Hi Matthew,
By now, you have received many posts telling you both how efficient and
inefficient TEVp is. You might be confused. This seeming contradiction
can be explained by a few events, among many others: Inaccessibility of
cleavage site, absence of reducing agents, and presence of detergents if
you are using them on membrane proteins.
Solutions are, respectively: (1) redesign the construct to give the
cleavage site some breathing room by adding a few residues around it,
(2) add 2-mercaptoethanol, DTT, TCEP, etc. to keep TEVp active; it is a
cysteine protease after all, (3) use a different detergent, enzyme, or
ridiculously high amounts of enzyme (see Mohanty et al, Inhibition of
tobacco etch virus protease activity by detergents, Protein Expression
and Purification, 2002).
It is true in my experience that TEVp is less "active" than thrombin and
the like. But it is extremely specific, and much more than thrombin, so
you don't have to worry about your protein being cleaved in unexpected
places.
Best,
Engin
On 5/24/10 12:43 PM, xiaohu mei wrote:
Hi Matthew,
TEV protease is very robust. I normally digest with 1:100 ratio
according to the OD280. I normally digest at 4C for overnight around
16-18 hours. Make sure your tev protease site are not inaccessible and
buried inside.
best
Xiaohu
On Mon, May 24, 2010 at 12:27 PM, Matthew Merski
<mer...@blur.compbio.ucsf.edu <mailto:mer...@blur.compbio.ucsf.edu>>
wrote:
Hello all,
I am working with a protein that is expressed as with an
N-terminal domain that is normally cleaved for activation of the
protein (and crystallization). For in vitro reasons I've needed to
switch the normal site to a TEV site. However, even though the TEV
site is in the same place as the original proteolytic site, I have
been unable to get cleavage despite using a lot of TEV at 37 C, pH
8.0. Has anyone been able to overcome a similar problem?
Matthew Merski
Post-doctoral researcher
UCSF
--
Engin Özkan
Post-doctoral Scholar
Howard Hughes Medical Institute
Dept of Molecular and Cellular Physiology
279 Campus Drive, Beckman Center B173
Stanford School of Medicine
Stanford, CA 94305
ph: (650)-498-7111
