(no subject)

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Mar 23 11:18:53 EST 2001


I'm in a similar situation.  I tried one vector to express in the
periplasmic space, pET25, which I got from Novagen.  It didn't really work
for me.  Seemed to me novagen was fairly realistic in it's claims regarding
periplasmic targeting-- sometimes it works, sometimes it doesn't.

Currently I'm trying to express in one of the strains that allows disulfide
bond formation (Origami).  So far I've been unable to recover transformants
from the DE3 lysogen of this strain, leading me to believe it's working but
that my protein is toxic.  I'm currently working on ways around that.  But
one of these "folding" strains may be helpful to you.

I've also gotten the impression the thioredoxin fusions are sometimes
helpful.  I think novagen (no affiliation) provides pretty good information
about many of these choices (although they do want you to buy their
products!).  Their pET system manual is available online at their website
www.novagen.com.

Hope this helps.

Mike



>Hi there folks,
>I need to produce a soluble native recombinant protein. At the moment
>its expressed in
>pET30a and is totally insoluble (the native protein is v. soluble). I've
>tried refolding but the protein has 3 di-sulphide bridges and refolds in
>many different forms (the multiple folded forms have been noticed by
>other groups).
>I want to try to get it soluble by expressing it in a vector that
>targets it to the perplasmic space so I would be very grateful if
>there's any one who has experience with these types of vectors (such as
>pET22b) could give me some advice as in what is a good vector to use,
>wether the manufacturer's claims are exaggerated about solubility etc.
>
>Thanks Nadia
>
>---


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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