-PAGE gel, placed in a pestle and mortar and pulverised in the presence of liquid nitrogen. 1 ml Freund’s complete adjuvant was added and the sample homogenised by passage through a syringe needle. Immediately before use, the sample was sonicated for five 5-second blasts. Approximately 150 pi of this suspension was injected intra- peritoneally into each of three Balb/c mice, which had previously been pre-bled from a tail vein. This was done by Michael Miles at LSHTM. After 2 weeks, another protein sample was prepared in the same way, except that Freund’s incomplete adju­

vant was used, and administered to the mice. This was repeated twice more at two week intervals. Blood samples were left to clot at 4°C overnight, then centrifuged for 10 min at 13,000 rpm in a microfuge. The serum supernatant was stored in 50%

glycerol, 0.02% sodium azide at -20°C.

2.4.5 Electrophoresis and blotting of proteins

SDS-PAGE. Parasite extracts were separated by discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a Mini-PROTEAN 3 electrophoresis cell (Bio Rad) according to standard procedures. 10% or 12% polyacrylamide Ready Gels (Bio Rad) were used as required. Samples were mixed with sample buffer (0.4 M Glycine, 2% (w/v) SDS, 5% (w/v) 2-mercaptoethanol, 0.1% bromphenol blue) and boiled for 5 min prior to loading. Protein from 2 x 10h cells was loaded per lane.

Precision protein standardsIM (Bio Rad) were loaded in parallel. Electrophoresis was performed at 100 V in lx running buffer (25 mM Tris/ 0.25 M glycine/ 0.1% (v/v) SDS). Gels were stained in 0.25% (w/v) Coomassie Brilliant Blue R 250/ 45% (v/v)

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methanol/ 45% (v/v) ddH20/ 10% glacial acetic acid and destained in 5% (v/v) methanol/ 7.5% (v/v) glacial acetic acid or by boiling in ddH20.

Western Blotting. Proteins separated by SDS-PAGE were transferred to Protran*- nitrocellulose membranes (Schleicher and Schuell), in a Trans-Blot Semi-Dry Elec­

trophoretic Transfer Cell (Bio Rad) according to the manufacturer's instructions, using Bjerrum and Schaefer-Nielsen transfer buffer (48 mM Tris, 39 mM glycine, 20% (v/v) methanol). Blotting was carried out at 15 V for 1 hour. Membranes were blocked in TBS/ 5% (w/v) milk powder for 30 min. Antibodies were diluted in TBS/

5% milk powder and incubated at r/t for one hour. Unbound antibody was washed off with three changes of TBS. Primary antibodies were anti Xpress™ (Invitrogen), diluted 1:5,000; mouse anti c-myc (9E10) (Santa Cruz Biotechnology), diluted 1:1,000; mouse antiserum to MET3 (section 2.4.4), diluted 1:500; P-tubulin antibody KMX1 (a gift from K. Gull), diluted 1:500. Secondary antibodies were goat anti­

mouse IgG horseradish peroxidase conjugate (Bio Rad), diluted 1:3,000. The blot was incubated with ECL Plus chemiluminescence substrate (Amersham Pharmacia Biotech) for 5 min prior to exposure to X-ray film for 1-5 min.

For detection of phosphoproteins, free binding sites on the membrane were blocked with Blotto B (TBS/ 1% (w/v) BSA/ 1% (w/v) milk powder) at r/t for at least 1 hour or at 4°C overnight. The following antibodies were used: Rabbit anti-Phosphoserine (PS) and Rabbit anti-Phosphothreonine (PT), polyclonal, 0.2 mg ml'1 (Zymed Labo­

ratories Inc.), and Mouse Anti-Phosphotyrosine (4G10), monoclonal, 1 mg ml'1 (Upstate Biotechnology UK). The relevant antibody was diluted 1:500 in TBS/ 1%

BSA and added to the membranes. Membranes were incubated either at r/t for one hour, or at 4°C overnight, with gentle agitation. Unbound antibody was washed off with three changes of TBS. The blots were then incubated as required with goat anti­

rabbit or goat anti-mouse IgG horseradish peroxidase conjugate (Bio Rad) diluted 1:3,000 in Blotto B for 1 hour at room temperature, and washed as above prior to chemiluminescence detection.

2.4.6 Immunofluorescence and confocal microscopy

T. cruzi cells were fixed in suspension with 2% paraformaldehyde (PFA) in PBS for 20 min at r/t. Fixed cells were pelleted and washed twice with PBS and re-suspended in PBS. 20 pi cell suspensions were spotted onto multitest glass slides (ICN Bio­

bodies were diluted in wash buffer/ 2% horse serum. Slides were incubated with the primary antibody for 45 min, and with the secondary antibody for 30 min, and rinsed in three changes of wash buffer. Primary antibodies were mouse anti c-myc (9E10) (Santa Cruz Biotechnology), diluted 1:200; mouse antiserum to MET3 (section 2.4.4), diluted 1:400; L1C6 monoclonal antibody (K. Gull, unpublished), diluted 1:200. Secondary antibodies were AlexaFluor 488- and 546-conjugated anti-mouse or anti-rabbit antibodies (as appropriate), all diluted 1:400. DNA was stained with 1 pM TOTO-3 (Molecular Probes) in PBS/ 0.1% saponin/ 10 mg ml'1 RNase A, for 20 min. Slides were washed twice in PBS and mounted in PBS/ 50% glycerol and viewed under a Zeiss LSM 510 axioplan confocal laser scanning microscope.

2.4.7 Protease assay

Detection o f cruzipain activity in gelatin gels. For detection of protease activity, 10%

polyacrylamide gels were prepared according to standard procedures, but 0.2% (w/v) gelatin was co-polymerised with the resolving gel (Greig and Ashall, 1990). Samples were diluted in sample buffer (0.4 M glycine, 2% (w/v) SDS, 0.1% bromphenol blue) without 2 -mercaptoethanol (2-Me) and were not boiled. Electrophoresis was at 100 V for 2 to 3 hours. After electrophoresis, gels were shaken in 200 ml o f 2% (v/v) Triton X-100 for 1 hour for SDS removal. For protease detection, gels were incubated in digestion buffer (50 mM sodium citrate, 50 mM HEPES, 50 mM

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sodium phosphate, 50 mM sodium Borate (Borax); pH 5.6) with 26 nM 2-Me for 18 hours at 37°C. Coomassie Blue staining was carried out as described in section 2.4.5 except that for destaining 10% (v/v) glacial acteic acid was used. Gels were photographed on a light box.