GST fusion: Solutions and more questions

Ann Loraine loraine at NATURE.BERKELEY.EDU
Sat Oct 8 14:54:06 EST 1994


Rod writes:

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


I have used the pRSETA plasmid from Invitrogen with pretty good success.
Others are available with different reading frames, etc., from a variety
of different companies.  However, they all contain a polyhistidine
sequence flanking a polylinker, allowing you to subclone into the
correct frame for producing (hopefully) mg quantities of your
protein-of-interest.

Purifying fusion protein from bacterial (or insect cell) lysates requires a
chromatography step over a zinc, nickel, cobalt (you get to pick which
one) bound to a metal-chelating resin.  You can buy the resin
ready-made (metal ion already bound) from a variety of sources.  Or, you
"chelate"  it yourself.  And you can buy metal-chelating resins suitable 
for FLPC & such other non-gravity flow chromatography systems.
The fusion protein is eluted from these columns either by washing with
imidazole, which competes with his residues for the metal binding sites,
or by washing with low ph buffer.


The only problem I've run into overexpressing his-tagged proteins was
with getting the fusion protein >80% pure, or so.  A higher-molecular
weight protein (about 80 kd or so..I run high-percentage minigels and so
it's kind of hard to judge sizes in that range) co-elutes with the
fusion protein.  This isn't too serious a problem, however, since the
fusion protein and contaminants can be separated by a second
chromatography step.  However, I wonder: has anyone who has tried both
the GST-fusion and the polyhis-fusion proteins systems found that
glutathione-sepharose chromotagraphy yields a much purer product?  This
has been my experience so far.  Another tip: contamination by bacterial
or insect cell proteins is greatly reduced by an ultracentrifugation
step prior to putting lysate over your column.  (You should do this
anyway if you are using F or HPLC...particularly if you are working with
a eukaryotic expression system :)

I hope this is helpful & that I haven't merely restated points from a
previous thread!

Ann L.



More information about the Methods mailing list