Thrombin Cleavage of GST

[Lyle Najita]

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>

Evan,

I used pGEX-2T to express portions of HDV delta 
antigen and found that I had to use about 5 times 
the amount of thrombin to cleave resin-bound fusion 
protein as what was recommended in Current 
Protocols in Mol.Biol.  I was using somewhere in 
the neighborhood of 100ug with gentle agitation at 
room temperature for 1.5 hours, adding another 
100ug at 3 hours and 4.5 hours for a total 
digestion of about 6 hours.

If you've already injected with your GST-fusion 
protein, you will already have a good response to 
GST, at least that's what I found with our fusions 
injected into rabbits.  Anti-GST Abs came up right 
away and with fairly high titre.  I eventually 
started boosting with just the domain of interest 
and was assaying by western.

Good luck,

Lyle Najita
University of California - Davis