Problem with protein expression

[Kyle Legate]

Peter Frank wrote:
> 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).
>
This may be a dumb question, but did you induce your cells? You didn't 
mention this step. There is an E. coli protein that reacts with the anti-His 
antibody (at around 60 kDa?), and you may be detecting this in your Western.

It's a good idea to compare some induced E. coli lysed directly with SDS 
loading buffer with the lysate you intend to use for purification to 
determine if your protein is soluble. Garbage in gives garbage out.

The Qiagen Ni-NTA matrix product manual is fairly comprehensive for this 
type of purification. It should be available as a pdf from their site.