GST-fusion thrombin cleaves but won't elute!

Dennis Templeton djt2 at po.cwru.edu
Wed Oct 16 17:08:02 EST 1996


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



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