PROCEDURE
1. In a 125 mL flask containing 10 mL of LB and 10 mL ofAmpicillin (100 mg/mL -for pGEX vectors, Life Technologies), transfer a single colony and Grow overnight
2. Transfer 5 mL of ON culture to a 250 mL flask with 45 mL of LB and 50 mL of Ampicillin (100 mg/mL)
3. Incubate at 28-37oC until A600 reaches 0.7 (best) - 0.8 (up to ~1.0). Induce with 10 mL of 1M IPTG/50 mL culture*. Incubate at 28oC (can also try at 37oC) for >2 hours at 250 rpms (longer incubations are OK if the GST-protein is stable and/or growing in BL21-DE3 proteolysis- cells)
*NOTE: varying the concentrations of IPTG may increase the yield of your protein (5 - 25mL of 1M IPTG/50 mL culture)
NOTE: growth of the expressed protein at 28oC after induction is supposed to reduce aggregation of the fusion protein
4. Centrifuge at 8,000 g for 10 mins at 4oC and discard the supernatant
Note: the pellet can be stored at -80oC for weeks
5. Resuspend pellet with 10 mL of cold buffer + 10 µL of 100 mM PMSF at 4oC.
BEST: in addition to the PMSF, other protease inhibitors should be added. A good cocktail of protease inhibitors is Complete (from Roche). This cocktail comes as a tablet that can be dissolved in 10 mL of buffer and provides good inhibition against 5 of the main proteases (at pH 7.8)
6. Lyze cells by 2-3 passages through a French press (4oC) at a pressure no lower than 1100 psi (keep pressure around 1100-1200 rpms) (cell lysis can be detected by clearing of suspension unless a high concentration of inclusion bodies is present)
NOTE: other means of cell lysis, like sonication, are not as good as a French press. In my hands, purifying the proteins I have purified, French-pressed samples yield more purified protein when compared to other extraction procedures. In a study I performed, 10 times more protein was obtained using a French-pressed sample as compared to a sonicated one
7. Add 0.5 mL of 20% Triton X-100/10 mL of lysate (to 1%Triton X-100). Mix on a rocker for 30-60 mins at 4oC (longer incubations may reduce the amount of inclusions bodies)
8. Centrifuge at 12,000 g for 20 minutes at 4oC and save the supernatant (can be stored at -80oC). This is termed total soluble protein = T
Optional: Inclusion Body (IB) proteins can be solubilized. Add 1 mL/50 mL culture pellet of IB buffer, sonicate (2-4x for 10-15 secs in a water bath somicator), resuspend and mix on a shaker for >30-60 mins at 4oC. Centrifuge at 12,000 g for 15 mins and save IB supernatant
9. Equilibrate a 50% slurry of Glutathione Sepharose 4B (Pharmacia Biotech, catalog # 27-4574-01) with PBS for 10 mins, spin for 2 mins at 500 g and discard wash. Add 10 mL of T / 2 mL of 50% slurry
Glutathione Sepharose 4B: store in 20% ethanol. 200-400 mmol Glutathione Sepharose per g dried substance. Spherical beads, 45-165 mm wet diameter. Spacer arm: 12 atoms (10 carbons)
10. Rock for >20 minutes at RT
11. Centrifuge at 500 g for 1 min. Save an aliquot of solution (Flow Through = FT)
12. Aspirate off supernatant withough getting too close to the beads and wash 4 times with 1X PBS/Wash solution each for <1 minute, namely invert the tube until the beads are resuspended (centrifuge for 2 mins at 500 g to pellet beads).
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