GST fusions - please, help

Dima Klenchin klenchin at macc.wisc.edu
Sat Oct 21 12:43:40 EST 1995


Dear all, 

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
better available?

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? 
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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