Trouble expressing small peptide
rafamaldo at yahoo.com
Mon Mar 14 05:35:43 EST 2005
Then I would think in message unstabilty. You better try with a
different vector. Small peptides are difficult to express, your problem
is no unsusual.
Brian Ballard wrote:
> 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.
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