25kDa-band in pET-System-Expression

A.P.N. de Boer thon at xs4all.nl
Tue Aug 11 15:43:29 EST 1998


In article <6q3shl$dmk$1 at gwsun.medinf.mu-luebeck.de>, "Lars Komorowski"
<larskomo at physik.mu-luebeck.de> 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 ?

It's a wild guess, but I can remember from some work I did on expressing
proteins that the B-Lactamase (Amp-r gene product) can sometimes be over
expressed. Check on the size of the Ap-r gene, it might be that.

Thon de Boer



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