Mr. J Hinshelwood wrote:
>> I am expressing a protein in E.coli using the pGex system. The fusion
> protein is expressed in high yield and can be purified using GST-agaroase.
> When I cleave my protein from the gst (using thrombin) on the GST-agarose
> I get a product that runs as a single band by SDS page plus a few minor
> contaminants. If I run this product on an IEF gel I see multiple bands and
> likewise if I try to purify this by ion exchange I get multiple peaks.
> Each of these peaks from the ion exchange run at the same hight on SDS
> PAGE and all are detected by my antibody in western blot. Could anyone
> explain what is happening.
> Thanks, Justin.
You may be getting unwanted cleavage reactions as thrombin is a
pretty bad enzyme in terms of specificity. Your best bet may be to
ditch the pGex vector and use the 6P1 vectors (which encode a
clevage site recognized by a highly specific viral protease which
Pharmacia sells). Works quite well in my hands and the MCS is the
similar so you may simply get away with ligating the insert from
pGex and inserting it into 6P1
HINT: if you do decide to switch to the 6P1 vector and use the viral
protease, do NOT believe Pharmacia about its efficiency... we use
somewhere near ten-fold LESS the amount recommended and get complete
cleavage in about 4 hours (guess they want to sell you more protease
than you need).