GST-fusions and proteolysis

ijiwaru at ijiwaru at
Tue Aug 6 14:29:26 EST 1996

In article <4tvq1j$2rb at>, biotec54 at (BIOTEC54)

> I've been working with several GST-fusion constructs that were built using
> the pGEX-2T vector. While I usually see good expression of my fusion
> protein....upon purification via GSH affinity...I also seem to co-purify
> material corresponding to the GST portion only. I'm wondering if the
> inclusion of the thrombin cleavage site at the fusion junction is being
> cleaved via a host protease? Has anyone removed the thrombin site and seen
> only full-length protein?
> Thanks

I've used pGEX-2T to express the whole and portions of hepatitis delta
antigen and found that there was what appeared to be some kind of
protease-sensitive site in the N-terminal domain.  We think this was the
case because the C-terminal domain isolated on GSH-agarose yielded
reasonably pure fusion protein with hardly any other proteins.  Both the
full-length and the N-terminal domain clones yielded fusion protein along
with something that ran about the size of GST alone.  We hadn't tried
removing the thrombin site because it appeared in our case the N-terminus
held the protease-sensitive site.  I tried to get around the problem by
trying to get the extraction/isolation procedure run all the way through
until the fusion protein was bound to the resin and then washed.  If it
got too late to continue at that point, I left it in the cold room with no
noticeable degradation of the fusion protein.

Good luck,

Lyle Najita
Plant Pathology
University of California - Davis

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