Problem with protein expression

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Wed Sep 29 10:01:12 EST 2004


Did you check if your protein might be 
accumulating as insoluble inclusion bodies, 
rather than in the soluble lysate?  Also, when I 
made a fusion protein with an N-terminal His tag, 
I was unable to purify it with Ni-NTA.  I found 
that my protein would not elute from the resin. 
When I boiled the resin in SDS-PAGE sample 
buffer, I found my protein was there.

I find the pET expression Manual, which can be 
downloaded from the Novagen web site, is a rather 
good resource for using pET vectors.

Hope this helps.

Mike



>Hi,
>
>I have some problems expressing a His-tagged protein. I cloned the
>sequence in-frame into the pET-20b(+) expression vector, which I used
>to transform E.coli BL21 (DE3) cells with. I thoroughly checked that
>insert and His-tag are in-frame. I grew the culture at 30 °C, and
>although it grew slowly, it did grow continuously as monitored by OD
>measurements. So, I assume the protein to be expressed is non-toxic. I
>lysed the cells completely. I purified some lysate using Ni-NTA
>magnetic agarose beads, which should highly enrich the His-tagged
>protein. I ran several SDS-PAGE gels with purified and unpurified
>lysates (with non-reducing and reducing sample buffer). The
>silver-stained gel did not show any band with significantly increased
>intensity (but then again there were many other bands of course that
>may obscure this).
>Two other gels were immunoblotted and a) incubated with an antibody
>against a peptide segment of the protein to be expressed as well as b)
>incubated with an anti-His-tag antibody. Western blot a) showed faint
>bands of the appropriate size whereas Western blot b) showed
>absolutely no bands for the purified lysates and bands of the wrong
>size for the unpurified lysates (too low MW).
>This is very confusing and I am not sure where the problem could be.
>The expression vector seems to be OK, but I cannot detect a His-tagged
>protein of the expected size using anti-His-tag antibody.
>
>Does anyone have any idea where the problem could be or can you
>recommend a good troubleshooting guide for prokaryotic protein
>expression?
>
>
>Peter


--

Michael L. Sullivan, Ph.D
Research Plant Molecular 
Geneticist
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5397 Phone
(608) 264-5147 Fax
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