TeV is great but it isn't always a magic bullet for producing native
N-termini: we've experimentally determined the obvious, that if you
bury part of its recognition site in secondary structure, it cleaves
very very slowly. We tried this on a protein where M1 is cleaved in
vivo and aa#2 is the beginning of a nice helix, but the sequence
itself is compatible with TeV sites. Adding a couple small flexible
residues between the cleavage site and the beginning of the folded
structure did wonders for the cleavage rate.
Phoebe
At 04:01 AM 3/2/2007, 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
===============================================
---------------------------------------------------------------------------------------------------------------------------
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
