Purifiying insoluble protein
bernard at elsie.nci.nih.gov
Wed Nov 16 16:35:44 EST 1994
In article <39vct7$9al at mserv1.dl.ac.uk>, ANDY PHILLIPS <andy.phillips at afrc.ac.uk> (Tel +44 275 392181 EXT 257) writes:
> We're trying to express a cytochrome P450 in E.coli to investigate its
> activity. We've more or less given up hope that we will get assembled, active
> enzyme, but we can get lots of insoluble protein, presumably in inclusion
[Tale trimmed for brevity]
> RFC-822 : ANDY.PHILLIPS at BBSRC.AC.UK : University of Bristol
Yeah, P-450 has been a pain to express in bacteria. Most people stick to
yeast, baculo or mammalian cells. Probably the most successful work has
been done by Zuyu Guo in Fred Guengerich's group, 'though success has also
been had by other groups expressing their favourite forms. If I remember
correctly, for good expression of active protein in bacteria you have to
trim off the N-terminal membrane anchor and Jud Coon's group has spent
some time looking at the aggregation level of the protein with different
N-terminal modifications. Some groups (eg. Mike Waterman) study the
activity of the protein in intact bacteria (apparently there's a flavodoxin
reductase that can substitute for P-450 reductase). I think His-tagging
is a favourite for purification of active forms.
There's a lot of good work out there so don't start from scratch!
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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