发信人: trissodium (Soso), 信区: Biology
标 题: Re: 求助！GST fusion protein cleavage-thrombin
发信站: Unknown Space - 未名空间 (Tue Mar 9 18:08:20 2004) WWW-POST

I had the same problem once. No good way to improve it. Two pieces of
suggestion:
First, try different amount of thrombin, different incubation time and
different temperature.
Second, try different enzyme.
My problem was solved by adding a TEV cleavage site between the GST and my
target protein. TEV is a very sequence specific protease. My protein was
purified by glutathione column and eluted with TEV enzyme. It worked.

【 在 leohawk (leohawk) 的大作中提到: 】
: read some books on protein purification, I don't really know the exact
: way, but there are a lot of standard protocols, you will have to
: find out which one is good for your protein.
:
: 【 在 iblicf (小虎。。) 的大作中提到: 】
: : Thanks, I'll try it.
: : We're now thinking if the GST-D doesn't not fold properly, that would be a
: : good explanation for the degradation right?
: : If I add Urea and cut it, how can I refold the protein after the cleavage?
: : 【 在 leohawk (leohawk) 的大作中提到: 】
: : : I belive you can add some detergent, like urea in your system to
: : : cut, thrombin will be fine in 3M(?) Urea, and denature of the protein
: : : ensures accessibility of thrombin to the cutting site.
: : : but if you can't denature your protein or your protein is subject to
: : : thrombin cut, there is really no good way to solve the problem --
: : : there may be other methods that I don't know of though.
: : 4
: : uncleaved
: : improve
:
:


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