Q: fusion protein expressin

Zhao Zhuoshen Mr mcbzzs at nuscc.nus.sg
Thu Dec 17 00:29:37 EST 1992

Dear fellow-netters,
I have had difficulty to express my gene(1.2kb) using GST-fusion protein
vector(P^GEX-2T) from pharmcia. What I did is as following:

1. construct the plasimd(P^GEX-2T with 1.2kb inframe insert), transform
it into E.coli(XL1-Blue strain) and choose the colony with 1.2kb insert.

2. grow up the bugs in 1XLB +amp medium o/n at 37C. add 10 ml the o/n
culture into 400 ml fresh 1XLB +amp,incubate at 37C for 4 hr.

3. Induce the fusion protein expression with 0.1mM IPTG and incubate it
at 37C for 3 hr. Then harvest the cells and resuspend the pellet with
15ml PBS+protease inhibitors. Sonicate and spin down it and pass the
supernatant through GST-sephorose column twice.

4.wash the column with PBS 4X before elute the fusion protein with 5mM
Glutathione(reduced). I normally collect 8 fractions 1ml each. But I can
not get any proteins by this protocol. Are there any of you know what is
wrong I did? Is there any people has a better protein expression and
purification method? Any suggestion is appreciated.

Thanks in advance.
mcbzzs at nuscc.nus.sg

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