pEt-Expression-Mystery

Frank Bartels fba at gbf.de
Fri Aug 7 16:22:44 EST 1998


Lars Komorowski wrote:
> 
> I am trying to overexpress a 17kDa-Protein in E.coli BL21 with Novagens
> pET24a Vector. I inocculate my LB medium from an overnight culture, let the
> culture grow until the OD reaches 0.6 and induce with 1 mM IPTG. After one
> night there is a band at 25 kDa in SDS-PAGE made with total cell protein.
> When I try to separate cytosolic proteins from the rest and make another
> SDS-PAGE with both fractions there is no band there.
> I have two questions:
> 1. If the protein band is not the desired protein what can it be ?
> 2. If the band represents the protein, is it possible that it is proteolysed
> within minutes ?


Hi Lars,

It is certainly possible that your 17 kD protein appears as a 25 kD on a
SDS gel since there are many reports about that (the reason can be that
your 17 kD protein is basic or acidic).
I dont know what your protocol is to separate the cytosolic proteins
from the rest but can it be that your expressed protein is insoluble
(mybe forming inclusion bodies)? If your prepare a crude cell extract
and centrifuge afterwards the protein may precipitate. Load the
supernatant and the pellet on the gel. The insoluble protein should
appear in the pellet.

Regards



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