Expression problems

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


Hi,

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,

jon.




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