Trouble expressing small peptide
NoReply at email.com
Thu Mar 10 13:40:47 EST 2005
No evidence to suggest the protein is toxic. Growth was pretty normal.
Transformation efficiency and colony size same as control, culture growth
maybe a little slower, but not dramatically. When expression is induced
with IPTG the culture continues to grow at similar rate. The full length
enzymatically active protein expresses at pretty high levels without any
problem, it's just this short C-terminal tail which will actually be
enzymatically inactive that's giving the problem.Wondered whether this
addition could be stopping the GST from folding and thus leading to its
rapid degradation, but there's no evidence of any degardation products
either. Being fused to the C-terminal end of the GST I hadn't anticipated
this being a problem even if this part didn't fold correctly.
In article <d0pdkr$u9q$1 at news.ua.es>, Rafael Maldonado
<rafamaldo at yahoo.com> wrote:
> Brian, I would say the protein is toxic to E. coli. Did you observe slow
> growth of the bug with the plasmid?
> Brian Ballard wrote:
> > I want to express the C-terminal 80 amino acids of a 250 amino acid
> > protein in E.coli. The full-length protein expresses very well.I have
> > tried it untagged, with a 6x His tag and also as a GST fusion but get no
> > expression at all. The most surprising failure was with the GST fusion.
> > The empty vector gives a nice stong band for GST on induction with IPTG.
> > When the 80 amino acids are fused onto the c-terminal end of the GST I get
> > absolutely nothing at all. Have tried changing the concentrtion of IPTG
> > for induction and have tried different temperatures from 3hrs to
> > over-night, but still see no fusion protein at all. Is this unusual to get
> > nothing at all? I'm not that experienced at coli expression, but
> > colleagues who are are a little surprised. Have checked the sequence of
> > the vector/insert boundary, but even if the insert was out of frame I'd
> > have expected to see at least a protein corresponding to the size of GST.
> > Any ideas?
> > Brian.
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