Biomedical Engineering Reference
In-Depth Information
Table 5.3
Control of expression of
chloramphenicol acetyltransferase
(CAT) in
E. coli
by three different
promoters. The levels of CAT are
expressed as µg/mg total protein.
Promoter
Uninduced level of CAT
Induced level of CAT
Ratio
l
P
L
0.0275
28.18
1025
trc
1.10
5.15
4.7
T7
1.14
15.40
13.5
When maximizing gene expression it is not enough
to select the strongest promoter possible: the effects
of overexpression on the host cell also need to be con-
sidered. Many gene products can be toxic to the host
cell even when synthesized in small amounts.
Examples include surface structural proteins (Beck
& Bremer 1980), proteins, such as the PolA gene
product, that regulate basic cellular metabolism
(Murray & Kelley 1979), the cystic fibrosis trans-
membrane conductance regulator (Gregory
et al.
1990) and lentivirus envelope sequences (Cunnin-
gham
et al.
1993). If such cloned genes are allowed
to be expressed there will be a rapid selection for
mutants that no longer synthesize the toxic protein.
Even when overexpression of a protein is not toxic to
the host cell, high-level synthesis exerts a metabolic
drain on the cell. This leads to slower growth and
hence in culture there is selection for variants with
lower or no expression of the cloned gene because
these will grow faster. To minimize the problems
associated with high-level expression, it is usual to
use a vector in which the cloned gene is under the
control of a
regulated
promoter.
Many different vectors have been constructed for
regulated expression of gene inserts but most of
those in current use contain one of the following
controllable promoters:
these promoters also carry the
lac
O operator and the
lac
I gene, which encodes the repressor.
The pET vectors are a family of expression vectors
that utilize phage T7 promoters to regulate synthesis
of cloned gene products (Studier
et al.
1990). The
general strategy for using a pET vector is shown in
Fig. 5.10. To provide a source of phage-T7 RNA
polymerase,
E. coli
strains that contain gene 1 of the
phage have been constructed. This gene is cloned
downstream of the
lac
promoter, in the chromo-
some, so that the phage polymerase will only be
synthesized following IPTG induction. The newly
synthesized T7 RNA polymerase will then transcribe
the foreign gene in the pET plasmid. If the protein
product of the cloned gene is toxic, it is possible to
minimize the uninduced level of T7 RNA poly-
merase. First, a plasmid compatible with pET vectors
is selected and the T7
lys
S gene is cloned in it. When
introduced into a host cell carrying a pET plasmid,
the
lys
S gene will bind any residual T7 RNA poly-
merase (Studier 1991, Zhang & Studier 1997). Also,
if a
lac
operator is placed between the T7 promoter
and the cloned gene, this will further reduce tran-
scription of the insert in the absence of IPTG
(Dubendorff & Studier 1991). Improvements in the
yield of heterologous proteins can sometimes be
achieved by use of selected host cells (Miroux &
Walker 1996).
The
P
L
, T7,
trc
(
tac
) or BAD.
Table 5.3 shows the different levels of expression that
can be achieved when the gene for chloramphenicol
transacetylase (CAT) is placed under the control of
three of these promoters.
The
trc
and
tac
promoters are hybrid promoters
derived from the
lac
and
trp
promoters (Brosius
1984). They are stronger than either of the two
parental promoters because their sequences are
more like the consensus sequence. Like
lac
, the
trc
and
tac
promoters are inducibile by lactose and
isopropyl-
λ
P
L
promoter system combines very tight
transcriptional control with high levels of gene
expression. This is achieved by putting the cloned
gene under the control of the
P
L
promoter carried on
a vector, while the
P
L
promoter is controlled by a
c
I
repressor gene in the
E. coli
host. This
c
I gene is itself
under the control of the tryptophan (
trp
) promoter
(Fig. 5.11). In the absence of exogenous tryptophan,
the
c
I gene is transcribed and the
c
I repressor binds
to the
P
L
promoter, preventing expression of the
λ
β
-d-thiogalactoside (IPTG). Vectors using
