>I wonder whether someone could give me some leads about how to express
>proteins
>with Se-Met for MAD phasing. I read the review by Ms. Doublie and Charlie
>Carter but
>that's it about. In particular, I'd like to know about suitable bug
>strains, new reviews and recipes, and
>good and bad stories, for example, how transferable protocols are that
>already worked
>in other E.Coli strains, and what about the oxidation issues.
For our system we were able to produce SeMet protein without the use of a
methionine auxotroph (see our crystallisation paper - Vos, de Jersey and
Martin due out soon in JStructBiol). The expression system we used is
pT7-7 heat induced expression and it gives loads of protein in about 2 and
a half hours. During this time, the cells are probably not able to
synthesise their own methionine so all the methionine is incorporated
asSe-Met supplied in the minimal media (better than 95% incorporation from
mass spec analysis). However if the expression is much slower as in
constitutive expression or induced expression over many hours or overnight,
the cells may have the opportunity to synthesise their own methionine (and
incorporation may be much lower than we got).
We did not have any problem with SeMet oxidation but did take the
precaution of adding 5mM DTT to all the purification and crsytallisation
protocols. The SeMet protein has been crystallised, the crystals diffract
to 1.8A and the data have been used in MIR phasing.
Siska Vos, John de Jersey and Jenny Martin
______________________________________________________________________
Dr Jenny Martin
J.Martin at mailbox.uq.oz.au
Centre for Drug Design and Development
University of Queensland
Tel: + 61 (0)7 3365 4942
Brisbane QLD 4072 AUSTRALIA Fax: + 61 (0)7
3365 1990
http://www.uq.oz.au/ddd/index.html
______________________________________________________________________
