Protocol 26 GSTBax 1171 protein

Method

11. Inoculate a colony of XL-1 blue cells carrying the pGEX-4T1-Bax (1171) plasmid into 1 litre of terrific broth medium supplemented with 1-2% glycerol containing 0.5 mg/ml cabenicillln, and grown at 37°C overnight with moderate agitation.

12. Allow the culture to cool to room temperature for 1 h, and add 10 jjiM IPTG.

13. Grow at room temperature for another 24 h with moderate agitation.

14. Recover cells by centrifugation in a GSA rotor at 5000 r.p.m. for 10 min. Store cells at-20°C until use. The expression level is monitored by SDS-PAGE (see Protocol 22).

15. Resuspend cells In 50 ml of 50 mM Trls, 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.

16. Sonicate on ice until viscosity is minimal.

17. Centrifuge samples at 27 500 g for 10 min and collect the supernatant. Also, save the pellet if planning to attempt solubilization In 6 M GuHCI and refolding as described above.

18. Incubate cell lysate with glutathionine resin (Pharmacia) at 4°C overnight. The amount of resin is based on the expression level as determined above. Using «=2-4 mg proteins per 1 ml beads. Samples should be tumbled gently during incubation in a 50 ml polypropylene centrifuge tube.

19. Pack beads in a column and wash with 20 mM Tris, pH 8.0, 150 mM NaCI, 0.1% Tween, 0.1% 2-mercaptoethanol until OD28o is less than 0.01.

10. Wash beads again with 10 vols of the same buffer without detergent.

11. Elute GST-Bax with 2 vols of washing buffer containing 20 mM glutathionine. Alternatively, beads can be collected and resuspended 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-Bax is collected after centrifugation at 2000 r.p.m. for 5 min.

12. 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 NaCI, 0.1% 2-mercaptoethanol, 0.1% Tween, 2.5 mM CaCI2 overnight. For 50 ml of 50% slurry, 23 mg thrombin is used. Elute Bax with 2 x resin volumes of the same buffer.

13. Dialyse the eluted Bax protein against 100 vols of 10 mM Tris-HCI, pH 8.0, 1 mM EDTA, 0.1% 2-mercaptoethonal at 4°C for 3 h. Inject into a Mono Q column (Pharmacia, HR10/10) that has been equilibrated with dialysis buffer, using a 2 ml/min flow rate. Wash the column with 50 ml of same buffer at 2 ml/min. Elute Bax with a linear gradient of 0-500 mM NaCI in 60 min at 2 ml/min. Collect the samples in 1.5 ml fractures and monitor purity on 15% SDS-PAGE.

14. Pool pure fractions and store at -20°C at a concentration of =«0.2 mg/ml.

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