suitable protease negative, lacIq E.coli

M. Alexeyev malexeyev at biost1.thi.tmc.edu
Fri Oct 27 09:39:09 EST 1995


In article <1995Oct26.143436.65026 at cc.usu.edu>, sl3wx at cc.usu.edu wrote:

> Hi Nettors,
> I would appreciate a recommendation for a protease negative strain of E.coli
> that is lac Iq and a suitable source supplying the same.
> I have my gene cloned into plasmid vector pIN-III-ompa 2 that has the 
> lpp5-lac promoter and suspect the reason I am not getting protein expression
> is proteolytic degradationof the expressed protein.
> Would welcome any thoughts on the subject.
> Thanks.
> 
> Ventris.

Dear Ventris:
I apologise if what I am about to write would seem trivial to you.
I beleive all E. coliB strains are naturally lon protease negative. You
can get one from ATCC. Sorry, can not be of much help with other
proteases. As regarding lacIq, you may consider introduction of the lacIq
allele into backbone of the plasmid or alternatively introduction it on a
compatible plasmid (say, pACYC derivative for all ColE1 vectors). Usually,
single-copy lacIq gene (like one located on F' in many common cloning
strains )is not sufficientis for full repression of strong lac-operator
controlled promoters located on multicopy plasmids (titration effect).
lacIq allele has an up-mutation in a promoter of lacIq gene that leads to
approximately 10-fold increase in expression ( I am not aware of any
changes in an affinity  of repressor to operator or mRNA/protein stability
that should be considered). Therefore, in the first approximation the
plasmid that has 10 copies per cell would   titer out an excess of
repressor. I am aware of at least one report that claims pBR322 copy
number to be about 55/cell. So, ideally you would have both gene of
interest and lacIq gene on the same plasmid. The lacIq gene should be
available from ATCC. Alternatively, you can PCR it out of one of the many
vectors.

Sincerely,
M. Alexeyev



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