The expression of Bid in bacteria is easily achieved. The recombinant BID protein is highly soluble at both neutral and acidic pH (37). The only problem associated with purification of Bid is that it is susceptible to digestion by thrombin. Therefore, performing partial digestions of GST-Bid is advised to reduce the degradation of Bid by thrombin. We typically digest GST-Bid under the same conditions as those described above for other Bcl-2 family proteins, except the digestion is stopped after 1-2 hours by addition of PMSF. The eluted protein is then dialysed against 50 mM acetate buffer, pH 4.8, 1 mM EDTA, 0.1% 2-mercaptoethonal, and Mono S ion-exchange chromatography is performed. Bid elutes at very low salt concentrations (—50 mM
NaCl), whereas the degraded contaminant elutes at high concentrations of salt. The recombinant purified Bid protein exists as monomeric protein at neutral and acidic pH (37).
Protocol 27. GST-Bid/Bid protein
11. Inoculate a colony of XL-1 blue cells carrying the pGEX-4T1-Bid plasmid into 1 litre of terrific broth medium supplemented with 1-2% glycerol and containing 0.5 mg/ml cabenicillin. Grown at 37°C overnight with moderate agitation.
12. Allow the culture to cool to room temperature for 1 h. Add 0.4 mM IPTG and grow at room temperature for 6 h with moderate agitation.
13. Recover cells by centrifugation in a GSA rotor at 4000 g for 10 min. Store cells at -20 °C until use. The expression level is monitored by SDS-PAGE (see Protocol 22).
14. Resuspend the cell pellet in 50 ml of 50 mM Tris, pH 8.0, 150 mM NaCl, 1% Tween, 0.1% 2-mercaptoethonal, 5 mM EDTA, complete protease inhibitor set (Boerhinger 1697498), 1 mM PMSF. Lyse cells with 0.5 mg/ml lysozyme at room temperature for 0.5 h.
15. Sonicate on ice until viscosity is minimal.
16. Centrifuge samples at 27 500 g for 10 min. Collect the supernatant and discard the pellet.
17. Incubate the cell lysate with glutathionine resin (Pharmacia) at 4°C overnight. The amount of resin is based on the expression level. Use =2-4 mg protein per 1 ml beads). Samples should be tumbled gently during incubation in a 50 ml polypropylene centrifuge tube.
18. Pack beads in a column and wash with 20 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween, 0.1% 2-mercaptoethanol until OD28o is less than 0.01.
19. Wash beads again with 10 vols of the same buffer without detergent.
10. Elute GST-Bid with 2 x resin volumes of washing buffer containing 20 mM glutathionine. Alternatively, beads are collected and resus-pended into a 50% (v/v) slurry in washing buffer containing 20 mM glutathionine in a 50 ml polypropylene centrifuge tube, incubated at 4°C for 1 h, and the supernatant containing eluted GST-Bid is collected after centrifugation at 760 gfor 5 min.
11. If the GST group will be removed by proteolysis, incubate the resin after step 9 (above) with thrombin (Boerhinger) at 4°C in 20 mM Tris, pH 8.0, 150 mM NaCl, 0.1% 2-mercaptoethanol( 0.1% Tween, 2.5 mM
CaCI2 for 2 h, and stop the reaction by adding 1 mM PMSF and 5 mM EDTA. For 50 ml of 50% slurry, 23 mg thrombin is used. Elute Bid with 2 x resin volumes of the same buffer.
12. Dialyse the eluted Bid against 100 vols of 50 mM acetate buffer, pH 4.8, 1mM EDTA, 0.1% 2-mercaptoethonal at 4°C overnight. Inject into a Mono S column (Pharmacia, HR10/10) that has been equilibrated with dialysis buffer using a 2 ml/min flow rate. Wash with 50 ml of the same buffer at 2 ml/min. Elute Bid with a linear gradient of 0-500 mM NaCI in 60 min at 2 ml/min. Collect the samples in 1.5 ml and monitor purity on 15% SDS-PAGE.
13. Pool pure fractions and store at -20°C at a concentration of »1 mg/ml.
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