GST fusions - please, help

[ijiwaru at wheel.dcn.davis.ca.us]

In article <46bbgc$16vi at news.doit.wisc.edu>, klenchin at macc.wisc.edu (Dima
Klenchin) wrote:

> 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?
BL-21 is good because of the lack of lon protease (if I remember
correctly).  I don't remember if JM109 has any of the same protease
deficiencies.  If you have been using an old transformation of your
expression construct in BL-21, you might try re-transforming, I have heard
from a number of people that stability of plasmids in this strain is poor.
YMMV

> 
> 2. I will need to remove thrombin after cleavage. Is there any highly specific
> affinity matrix that will stick thrombin selectively? 
I don't know if your amount of expression will allow it but, if you can't
find a suitable affinity matrix, how about using 30K MWCO Ultra-free type
spin filters to get the thrombin away from your 70K?  Again YMMV.

> 
> 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 
> /________\ \ \    
> \___________\/
> 
>