jonner at NOSPAM.bigfoot.com
Tue Oct 9 05:09:11 EST 2001
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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