Problems with expression in E.coli

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Mar 22 09:38:09 EST 2001


>Hi,
>
>I am trying to express a protein in E.coli as a HIS-tagged fusion
>protein to produce an immunogen. I have done is already several times
>but this time I have run into a major problem .... no expression at all
>!!
>
>I cloned my protein of interest into pET16 and pET28 using the NdeI site
>and the ATG of my protein. Sequence (and experience with other
>construcs)tells the cloning should be perfectly in phase. Also, all my
>expression controls work fine.

Paul,

By what criteria are you determining there is no expression?  I have a
his-tagged construct in pET28, and while I found it difficult to see
induction just by staining, it was very obvious when I ran a western blot
using anti 6Xhis antibody.  Also, my protein turn out to be insoluble, and
once I made soluble/insoluble fractions, it was very obvious protein was
being expressed.  Have you tried these approaches yet.


Mike


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

---




More information about the Methods mailing list