Expression library screening with fusion proteins

andre nantel at biotech.lan.nrc.ca
Tue Aug 15 13:54:11 EST 1995


In article
<Pine.ULT.3.91.950814150135.6767C-100000-100000-100000-100000 at zombie.oul
u.fi>
"Petri T. Kursula" <pkursula at zombie.oulu.fi> writes:

> I am starting to look for proteins that bind a specific protein domain 
> that we are producing as a GST fusion protein. I wonder if anyone has any 
> experience in screening an expression library with such a fusion protein, 
> using anti-GST anibody and a secondary antibody coupled with alkaline 
> phosphatase to visualize the positive clones, i.e. the ones that bind the 
> fusion protein. 
> 
> What kind of conditions should one use to make the fusion 
> protein bind the ones on the filter made from the expression library 
> plates? Also, how easy would you think it would be to totally wash such a 
> filter so you could use a different fusion protein to screen the same 
> filter? What kind of conditions should you absolutely NOT use? Any help 
> will certainly be appreciated!

A lot of this depend of the type of protein that you plan to use as a
probe. Anmyway, I'd be surprised to see where you hope to find (and pay
for) enough anti-GST antibody for such a screen. I would rather suggest
using the pGEX-2tk vector which contains a phosphorylation site for the
commercially available cAMP-dependent kinase from heart muscle. The
exact labeling and screening conditions are in a 1993? Biotechniques
paper by Ron & Haberer.

Personnaly, I've successfully used biotinylated MBP-fusion proteins to
isolate plant genes encoding novel bZIP factors. The exact protocol
should be out by now in Plant Molecular Biology Reporter.

Andre Nantel
Biotech Research Institute
National Research Council Canada
Montreal, Quebec



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