Trouble expressing small peptide
rafamaldo at yahoo.com
Thu Mar 10 07:12:56 EST 2005
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?
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