GST fusions - please, help
klenchin at macc.wisc.edu
Sat Oct 21 12:43:40 EST 1995
I would greatly appreciate, if you can clarify for me the following.
I need to overexpress 70K protein as a GST fusion. It is cloned into
Pharmacia's pGEX-2T. As usually, expression level and solubility is a problem.
1. What strain should be used? Pharmacia recommends BL21 that we don't have
right now. Does it really offer any advantages over, say, JM109? Is anything else
2. I will need to remove thrombin after cleavage. Is there any highly specific
affinity matrix that will stick thrombin selectively?
3. Increasing solubility. I know of the following cheap tricks: low IPTG
concentration/room temperature growth. I remember someone's suggestion
to heat shock bacteria before induction at 25C. Does this really improve results?
What else could be tried?
Thanks you very much,
/ /\ Dima Klenchin
/ / \
/ / /\ \ klenchin at macc.wisc.edu
/ / /\ \ \ tel. (608)262-4380
/ /_/__\ \ \ FAX (608)262-4570
/________\ \ \
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