I'd like to third TeV & second 3C ("PreScission").Both have high specificity, good processivity and I have had a lot of success with 3C. I have _never_ got Thrombin to cut cleanly - but I guess I could have been unlucky... Dave On 05/03/07, Cynthia Kinsland <[EMAIL PROTECTED]> wrote:
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
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