Dennis J. Templeton
djt2 at po.CWRU.Edu
Thu Dec 10 17:49:55 EST 1992
In a previous article, PHILLIPSA at LARS.AFRC.AC.UK (Andy Phillips) says:
>I have a number of cDNAs which I need to express in E.coli, some
>for antibody production, and others for enzyme assay and characterization.
>I've been sent some blurb by Invitrogen about their Xpress system.
>The vectors use a polyHis metal binding tract (for purification) and
>an enterokinase (=enteropeptidase) site for subsequent cleavage. This
>looks, on paper, a neat system. Does anyone out there have any comments or
>experience, good or bad, with this?
>Secondly, one of the cDNAs I need to express is an intrinsic membrane
>protein. Are there specialist vectors for use with membrane proteins
>or would the system above work OK? I realize that the protein may
>not fold/insert properly and that I may need to do the purification
>in detergent and/or denaturant.
>All comments appreciated.
>Long Ashton Research Station
>PHILLIPSA at LARS.AFRC.AC.UK
Andy; In general the His-purification system works ok, though my experience
is that it doesn't yield perfectly pure protein.
I would suggest that you look at the pET vectors from Novagen (800 526 7319
or 608 238-6110 or FAX 608 238-1388) They will send spec sheets by fax.
While I haven't used the His-tagged version they sell, the general vector
design (Based on Studier's vectors) is time tested. It is a Lac-T7 system.
They also sell a verstion with an ompA leader that might handle your
membrane protein well; normally it is used to express the protein into the
extracellular space, but it might actually orient a trans membrane protein.
Again, I have not used this.
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