protein expression in PLysS

[Cornelius Krasel]

"kandasamy ravi" <ravi77 at MAILCITY.COM> wrote:
> I have cloned my gene in pET15b transformed in to DH5 and isolated DNA and
> retransformed in to pLysS BL21(DE3).checked the sequences(it is in right
> frame) But i don't get overexpression just little increase in expression.and
> the uninduced one is expressing as good as induced.I tried different IPTG
> concentration but no use and even the story is same in C43/C41 cell lines.

I remember that someone on this list reported previously that he ran into
a similar problem. Finally he found out that his LB contained lactose. So,
one possibility would be to obtain a different batch of peptone/yeast extract
or alternatively use a defined medium for growing the bacteria.

You could also try to switch to pLysE cells instead of pLysS, but if
you can already see expression of your construct without IPTG, I somewhat
doubt that leakyness of the T7 promoter is the problem.

--Cornelius.

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/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
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