GST-fusion expression of a small protein

Esme esmeh at cyllene.uwa.edu.au
Thu Jan 31 03:03:09 EST 2002


Hi,

I am currently attempting to express a small (100aa) protein in the pGEX-6P2
vector in BL21 Codon + bugs.  I have sequenced the plasmid and there are no
mutations, and the protein is in frame.

I have had successful (GST-fusion) induction of my protein (with protease
inhibitors) over a 3 hour period, at temperatures from room temp, to 30 C,
to 37 degrees C, each time with the same results on a 12% protein resolving
gel.  The protein does occur in the soluble fraction.

When I induce the protein it runs 3-5 KDa higher than expected.   

My problem however, is that when I induce, not only do I get my protein, but
also a large amount of GST alone being induced.  Thus when I attempt to put
the protein mix on the beads, GST appears to outcompete the protein and I
end up with purified GST with no protein at all.

I have no antibody for this protein, and have tried to purify it a number of
times.

Has anyone seen this before, or does anyone have any ideas? 

Thanks a lot,
Esme. 


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