Thrombin Cleavage of GST

[Dr A.H.M. Van Vliet]

eburkala at CENTRAL.MURDOCH.EDU.AU (Evan Burkala) wrote:
>Hi,
>   I have a fusion protein tagged to GST and am trying to cleave the GST 
>fusion partner off with thrombin. It does not seem that cleavage is 
>efficient if at all. Could anyone recommend a protocol that they know 
>work? I would be extremely grateful.
>
>Another thing, is it necessary to cleave the GST off if wanting to inject 
>into rabbits for antisera to the recombinant. I subsequently want to use 
>the fusion protein (uncleaved) for ELISA. Will I get too high a 
>background in the ELISA to the GST? Does anyone recommend raising 
>antisera specifically against the recombinant? 
>
>Thanks in advance,
>Evan Burkala
><eburkala at central.murdoch.edu.au>
>

Hi Evan,

I have used GST-fusions in ELISA. I expressed a rickettsial protein antigen fused with GST and 
used it for serodiagnosis in cattle, goats and sheep. This was fine using experimental animals, 
but when we started to test field sera, we got 10-15% sera positive with GST. When we cleaved 
GST off using factor Xa, this destroyed the epitope for an unknown reason. Due to these 
problems we switched to the QiaExpress system (6 His fusions), and that didn't give us any 
problems.

hope this helps,

Arnoud