[Arabidopsis] Chloroplast envelope proteins

jbrumos from ncsu.edu via arab-gen%40net.bio.net (by jbrumos from ncsu.edu)
Wed May 11 16:04:09 EST 2011


Hello,

I would like to ask for the advice of those of you who had the experience
with expressing plant chloroplast envelope proteins in E. coli.

* Does a predicted transit peptide need to be removed from the E. coli
construct?
* Do codons need to be modified to reflect optimal E.coli codon usage?
* Would the plant protein be targeted into the E.coli cell membrane by
default or
does a signal sequence need to be added?
* Is there any reliable way to predict the topology of the protein in the
membrane,
i.e. which way (in or out of the cell) the N- and C-termini would face?
* If the chloroplast inner membrane protein works as a transporter and the
hypothesis is that in plants it pumps the substrate out of the plastid, is
there a
way to predict the direction of substrate flow in E. coli (assuming the
transporter
is functional in bacteria)?
* If the plant chloroplast protein (minus the transit peptide) is
expressed in
yeast, does all of the above hold true?

Thank you for your help,

Javier Brumos



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