protein expression systems

Wolfgang Wegloehner wegloehner at mpimg-berlin-dahlem.mpg.de
Sun Aug 8 06:43:07 EST 1993


In article <CBAnuz.Lt6 at news2.cis.umn.edu>, paul-b at molbio.cbs.umn.edu (Paul Bucciaglia) writes:
> Hello folks,
> 
> 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 Bucciaglia
> 
> paul-b at molbio.cbs.umn.edu
>  
> 

Dear Paul,

I've got exactly the same problem. I've tried to overexpress an nuclear encoded
chloroplast ribosomal protein in E.coli with lots of different expression
systems (lambda, trc, tac, and the pET-vectors) with absolutely no visible
expression on coomassie (but the protein was detectable by western blot
analysis). Because I had no time to establish the yeast or baculovirus system I
gave E.coli a last chance. I used an pET derived vector who secretes your
protein into the periplasm. It works wonderful. I can see a very faint new band
occuring on total cell extract SDS-PAGE, but if I analyze the periplasmic
fraction I have only two main components: beta-lactamase and my protein. In
addition I used the BL21(DE3) strain witch is deficient in outer membrane
protease (can see no degadation at all). By a simple two step-purificatin (IEC
and HPLC) I can get now about 10 mg of protein from one liter. The only problem
is that with this system it is rather difficult to handle large culture volumes
(have a look in Current Protocols [16.6.7]). I would give coli another chance.
Hope this helps

Wolf



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