two proteins in E. coli

Mr. Chris Hoyle bmbckh at biovax.leeds.ac.uk
Wed Jun 26 01:42:20 EST 1996

In article <4qo2p5$3sl at news.Belgium.EU.net>
Jurgen Vanhauwe <jvanhauw at janbe.jnj.com> writes:

> As I want to study the interaction of two proteins in E. coli, I'm
> looking for some advice. I already expressed one protein in E. coli and
> now I'm up to the second protein.
> My question is: Wchich is the most feasible method to make this proteins
> interact in E. coli inner membranes? Is this coexpression by means of
> compatible plasmids or polycistronic plasmids or even genomic integration
> of one of the proteins, or might it be more intruiging to try to
> reconstitute that interaction by fusion of phospholipid vesicles
> (containing the purified second protein from a mammalian source) and E.
> coli membranes (or even lysozyme treated bacteria). In the last case I
> have the advantage that the second protein is posttranslational modified,
> but it hasn't really been shown that this modification is necessary for
> both proteins to interact.
> Since this is a very critical decision to make, I'd really appreciate any
> comment or suggestions.
If you can easily purify both proteins, due so. Assuming your proteins
are from a eukaryote (?mammalian) an experiment you may already be able
to do is as follows:
label (e.g. 35S-met) the protein expressed in E. coli and purify.
Add to second protein preparation, presumably in vesicles.
Separate total protein by native-PAGE and detect using phosphorimager
or equivalent.
Compare with labeled protein only.
You may, however need to treat with a crossliking reagent.

Alternatively you may be able to carry out the above in detergent
instead of in vesicles. 

Just a suggestion. Looks like a good project.  

Chris Hoyle

   Department of Biochemistry and Molecular Biology
University of Leeds             phone +44 0113 2333172
Leeds LS2 9JT                   FAX   +44 0113 2333167
Great Britain           e-mail BMBCKH at biovax.leeds.ac.uk

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