GST-fusion thrombin cleaves but won't elute!

ijiwaru at wheel.dcn.davis.ca.us ijiwaru at wheel.dcn.davis.ca.us
Wed Oct 16 15:41:00 EST 1996


In article <djt2-1610961808110001 at path22285.path.cwru.edu>,
djt2 at po.cwru.edu (Dennis Templeton) wrote:

> We have had a strange problem several times using the vector pGEX-KG to
> express fusion proteins that can be cleaved using thrombin. In most
> instances we get pretty good yield, and have tried the strategy of
> purifying the protein on GSH-agarose, then cleaving the protein in situ,
> hoping to collect the cleaved protein without the GST in the soluble
> phase. Judging from coomassie gels of the protein, the thrombin cleaves
> the protein efficienly, more than 90%. However, only a small minority of
> the cleaved protein is removed from the gel, about 5-10%! We get good
> elution using GSH (about 75%).
> 
> This has happened to us with 4 different fusions (all kinases) and we
> thought we might be seeing some sort of dimerization that holds a network
> of proteins on the bead, but our neighbor came to us with a similar
> problem expressing an unrelated protein. We use home-made GSH beads from
> an epoxy link protocol from Kurt Ashendel (thanks Kurt!) but our neighbor
> had the problem using his tried and true beads from Sigma. The loading of
> the protein in our case is quite high, probably 5 mg/ml.
> 
> We have tried eluting in 1% NP40; 20 mM DTT 37dC; and 0.5 M NaCl to no
> avail. It comes off (with the GST moiety) in 0.1% SDS, but that is not
> helpful.
> 
> Any suggestions?
> 
> Dennis Templeton
> Institute of Pathology
> CWRU School of Medicine
> djt2 at po.cwru.edu
> http://129.22.87.13

I've used pGEX-2T and done similar cleavages with thrombin, but haven't
had the same experience you had.  I did find that I had to cleave for a
much longer period of time with much more enzyme when the fusion protein
was resin bound than when it was soluble.  I was cleaving with something
like 100mg (total) added over a period of about 4 hours with mixing at
room temperature.  My fusion partner, different parts of HDV delta antigen
(which has been shown to forms dimers) came off just fine.  I think I did
this in PBS (sorry for the swiss cheese memory recall).  When the fusion
protein was soluble, complete cleavage occurred with something like 20ug
of thrombin in 2 hours as determined by gel electrophoresis.  There was no
indication that anything stuck to the resin, obtained from Sigma.  I'd be
interested in hearing what you come up with as a solution to this
situation.

Lyle Najita
Plant Pathology
University of California - Davis



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