Hi Matthew,

 

TEV is probably the least robust protease among those commonly used for tag removal. Here’s a common unit definition of TEV. One unit (corresponding to 0.1 ug TurboTEV) cleaves ≥85% of 3 μg control substrate in 1 hour at 30C. You need to use really a lot of TEV. Information collected from our customers shows large variation in quality of home-made TEV proteases. Some used 1:5 w:w ratio.

 

Adding a little bit EDTA to Ni pool helps TEV digestion. You can find a general protocol at http://www.accelagen.com/TurboTEV-protocol.htm.

 

Cheers,

 

Chun

Accelagen

 

 

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