dkim at nmsu.edu
Tue Jun 25 11:59:32 EST 1996
In article <4qo2kv$3rq 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.
>Jurgen Vanhauwe, Ph. D. student
Let me get this straight . . . You have two proteins that are bound to
the inner E. coli membrane (projecting into or out of the cell?) and you
want to know if they associate with each other in vivo?
There is a recent paper (well, 1995) that addresses a similar question:
Kolmar, H., et al.
"Membrane Insertion of the Bacterial Signal Transduction Protein ToxR
and Requirements of Transcription Activation Studied by Modular
Replacement of Different Protein Substructures"
EMBO J. 14(16):3895-3904. 1995
The Vibrio cholerae protein ToxR is an integral membrane protein that
acts as a transcription activator i response to environmental signals.
Transcription activation starting at the ctx promoter depends on
dimerization of the periplasmic ToxR domain.
If the periplasmic domain of ToxR is replaced by a domain that dimerizes,
a reporter gene controlled by the ctx promoter is turned on (in E. coli).
somewhat equivalent to the Yeast two-hybrid system for deterimining in
vivo protein interactions.
You might want to look into this system as a possible model for your own
dkim at nmsu.edu
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