Foreign protein expression in E. coli
Mark D. Garfinkel
garfinkl at iitmax.iit.edu
Sun Feb 6 10:53:30 EST 1994
In article <9402050924.AA08685 at pclsp2>
vinz at PCLSP2.KUICR.KYOTO-U.AC.JP (Vincenzo Nardi-Dei) writes:
>I have recently cloned an hydrolase of 35,000 M.W. from Pseudomonas
>in to E. coli XL1 Blue, provided from Stratagene.
>The plasmid used is pUC118, the insert is about 1,200 bp
>and the expression is around 10 % of the cell crude extract
A wild guess is that the pUC118 plasmid's high copy number
is titrating out the lac repressor produced by the XL1-Blue F'-lacI(Q)
episome. In effect you have constitutive high level synthesis of the
hydrolase that is deleterious. I think the problem may be with the
*vector* and not the host. Try subcloning the insert into another
plasmid, say a TrpE fusion vector of the pATH series. The insert will
therefore be under *positive* transcriptional control that you can
initiate by adding indoleacetic acid to the medium when the cells are
at the right density for your needs.
The newsgroup bionet.molbiol.methds-reagnts is designed for
discussing these questions. You might get better advice there.
Mark D. Garfinkel (e-mail: garfinkl at iitmax.acc.iit.edu)
My views are my own, which is why they're copyright 1994
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