I have been trying for months to overexpress a plant protein in e. coli. After
three vectors, several media and growth conditions and more hosts than i can
count on my fingers and toes, i am beginning to wonder if there ain't a better
way. Results from pulse labelling experimets in aT7 expression system show that
protein is being synthesized (and is soluble) but it does not accumulate to
detectable levels on Coomassie stained gels. Similarly, only very low levlels
of total protein (again about half soluble) were produced with pFLAG vectors as
detected by monoclonal antibodies to the flag epitope. I beleive the protein is
unstable in e. coli; alternatively the protein might be toxic to e. coli however
no lysis of induced cultures appears and they grow almost as well as induced
controls (vector only).
*****I would love to hear from those who have tried other expression systems,
expecially yeast and baculovirus. Any suggestions as to vectors, hosts and
growth conditions would be appreciated. ******
I am particularly interested in yeast vectors which express epitopes such as
the flag (yeasts contain similar enzymes as the plant enzyme i wish to
oeverproduce) to aid in purification; this would save me the step of having to
create my own vector/epitope/cDNA construct.
Thanks in advance!
paul-b at molbio.cbs.umn.edu