Hi all, Summary points for choice of proteases:
1. Most favoured protease is TEV protease for precision and cutting efficiency (commercial source or expression vector widely available). 2. 3C/PreScission protease has not been used as frequently, but is also strongly recommended. 3. Thrombin, factor Xa, enterokinase have had mixed reviews, with some potential problems with secondary cleavage of your protein. Using thrombin may require careful optimization including testing multiple suppliers. 4. Other proteases: SUMO protease and TVMV protesase. The latter is similar to TEV, but with a different specific excision site (vector available from www.addgene.org).
5. Adding the same purification tag to your protease as you are using to purify your protein is advocated for ease of removing the protease afterwards. 6. For membrane and secreted proteins careful consideration of where to put your N-terminal tags and protease sites after the signal peptide is emphasized, as is avoiding putting a protease cleavage site within predicted secondary structure. 7. Awareness of the excision site residues left after cleavage, particularly for protease sites at the C-terminus. 8. Most of the proteases and their excision sites are nicely tabulated here: http://stevens.scripps.edu/webpage/htsb/cleavage.html
I need to tag up to three subunits that I can selectively excise, so I have decided to go for TEV, 3C/PreScission protease and TVMV.
Thank you for your helpful advice. 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 ===============================================
