inprove E coli expression

Mark Bowen mbo at slac.stanford.edu
Wed Jan 3 08:46:21 EST 2001


If you are geting good levels of expression of MBP/GST without the
hormone attached, I'd suggest you are either getting premature
termination or are having a proteolysis problem.
Many peptide hormones lack regular structure.  Try doing a time course
of expression (eg sampling every30 min with SDS-PAGE on the lysate) to
see if you can see degradation to the smaller product during the
expression run.
You could also look at the codon usage to see if you have rare codons
(i.e. codons not used by coli).

Gramscale is really a lot to expect from a fusion protein expression
particularly if this hormone is small relative to GST/MBP.  If the
sequence is less than 50aa i'd suggest having it synthesized rather than
expression.

--
Mark Bowen








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