Phage Display of cytosolic Protein

ijiwaru at wheel.dcn.davis.ca.us ijiwaru at wheel.dcn.davis.ca.us
Thu Sep 19 12:18:49 EST 1996


In article <324144EF.3427 at bway.net>, tomweber at bway.net wrote:

> Hi there,
> I want to display a cytosolic protein, and thereafter random mutants 
> of it, as a fusion protein with a protein of a filamentous phage, much 
> like petide libraries.
> 
> Here my questions:
> 
> has anyone of you out there ever done this and what was your 
> experience with the nonreducing environment that the usually cytosolic 
> proteins are now exposed to ?
> 
> What is the best vector (phagemid) to use and where can I get it from 
> ?
> 
> Did any of you ever use the expression module of the Pharmacia 
> Recombinant Phage display Antibody System (RPAS) for a similar 
> purpose, and how are your experiences ?
> 
> Thanks for your input
> 
> Thomas

Thomas,

I've modified Pharmacia's RPAS because I had to.  Pharmacia very nicely
supplies the pCANTAB5E vector as a NotI-SfiI isolated, large, linear DNA
molecule so it cannot be propagated without ligating something into it. 
If you do intend to make modifications, you may want to check to make sure
you design in a functional signal peptidase cleavage site (that part is
missing without the NotI-SfiI fragment).  Keep the plasmid in a lacIq
background and under glucose repression to insure that the promoter is as
shut off as you can get it, I've found that the Plac is very leaky and
I've gotten very good expression of the clone in XL-1 in plain LB.  I've
not used RPAS for antibody expression, that's why I wanted to modify it,
e-mail me if you want more details.

Good luck,

Lyle Najita
Plant Pathology
University of California - Davis



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