pGEX/GST gene fusion problems-> HELP!

Michael DiDonato michael.didonato at utoronto.ca
Tue Aug 13 09:58:12 EST 1996


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.

We had this problem with one of our fusion proteins also.  PMSF will
inhibit both thrombin and Factor Xa the fact that you are still getting
degradation suggests that it is not due to these enzymes.  In our case it
seemed that the fusion was auto-cleaving right at the junction of the GST
and the protein, we never did figure it out.  You could do amino terminal
sequencing of the degradation products to determine where it is cleaving.

Mike
Department of Biochemistry Research
Hospital for Sick Children



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