Hi,
I'll second the TEV protease suggestion. We use it routinely because
it is highly specific and easy to make ourselves (and, therefore,
cheap). We have never seen it cut non-specifically and, since it is
cheap, we just chuck in a bunch and let it go.
The Prescission protease is also very specific and also available for
home preparation (it is the 3C protease...prescission is a marketing
name). I don't have as much experience with it, but it has behaved
for me so far and I know that a number of people use it routinely
with great success.
Another nice thing about having the clones around to make your own is
that you can make the protease with the same tag that you intend to
cut off (say, His or GST or whatever your favorite is). Then, you
can remove cleaved tag, uncleaved fusion protein and the protease all
in one post-cleavage step.
In our case, we almost always have a HisTag (often as part of some
larger fusion) so our TEV is His-tagged. We have some of the GE pGEX
vector for Prescission protease, so our 3C clone has GST on it.
Best of luck,
Cynthia
On Mar 2, 2007, at 5:01 AM, Rene Frank wrote:
Hi,
A non-ccp4 Q. Sorry.
I would like to use a cleavable purification tag at the N-terminus/
extracellular end of my membrane protein for purification. Before I
start, I wonder if someone could recommend a particular protease
site that I can engineer between the tag and my protein? How about
a proprietary cleavage system such as the PreScission protease (GE
Healthcare)? I would be grateful to hear success and horror
stories in this area.
Best wishes,
Rene
================================================
Dr R.A.W. Frank, PhD
Royal Commission for the Exhibition of 1851 Research Fellow
Prof Seth Grant Lab / Genes to Cognition
Wellcome Trust Sanger Institute
Hinxton
Cambridge CB10 1SA
Work Tel: 0044 (0)1223 834244 ext. 7318
Cell No.: 0044 (0)7870 208280
===============================================
____________________
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
