pGEX/GST gene fusion problems-> HELP!

Brandon Harder bharder at u.washington.edu
Mon Aug 12 18:44:35 EST 1996


  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.


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		 \  Brandon Harder bharder at u.washington.edu  /
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