Critical decision!!!

D. KIM 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).  
this is 
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 
experiments.

Daniel Kim
dkim at nmsu.edu



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