Need help with GST fusion protein purification
H A Lacey
H.A.Lacey at shef.ac.uk
Thu Nov 14 04:46:49 EST 1996
Edward B. Arias wrote:
> I'm having a problem purifying a 104 kDa GST fusion protein with the
> Pharmacia kit. I've tried most of the trouble shooting tips to no
> avail. One ref in the manual takes about losing GST fusion
> protein:glutathione sepharose beads affinity if your fusion protein is
> 80+ kDa. I'm a grad student and this is the last thing I must do to
> graduate...that is purify out some of my protein which is 75 kDa (+ the
> 29 kDa GST = 104 kDa fusion protein which gets bound to the beads).
> Any help or comments with those using the GST fusion protein kit from
> Pharmacia with similar problems is greatly appreciated.
> Edward B. Arias - "Just Say Know"
> UIC Dept of Physiology & Biophysics
> 835 S. Wolcott Ave.
> Chicago, IL 60612-7342
> Tel: 312.996.4161
> FAX: 312.996.1414
> E-mail: earias1 at uic.edu
> WWW: http://www.uic.edu/~earias1
I have had similar problems with a protein of 102kDa. I think a lot of
the problem is the size range. The highest molecular weight protein
purified using the GST beads is I think 98kDa. I think you are lucking
out on the binding ability due to the fact that the protein is probably
either folding in around the tag not due to the natural folding of the
protein but the shear siz of it with the tag or it could be that when you
are purifying it the GST tags dimerise and therefore will not be avaiable
for binding to the beads!
Have you tried the following: changing growth temp, induction temp, time
of induction, IPTG concentration, using carbenicillin instead of amp in
your growth medium, reducing sonication times, french press, use of
lysozyme before sonication.
If you need any more help, not that the above is then please contact me
H.A.Lacey at sheffield.ac.uk
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