thrombin for GST prot cleav

Achim Recktenwald achim at ibex.ca
Mon Mar 18 15:32:31 EST 1996


Cornelius Krasel wrote:
> 
> GLUXLAB (Laurette at msvax.mssm.edu) wrote:
> >       We are currently using highly purified human thrombin
> > 4,000 Units/mg [sigma #T-3010] for cleaving our fused protein
> > from glutathione S transferase.  the price is becoming
> > prohibitive ~$300/1000 units. is there a better source for
> > thrombin, and not neccessarily human that will work as well.
> > our end product id being crystallized for structure determination
> > as well as for nmr characterization.
> 
> I have been told that it is fairly easy to make large amounts of
> thrombin from bovine blood but I haven't tried to do it myself.
> Generally, less pure preparations may be cheaper but you introduce
> additional contaminants and maybe additional proteases.
> 

About two years ago I helped a friend to purify thrombin from a 
commercially available crude preparation. He needed the highly purified 
form for inhibition kinetics. I think he got the crude stuff from Sigma. 
We did a single step, an ion-exchange chromatography; it worked out very 
well at our first trial and was rather easy. As far as I can remember he 
did an HPLC run of the product and the protein was homogeneous.

I cannot remember exactly what we did in detail, I also lost contact 
with him, but it was surprisingly easy.

Achim


________________________
Achim Recktenwald, PhD.
IBEX Technologies, Inc.
5485 rue Pare
Montreal, PQ, H4P 1P7
Canada




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