Bacterial Expression of IRS-1 containing
GST-fusion Proteins
1. Grow cells in
5ml (or more) of LB-Amp overnight for a starter culture.
2. Grow larger
culture using the overnight culture as a seeding culture. The culture is grown in LB-Amp medium. Aerate well with shaking at 37 C until OD at
600nm is ~0.6.
3. Add the
appropriate amount of IPTG stock solution to the culture to obtain a final IPTG
concentration of ~2mM.
4. Continue shaking for several hours up to overnight.
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 sonication at 4 C.
7. Add Triton
X-100 to 1% final concentration.
8. Centrifuge at
20 - 30 K rpm in ultracentrifuge at 4 C for 30 min.
9. Filter sample
through a 0.45m pore size membrane. Mix this
filtered supernatant with glutathione Sepharose 4B which has been washed with
PBS-CMF, 10mM DTT, 0.5M NaCl.
10. Rotate the filtered supernatant/sepharose at
4 C for 30-60min.
11. Spin, remove liquid and wash the sepharose
2x with PBS-CMF, 10mMDTT, 0.5M NaCl.
12. Elute the fusion protein from the sepharose
3x with 10mM glutathione, 50mM Tris pH8.0, 10mM DTT, 0.5M NaCl.
13. Dialyze fusion protein against 50mM Tris pH8.0, 10mM DTT, 0.5M NaCl.