GST-fusion expression of a small protein

Frank O. Fackelmayer Frank at Fackelmayer.de
Fri Feb 1 08:04:31 EST 2002


Dear Esme,
The (quite common) problem is that GST is extremely stable, and mostly
remains untouched when other proteins are already degraded. The "free
GST" that you see is actually the remnant of your fusion protein, but
with your protein digested away. GST also does not outcompete your
protein when binding to the column, but rather remains bound when the
fusion partner gets chewed up by contaminating proteases. What you elute
later is the free GST. 

The best workaround is to purify your protein quickly and in the cold.
Also the addition of protease inhibitors can help, but obviously they
are not sufficient in your case. 
Also, shorter expression times can help, because much of the digestion
may already occur in the bacteria. We had that with one of our
constructs that could be purified only after 30min of induction, but was
completely gone later. It took us quite some time to figure out...


Frank




Esme wrote:
> 
> 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.
> 
> <http://www.biowww.net/forum/read.php?f=1&i=5292&t=5292>




More information about the Methods mailing list