GST fusion: Solution and more questions

Rodney Tweten Rod-Tweten at uokhsc.edu
Thu Oct 6 18:14:35 EST 1994


In article <3679f2$8ih at apakabar.cc.columbia.edu> Pavel Sova,
ps44 at konichiwa.cc.columbia.edu writes:
>I posted here couple of months ago a question about isolation of 
>GST-fusion protein. My problem was that 23-kD protein hooked up behind 
>GST wouldn't bind to a G-Sepharose, after I was able to solubilize it 
>from inclusion bodies (which contained 100% of the fusion protein) by
1.25% 
>Sarcosyl in Tri-ethanolamine buffer. The problem was that I 
>omitted final step before binding it to G-Sepharose, that is bringing 
>adding Triton X-100 to 2% concentration to solubilized protein. This 
>step, I suppose, should sequester the SDS-like compound, Sarcosyl, to 
>micelles and thus remove it from protein, which is in my case, probably 
>denatured by Sarcosyl.............................................

You might want to look into moving your protein to another system such as
the pET vectors from Novagen.  The pET vectors allow you to fuse your
protein to a polyHistidine sequence at either the carboxy or amino
terminus of your protein.  They are then purified by passage over a
Zn-chelate column to which the polyHistine sequence binds and is eluted
by various means.  The advantage of this system is that the fusion
protein can be dentaured in things as harsh as 6M guanidine-HCl or 8 M
urea and it will still bind to the Zn-chelate column.
I have no connection with Novagen.
Cheers,
Rod Tweten



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