How to express toxic proteins in bacteria - correction!

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Thu Sep 14 15:01:46 EST 2000


The previous post had some errors, so let me summarize the correct answer. In
strain BL21(DE3), the T7 RNA polymerase gene is under the control of the
lacUV5 promoter. Since the lac promoter cannot be shut off 100% (induction of
the lac operon requires the presence of beta-galactosidase), there will always
be some T7 polymerase synthesized in these cells. To overcome this, there are
three common approaches.

In the first, one uses the host cells BL21(DE3)pLysS or BL21(DE3)pLysE. These
synthesize low (pLysS) or high (pLysE) levels of the phage T7 lysozyme
protein. T7 lysozyme binds to and inhibits T7 RNA polymerase (it does not
degrade the polymerase!). The concentrations of lysozyme in pLysS are low
enough that when the T7 polymerase gene is turned on, normal expression of the
target gene is observed. We have use BL21(DE3)pLysS for all our T7 expression
work; we see little if any target protein before induction, and up to 50% of
total cell protein after induction. In BL21(DE3)pLysE, the concentrations of
lysozyme are high enough that--according to the literature and supplier's
information--synthesis of the target protein will be reduced. A side advantage
of using the pLysS host is that the cells are easily lysed by three cycles of
freezing at -70C followed by thawing. (A disadvantage of the pLysS plasmid is
that because it's a pACYC184 derivative which confers chloramphenicol
resistance to cells, it's incompatible with other pACYC-based or CamR
plasmids.)

The second method is to use a hybrid T7/lac promoter, in which the lac
operator has been placed just downstream of the T7 polymerase start site. This
promoter is claimed to give tight control alone, and exceptionally tight
control in conjunction with the pLysS plasmid.

One must note that the chromosomally-integrated T7 polymerase gene cannot be
down-regulated by growth of cells in medium containing glucose. The lacUV5
promoter does not bind CAP (cAMP-receptor protein), so is not subject to
catabolite repression.

A third method is to use a host cell lacking the T7 polymerase gene, and
induce target protein synthesis by infection with a lytic phage carrying the
T7 polymerase gene. The phage is available from Novagen and other sources.

Other: As far as I know, the T7 expression system gives the highest levels of
protein synthesis, but the protein is more susceptible to aggregation. This
can be minimized by slowing down the rate of protein synthesis by i) inducing
cells at 15-20C, and ii) inducing with a low concentration of IPTG (25-100
microM, rather than 1 mM).  Also, once the T7 RNA polymerase gene has been
induced, the cells stop growing because all of their energy is spent
synthesizing T7-directed mRNA and protein. Cessation of growth is a test that
the expression system is working.

-=-=-=-=-=-=-=-=-=-
THe original question was:

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

Emir's answer said, in part:

ð there is a
ð well-tested way to deal with the basal T7 RNAP expression. Another plasmid,
ð pLysS,  is introduced into the cells that encodes lysozyme from the same T7
ð phage as the RNAP. Lysozyme selectively digests RNAP in uninduced cells,
ð allowing normal cell growth.
<snip>
ð benefit is that your protein appears in the cell only for a relatively short
ð induction period. By that time you already have a well-grown culture and
ð don't have to care whether the cells do grow after addition of IPTG.

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