kandasamy ravi 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.
> what do i do to get over expression?and why there is a leaky expression in
> uninduced control.
> somebody help me please...
> All suggestion welcome
>> Thank you
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Are you sure you're inducing at all? I doubt the pLysS cells are leaky enough to have
an uninduced and induced sample with the same level of expression. Did you determine
the optimal parameters for induction, or are you just growing the cultures and throwing
in some IPTG after a few hours? In my experince (with pQE, pET-15b, and my homemade
vectors), nearly every 6-his protein needs to have induction parameters worked out
Remember also that the BL21(DE3)pLysS will give a lower induction than regular
BL21(DE3) will, because of the lysozyme inhibition. Are you also sure you're looking
at the correct protein in youre samples? Lysozyme is about 17 kDa, and the lacI is
about 39 kDa. You could be seeing these proteins and think they're yours.
--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
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