How to express toxic proteins in bacteria

Emir Khatipov ekhatipo at NOSPAMmidway.uchicago.edu
Thu Sep 14 01:50:00 EST 2000


It is a well-known disadvantage to the standard, IPTG-inducible, BL21(DE3)
system that the very low expression of T7 RNAP that occurs in the absence of
inducer can be deleterious in the presence of extremely toxic target genes.
If you are not ready to reclone your gene into something better that pET
(e.g., NEB's pBAD which is arabinose-inducible system), there is a
well-tested way to deal with the basal T7 RNAP expression. Another plasmid,
pLysS,  is introduced into the cells that encodes lysozyme from the same T7
phage as the RNAP. Lysozyme selectively digests RNAP in uninduced cells,
allowing normal cell growth. Induction with IPTG will rise T7 RNAP levels
hundreds of times, so the effect of lysozyme will be negligible. The main
benefit is that your protein appears in the cell only for a relatively short
induction period. By that time you already have a well-grown culture and
don't have to care whether the cells do grow after addition of IPTG.
Hope that helps.
Emir

"Hui Wang" <wang.508 at postbox.acs.ohio-state.edu> wrote in message
news:39bf27e6.5658660 at nntp.service.ohio-state.edu...
> Hi,  I am trying to express my favorite gene cloned in pET vector in
> E. Coli BL21. The N-terminal truncated protein (missing 13AAs) can be
> expressed to high level after 1mM IPTG induction for 2 to 4 hours.
> However the full length protein can not be expressed at all. After the
> 2.5 ml overnight culture (some times this culture  grew fine) was put
> into 50 ml LB, the bacteria grew very very slow as compared with the
> truncated clone. I guess that the full length is toxic to bacteria,
> but why its expression is not suppressed before adding the IPTG? How
> can I completely suppress its expression (adding glucose or others?
> how much?) so that the bacterial density can be high and then by
> adding IPTG to expression my full length protein. I need your kind
> help. Thanks
>
> Hui Wang







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