Hi Rene We use Tobacco Etch Virus (TeV) protease - its pretty active and very specific - As long as you are not using it for commercial purposes (i.e. selling it), it is possible to source the clone for it and readily express buckets of it (or alternatively just get the gene synthesised and bang it into the appropriate expression system). We have some success in using TeV in automated ACTA-XPRES systems for cleavage after the affinity step. I have heard good things about prescission but not used it myself. Disadvantages of both are if you have a C-terminal tag you end up with a relatively long tail because of the position of the cleavage site in the specificity sequence.
My advice is to give thrombin / factor Xa a wide berth - they are just too non specific and can carve up your protein to bits. We have used the oddly named protease "enterokinase" with some success, but again, it can readily munch your protein to bit - to be honest the commercial TeVs etc do have the advantage of being super specific Hope that helps J Rene Frank <[EMAIL PROTECTED]> 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 > =============================================== -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
