sergioal at bbm1.ucm.es
Tue Oct 9 06:37:31 EST 2001
It seems hard to detect where the problem is, since you didn't change
anything. So, just some hints:
have you checked the amount of plasmid/cell? maybe an old antibiotic stock
allows cells without plasmids or with low copy number plasmid to survive.
are you sure the induction is working? try the IPTG over another
overexpression clone or over a Plac-lacZ plasmid and check the miller units.
If you don't trust on protease inhibitors, you could lyse teh cells directly
in the PAGE loading buffer, and check whether they are really overexpressing
the protein, before performing the purification.
a good supposed_to_be_positive control could be to use a pMAL or pGST vector
and check the plasmid copy number, whether you can see the bands these
vectors produce after induction with IPTG (loading the heat lysed cells),
after lysing using the sonicator/french press/whatever you use, and after
passing through your columns (to which they obviously shouldn't bind).
jon ha escrito:
> We have a pET15b construct that we have been using routinely to express
> an N-terminally His-tagged protein in E. coli. Although there has been
> some variation in absolute expression levels and yields between preps,
> the overall expression has been good. Recently, however, the yield has
> plummeted, leaving us confused as to what could be going wrong.
> The plasmid prep used to transform the expression cells has been the
> same tube all along. Cells are BL21(DE3)pLysS, which are
> freshly-transformed before each prep. Nickel chelate columns are being
> used to purify the protein. Whereas before, about 1ul of the peak
> fractions would produce a huge band on a coomassie-stained gel, now,
> about 5 times more is only just visible on the gel. All growth
> conditions are the same as previously, as are methods of lysis, protease
> inhibitors, and purification, etc.
> Any advice would be greatly appreciated.
> Thanks in advance,
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