Purification of protien of interest-GST

D.K. dk at no.email.thankstospam.net
Tue Apr 30 07:07:27 EST 2002


Chan Shzu Wei <mdccsw at nus.edu.sg> wrote:
>Dear colleagues,
>
>I have problem in purifying my protein of interest  from the GST from
>using GST column. I have cut my protein of interest (~25.7kDa) from the
>GST fusion protein (26 kDa) with thrombin protease, and as in theory, I
>should get my protein of interest in flow through, but it didn't. I
>found that it came out together with the GST when I eluted with the GST
>elution buffer. Please give some suggestion to purify my protein of
>interest from the GST protein. Thanks.

If you know for sure that cleavage has worked then obviously your
protein binds to glutation. This is charged tripeptide and therefore, 
if interaction of your protein with glutation is "non-specific", you 
can try increasing salt, or adding detergents or both. If you are 
very lucky, then your protein may elute pH lower 6.5-7.0, where
GST elutes inefficiently. If none of this works, I'd switch to 
another fusion. 

DK




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