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Autoinduction
of Expression in the T7 Expression System
Revisions
5/13/2002: The NPS recipe was corrected.
Rationale
The T7 expression system present in the pET
vectors from Novagen (pdf
manual) was originally
developed by Studier
at Brookhaven National Laboratories (Studier
and Moffatt, 1986). The pET plasmids contain an expression cassette
in which the gene of interest is inserted behind an
extremely strong promoter from the E. coli bacteriophage T7.
In the absence of
the T7 polymerase, this promoter is completely shut off. For
expression, the pET plasmids are transformed into bacteria strains
that typically
contain a single copy of the T7 polymerase on the chromosome
in a lambda lysogen (the most commonly used lysogen is known
as DE3).
The T7 polymerase
is under the control of the Lac-UV5 lac promoter. When cells
are grown in media without lactose, the lac repressor (lacI)
binds
to the lac
operator and prevents transcription from the lac promoter. When
lactose is the sole carbon source, or when the lactose analog
IPTG is added
to the media, lactose (or IPTG) binds to the repressor and induces
its dissociation from the operator, permitting transcription
from the promoter. Finally, addition of glucose to the culture
media
contributes
to repression of the T7 RNA polymerase via the mechanism of catabolite
repression, as seen in Figure 1 below.
Figure 1. Transcriptional control of T7 gene
1 in lambda-DE3 lysogens
Transcription of T7 gene 1 (encoding T7 RNA polymerase) in pET
System expression hosts (?DE3 lysogens) is controlled by the
L8-UV5 lac
promoter. T7 gene 1 is transcribed as the second gene in a
bicistronic mRNA (the first gene contains an N-terminal fragment
of lacZ that
includes the ?-peptide coding region). Positions of the three
mutations of the wild type lac promoter region are indicated
by colored circles.
The lac repressor (lacI gene product) binds to lacO1, and then
interacts with pseudo-operators lacO2 and lacO3 to prevent
transcription by E. coli RNA polymerase. The inducer IPTG binds
to the repressor,
reducing its affinity for lacO1 and thus enabling transcription
to occur. When cAMP levels are sufficiently high (e.g., in
the absence
of glucose)
the CAP/cAMP complex is formed and binds immediately upstream
from the promoter to fully stimulate transcription. In the presence
of glucose, CAP/cAMP is not formed and transcription is
decreased. This is called the glucose effect, or catabolite repression.
(Figure and caption form Novy
and Morris, Novagen, Inc.).
In the classical protocol for induction of protein expression in
T7 systems, bacteria are grown in complex media (such as LB) to mid-log
phase, and expression is induced by the addition of IPTG. This protocol
requires careful monitoring of the growth of the cultures, so that
they can be induced before they reach saturation. In addition, once
they are induced, bacterial proliferation shuts off, as the cells
now become devoted to production of proteins from the T7 promoter
on the plasmid.
The goal of this new protocol is to grow the bacteria
in a defined media in which expression of the T7 polymerase is
automatically induced in late log-phase growth due to the depletion
of carbon sources
other than lactose. As the other carbon sources become depleted,
the cells are forced to use lactose, at which point synthesis of
the T7 polymerase is turned on. In addition, once glucose is depleted,
cAMP levels rise and catabolite repression is relieved. The advantages
of this revised protocol are:
- It is no longer necessary to carefully monitor the bacterial
cultures before addition of the synthetic inducer.
- Cultures grown under the modified conditions yield a greater
cell mass-and concomitantly greater yield of recombinant
protein-since the media is well buffered and the T7 polymerase
is induced in
late
log-phase.
- Protein production cultures can be incubated overnight, since
they do not need to be manipulated.
- It avoids the high cost of IPTG.
Overview
The basic steps of the autoinduction protocol are as follows:
- Transform the expression plasmid into the BL21(DE3)
strain of E. coli, and select transformants using agar plates
with appropriate
antibiotics.
It is also possible, and in fact desirable to use modified
versions of BL21(DE3) that also carry a plasmid coding
for tRNAs that
are common in mammalian cells but rare in bacteria. Among
the available
strains are "Rosetta(DE3)" from
Novagen and "BL21(DE3)RP" or
"BL21(DE3)RIL" from Stratagene.
- Transfer a single colony of bacteria into an Inoculum Culture
in non-inducing media that does not permit expression of
the T7 polymerase. The Inoculum Culture is typically started
first
thing
in the morning,
and is ready by the end of the day.
- Transfer a small aliquot (~150 µl) of the Inoculum Culture
into flasks containing the autoinduction media.
Typically, these will be 2-liter flasks that contain 400
ml of media. If higher volumes of media are used, the
cultures will
not be sufficiently
aerated, and they will reach saturation at a lower
cell density. This defeats the purpose of using a larger volume
of
media. - Grow the Induction Cultures
overnight with shaking at 300 rpm at 37°C.
- The next morning, measure the density of the cultures,
save an aliquot for analysis of total proteins by
SDS-PAGE, and
harvest the
bacteria by centrifugation.
- Immediately after harvest, process the cells using
the Inclusion Body isolation procedure.
Do not freeze the bacteria after
protein production unless absolutely necessary.
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