This the a summary on a survey on the usage of pGex vector.
|> Dear All:
|>|> I am a pGex-2T user and I want to ask two questions. I will post a
|> summary on these questions later.
|>|> 1. Do you add protease inhibitor(s) in your E. coli lysis buffer? I used 10
|> EDTA and 0.1mM Benzamidine but still got quite a lot of degradation
|> even though I did all the steps in cold room or on ice.(centrifuge,
|> resuspend, sonication, affinity column.... I think the cleavage is
|> since I could see intact protein band in total cell lysate but it was
|> 2.How do you wash your GST agarose before eluting your fusion protein by
|> Glutathione? Can I use up to 0.5 NaCl or up to 1.0 M NaCl in my wash buffer
|> to remove non-specific proteins before elution? I could see other
|> proteins in SDS-PAGE after elution.
|> Thanks in advance.
|> Pls post to my account if possible so that I can summarize easily.
|> Wallace Lim
|>BLLIM at molbiol.ox.ac.uk
I use BL21 cells, which are protease deficient, for my pGEX work. In
addition to the protease deficiency of the cells, however, I also use PMSF as
inhibitor. We definitely notice a difference without it! One point - I add
PMSF after each sonication burst (I do three 30 sec bursts) to mop up any new
proteases that are released. I don't carry out the affinity step at 4C....
just as good at room temp. and MUCH more comfortable!
I wash with 2 column volumes of TTBS (50 mM Tris-HCl pH 8.0 containing
150mM NaCl and 0.1% TritonX-100) then 5 volumes of TBS (same, no Triton). Then
I elute the protein! There is occasionally some other material (about 90kDa)
is in the eluted material, but as I subsequently use both a gel filtration and
ion exchange step, it doesn't worry me.
Hope this was of some use!
Craig Morton ][
Research Assistant, ][ If you're not part of the solution,
Oxford Centre for Molecular Sciences, ][ You're part of the precipitate,
South Parks Road, Oxford. ][
I've had some experience with purifying large GST proteins and have found that
the yield is lower and that their is always some degradation. I have
found that a combination of protease inhibitors helps - PMSF, Leupeptin and
Pepstatin. These three cover most proteases that may be active in your
lysates. I think the contaminating bands you are isolating are probably
breakdown products of the full size fusion protein and probably can not
be removed simply by washing.
Univ. of Guelph,
Guelph, Ontario, Canada.
e-mail: ugg00489 at vm.uoguelph.ca
We use pGEX-3X to produce a fusion protein of about 70kDa. The lysis
buffer includes 10mM EDTA, and 1ug/ml Leupeptine + 1ug/ml aprotinin
+2ug/ml pepstatin. Even with these I still can see twin bands at 30kDa
although less than 10% my predicted major band. The affinity matrix is
washed extensively before competed elution, all in cold as well, and
with 10mM DTT. The M.W. were evaluated by SDS-PAGE.
We therefore rely on other separating method. My personnal idea is
that the E.coli buggs might have GSH binding proteins, whatever they are,
but do not have any detailed data on that.
Andre White (awhite at alpha.oci.utoronto.ca)
We have used pGEX extensively. We find the system variable with different
Small proteins tend to work better than large proteins. Hydrophobic proteins
are a pain. If you are expression the protein for antibody production, you
try expressing a different region of your protein. Also, using different
strains of E. coli may help but this rarely makes a difference for us.
We don't use any proteinase inhibitors. We just use PBS (150 mM NaCl)
suplementedwith 0.1% tritonX-100 for column binding and washing. A trick we
like is to digest the protein with thrombin on the glutathione beads and elute
fragment of interest while leaving the GST attached to the glutathione beads.
Rarely, the cleaved fragment binds to beads and then we use the more
technique of cleaving the protein after elution with glutathione and gel
purifying the protein.
lassner at calgene.com
FWIW I have developed a set of baculovirus vectors which make GST fusions in
insect cells to very high levels, with the obvious advantages of eukaryotic
processing, and the additional advantage that GST is a soluble cytoplasmic
protein in insect cells and all fusions made to date are equally soluble. No
detergent at all is needed to release them
They are just about to be published in the August issue of Bio/technology - I
can send you a reprint if you like.
Could you email me at adavies at cgl.ucsf.edu rather than the Oxford address? I
have just moved to the States, but still use my Oxford account for News etc..
Thanks a lot - best regards -
Department of Pathology
University of California
When I tried to use the pGEX system the biggest problem was in just getting
some amount of protein expressed. Are you sure that the protein that you see
in the total cell lysate isn't from inclusion bodies? If they are inclusion
bodies you could expect that the proteins would be protected and no
would be detected. To reduce the amount of protein that is being lost to the
inclusions you might try to lower the temp to about 25C, for my expressions
I see less degradation and more soluble protein. Good Luck.
Thank you for all of the above persons who contribute.
BLLIM at molbiol.ox.ac.uk