Paul.xu at 20:02, 26 February 2013

2013-02-26T20:02:19Z

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1. Freshly transform the 1613 pET28-cold-GST-3C construct into E. coli BL21 Star (DE3) pRARE2-pLysS. Plate out on LB-agar containing Kan (50 μg/ml) and chloramphenicol (33 μg/ml). Incubate overnight at 37°C.

 2. Pick 3 colonies from the plate to inoculate 100 ml LB medium containing Kan (50 μg/ml) and chloramphenicol (33 μg/ml). Incubate overnight at 37°C in a shaker/incubator.

 

3. Use the overnight cultures to inoculate 8 x 1 L LB medium containing Kan (50 μg/ml) and chloramphenicol (33 μg/ml) in 2 L culture flasks.

 

4. Grow the cultures at 37°C until the OD600 is approx. 1 to 1.5. Then lower the temperature of the shaker/incubator to 20~25 °C, add 1 M IPTG to 0.1 M final conc, and incubate the cultures overnight.

 

5. Harvest the cells by spinning for 5 min at 6,000 rpm. Weigh pellet.

 6. When not immediately used store the pellet at -20°C.

 7. Resuspend the pellet in 15 ml/g wet pellet Lysis Buffer

 8. Freeze-thaw at least 3 times to lyse the cells.

 9. Break DNA using a homogenizer.

 10. Spin the cell lysate for 20 min at 12,000 rpm using a SS-34 rotor at 4 oC. Collect supernatant. Wash pellet with 10 ml lysis buffer per gram of pellet, homogenize and spin again. Collect and combine supernatant.

 12. Spin supernatant at 25000 rpm for 15~25 min, 4 oC, using ultracentrifuge (be very careful).

 13. Combine and apply the supernatant to 15~25 ml Ni column. Collect flow through. Apply flow through to the column again.

 14. Wash column with 400 ml washing buffer 1.

 15. Elute protein with 12 X 5 ml elution buffer 1 (Buffer B, with 1 mM b-ME, 300 mM imidazole).

 16. Load eluate to 10-ml glutathione-agarose column.

 17. Wash with 150 ml PBS or 20mM Tris-HCl, with 150 mM NaCl, pH 7., until no protein is  eluted (monitored using OD280). 

 18. Elute GST-3C with 10 mM reduced glutathione in 20 mM TBS 20mM Tris-HCl, 150 mM NaCl, pH 7.5 (elution buffer 2)

 19. Collect the eluate in 2 ml fractions. Measure protein conc in each fraction. Combine fractions containing GST-3C. Run SDS-PAGE to check purity, if not pure, purify further with TSK gel filtration column after concentrating using spin column (step 20). 

 20. Concentrate protein using Pierce 10kDa spin column, spin at 2500 rpm for 45 min, at 4 °C cold room.

 21. Transfer concentrated protein solution to a dialysis tube, dialyze overnight against 40 ml storage buffer at 4°C.

 22. Dialysis again with 40 ml Storage Buffer at 4°C.

 23. Measure protein concentration, adjust DTT concentration to 1 mM final conc using 1 M or 2 M DTT. Transfer 1.5 ml to microcentrifuge tubes, print label (name, clone id, conc, lot number), store the aliquots at -80°C. ''' '''

'''Lysis Buffer''' 

50 mM Tris-HCl pH 8.0 300 mM NaCl 10% (v/v) glycerol 0.2% (v/v) NP-40 

10 mM Imidazole 1 mM b-ME 

 

'''Wash Buffer ''' 

20 mM Tris-HCl pH 8.0 300 mM NaCl 

10 mM Imidazole 5% (v/v) glycerol (optional) 1 mM b-ME 

 

'''Elution Buffer 1''' 

20 mM Tris-HCl pH 8.0 300 mM NaCl 300 mM Imidazole 

10% (v/v) glycerol 1 mM b-ME 

 

 

'''Elution Buffer 2''' 

50 mM Tris-HCl pH 8.0 300 mM NaCl 10 mM reduced glutathione 10% (v/v) glycerol 1 mM b-ME

 

'''Storage Buffer''' 

50 mM Tris-HCl, pH 8.0 150 mM NaCl 10 mM EDTA 50% (v/v) glycerol 0.1 mM DTT

GST-3C Protease Purification - Revision history

Paul.xu at 20:02, 26 February 2013