Bacterial Expression of GST-fusion Proteins
1. Grow cells in 5ml (or more) of LB-Amp overnight for a starter culture.
2. Grow larger culture (100x volume of starter culture) using the overnight culture as a seeding culture. The culture is grown in LB-Amp medium. Aerate well with shaking at 37o C until OD at 600nm is ~0.6 (usually about 4 hrs).
3. Add the appropriate amount of IPTG stock solution to the culture to obtain a final IPTG concentration of ~2mM. (Add 4mls water to an unopened bottle= 1M. Induce 1L culture with 2mls.)
4. Continue shaking for 4-5 hours. (keep in mind that some proteins get insoluble or degraded if induction goes too long).
5. Spin down cells and resuspend pellet in 1/100 initial volume of culture into lysis buffer -- PBS-CMF, 2mM EDTA, 10mM DTT, PMSF, benzamidine, and 0.5M NaCl.
6. Cells are lysed by sonicating 3x15 seconds at 4o C.
7. Centrifuge at 20 - 30 K rpm in ultracentrifuge at 4o C for 30 min.
8. Mix supernatant with 3 ml/L glutathione Sepharose 4B which has been washed with PBS-CMF, 10mM DTT, and 0.5M NaCl.
9. Rotate the filtered supernatant/sepharose at 4oC for 30-60min.
10. Spin, remove liquid and wash the sepharose 2x with PBS-CMF, 10mMDTT, 0.5M NaCl. Beads can be stored at 4oC overnight and/or test an aliquot on a protein gel.
11. Elute the fusion protein from the sepharose 3x with 10mM glutathione, 50mM Tris pH8.0, 10mM DTT, and 0.5M NaCl.
12. Dialyze fusion protein against 50mM Tris pH8.0, 10mM DTT, 0.5M NaCl.
Lysis Buffer 50mls
PMSF 35mg/ml in EtOH 500ml
Benzamidine 40mg/ml 500ml
DTT (1M stock) 500ml
EDTA (0.5M stock) 200ml
NaCl (5M stock) 5ml
Make up the rest of the volume with PBS-CMF