Protocol 22 GSTBcl2 1218 protein

Method

11. Inoculate a colony of XL-1 blue cells carrying the pGEX-4T1-Bcl-2 (1218) plasmids into 1 litre of Luria-Bertani (LB) medium containing 100 (jig/ml ampicillin and grown at 37°C overnight with moderate agitation.

12. Dilute the culture by half in fresh LB medium containing 100 (xg/ml ampicillin and allow to cool to room temperature for 1 h.

13. Induce GST-Bcl-2 protein production by addition of 0.4 M IPTG (isopropylthio-p-D-galactoside) to medium at 25°C from 6 h to overnight.

14. Recover cells by centrifugation using a Sorvall GSA rotor at 4000 <7 for 10 min. Store cells at -20°C until use. The expression level is monitored by SDS-PAGE (100 jjJ of the culture is resuspended in 25 |xl Laemmli-SDS sample buffer, and 12.5 iaI is loaded into a 15% SDS-PAGE minigel to verify protein induction: 1 jjig induced band equates to «=20 mg/l protein).

15. Resuspend cells in 50 ml of 50 mM Tris, pH 8.0, 150 mM NaCI, 1% Tween, 0.1% 2-mercaptoethonal, 5 mM EDTA, complete protease inhibitor set (Boerhinger 1697498), 1mM 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 27500 g for 10 min and collect both supernatant and pellet.

18. To purify the soluble GST-Bcl-2 found in the supernatant, incubate the cell lysate with glutathionine resin (Pharmacia) at 4°C overnight. Samples should be tumbled gently during incubation in a 50 ml polypropylene centrifuge tube.

19. Pack beads in a column and wash it with 20 mM Tris, pH 8.0, 150 mM NaCI, 0.1% Tween, 0.1% 2-mercaptoethanol until the OD28o (optical density at 280 nm) is less than 0.01.

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

11. Elute GST-Bcl-2 with 2 vols of washing buffer containing 20 mM glutathionine. Alternatively, the beads are 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-Bcl-2 is collected after centrifugation at 760 g for 5 min.

Protocol 23. Hisg-Bcl-2 (1-218)

Method

11. Inoculate a single colony of cells into 1 litre of LB medium containing 100 jig/ml ampicillin and grow at 37°C overnight in a 2 litre Erlenmeyer flask with vigorous agitation.

12. Dilute the culture by half in fresh LB medium containing 100 ng/ml ampicillin and allow to cool to room temperature (RT) for 1 h.

13. Induce HiSs-Bcl-2 protein production using 1 mM IPTG for 6 h at RT, with vigorous agitation.

14. Recover cells by centrifugation in a GSA rotor at 4000 g for 10 min. Store cells at -20°C until use. The expression level can be monitored by SDS-PAGE (see Protocol 22).

15. Resuspend cells in 50 ml of 50 mM phosphate buffer, pH 8.0,150 mM NaCI, 1% Tween, complete protease inhibitor set (Boerhinger 1697498), 1mM 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 gfor 10 min and collect the pellet.

18. Wash the pellet by resuspending and then centrifuging three times in 200 ml of 50 mM phosphate buffer, pH 8.0, 150 mM NaCI, 1% Tween to remove soluble contaminant proteins.

19. Resuspended pellet in 10 ml water by sonication. Add 8 M GuHCI and 1 M phosphate stock solution to give final concentrations of 6 M GuHCI, 50 mM phosphate. Adjust pH to 6.8 with NaOH.

10. Centrifuge at 27 500 grfor 10 min and collect supernatant.

11. Add nickel resin at ~6-8 mg His6-Bcl-2 per 1 ml resin. Add imidazole at 25 mM final concentration. Adjust the pH to 6.8.

12. Incubate at 4°C from 3 h to overnight.

13. Pack beads in a column and wash it with 50 mM phosphate buffer, pH 6.8, 4M GuHCI, 25 mM imidazole until OD280 is less than 0.01.

14. Elute His6-Bcl-2 protein with 2 X resin volume of 0.2 M acetic acid, 4 M GuHCI.

15. Dialyse the eluted HiSe-Bcl-2 against 100 X volumes of cold 25 mM acetic acid, 1mM EDTA, 0.1% 2-mercaptoethonal at 4°C for 4 h, three times.

16. Store protein at -20°C until use.

17. The eluted sample is generally >90% pure. It can be further purified by gel filtration under denaturing conditions (4 M GuHCI) or, after refolding (step 15), by ion-exchange chromatography using acidic pH solution (Mono S HR10/10 at pH 4.5) (Pharmacia).

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