GST-fusion problems

Kevin Brady rcxkb at unix.ccc.nottingham.ac.uk
Mon Mar 5 06:40:35 EST 2001


Hi,
I'm trying to purifying a GST fusion product which has a MW of 37kDa,
using Glutathione-Sepharose from Pharmacia. I'm using 10mM GSH in
50mM Tris for elution. When I run fractions out on an SDS-PAGE gel, I
can see the 37kDa band, but also about 6 smaller proteins ranging from
about 35-22kDa. These dont appear in the wash steps at all, and are
specifically eluted with GSH. I'm using BL21(DE3)pLysS E.coli with
pET42b for expression. Has any one else seen these sorts of proteins
appearing? I've read about the possibility of co-elution of HSP's from
E.coli in this system, but aren't they usually about 50-60kDa?
Cheers for the help
Kev

--
Dr Kevin Brady
Organic Chemistry Section
School of Chemistry
University of Nottingham
Nottingham
UK
NG7 2RD
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Email : Kevin.Brady at nottingham.ac.uk or
        kevin at basil.chem.nott.ac.uk
URL   : http://www.nottingham.ac.uk/~pcxsc/thomas
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