pGEX/GST gene fusion problems-> HELP!

ijiwaru at wheel.dcn.davis.ca.us ijiwaru at wheel.dcn.davis.ca.us
Tue Aug 13 17:07:55 EST 1996


Brandon,

not knowing what your expressions conditions are, I'm going to throw my 2
cents into the fray and hope it helps.  We had similar problems with a
protease (undefined) sensitive site near the N-terminus of hepatitis delta
virus delta antigen.  Both full-length and just the N-terminal half when
fused to GST would yield loads of degradations products.  We managed to
get reasonable amounts of intact fusion protein (enough for rabbit
immunizations) by keeping the induction period short (2-4 hours).  Since
then, based on what I've seen on this group and from other protein
expressions I've tried, I would recommend the following. 
1)  work off of a reasonably fresh transformation of your plasmid,
especially in BL-21.  No more than one month old.  Don't even bother with
glycerol stocks, they may not keep.
2)  When you do propagate, grow in the presence of glucose to keep your
expression as shut off as possible.  I've found good shut off of Plac with
0.25-0.5%.
3)  Grow cells reasonably dense, OD600 ~0.6, in media with glucose, pellet
cells, place in media with inducer (1mM IPTG, for example) and grow for
maximum of 4 hours.
4)  Harvest cells, lyse in presence of protease inhibitors, run extract on
GSH-agarose and wash extensively before you even think about stopping for
the day.

Good luck,

Lyle Najita
Plant Pathology
University of California - Davis

In article
<Pine.OSF.3.92a.960812163818.16112A-100000 at saul7.u.washington.edu>,
Brandon Harder <bharder at u.washington.edu> wrote:

>   Howdy, I'm using a GST fusion system and am getting a lot of fusion
> protein cleavage, which is highly undesireable.  The GST is attached to
> the cytoplasmic tail of a eukaryotic cell receptor.  I've tried using the
> protease inhibitor PMSF at a 1mM concentration with no effect.  I'm not
> sure if cleavage is occuring in the GST portion or the fusion portion of
> the protein.  I am getting a fusion protein, but also get equal to greater
> amounts of a degradation product.  If you have any leads or ideas, please
> let me know.  I also am unable to think of an experiment to determine
> where the cleavage is occuring.  I have the option of switching to a
> different GST vector but am unsure whether or not it would help.  Oh, the
> proteins are being grown up in E. Coli BL21 cells (tried DH5 cells also,
> no difference) and use IPTG as an inducer.
> 
> 
>                  \\\\\\\\\\\\\\\\\\\\\\*//////////////////////
>                  \  Brandon Harder bharder at u.washington.edu  /
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