Biomedical Engineering Reference
In-Depth Information
the glucose and arabinose content of the growth
medium (Fig. 5.12). According to Guzman
et al.
(1995), the pBAD vectors permit fine-tuning of gene
expression. All that is required is to change the sugar
composition of the medium. However, this is disputed
by others (Siegele & Hu 1997, Hashemzadeh-Bonehi
et al.
1998).
Many of the vectors designed for high-level ex-
pression also contain translation-initiation signals
optimized for
E. coli
expression (see Box 5.1).
(a)
O
2
C
AraC dimer
N
N
C
No transcription
I
1
I
2
pBAD
+ arabinose
Vectors to facilitate protein purification
Many cloning vectors have been engineered so that
the protein being expressed will be fused to another
protein, called a
tag
, which can be used to facilitate
protein purification. Examples of tags include glu-
tathione-
S
-transferase, the MalE (maltose-binding)
protein and multiple histidine residues, which can
easily be purified by affinity chromatography. The
tag vectors are usually constructed so that the
coding sequence for an amino acid sequence cleaved
by a specific protease is inserted between the coding
sequence for the tag and the gene being expressed.
After purification, the tag protein can be cleaved off
with the specific protease to leave a normal or nearly
normal protein. It is also possible to include in the
tag a protein sequence that can be assayed easily.
This permits assay of the cloned gene product when
its activity is not known or when the usual assay is
inconvenient. Three different examples of tags are
given below. The reader requiring a more detailed
insight should consult the review by LaVallie and
McCoy (1995).
To use a polyhistidine fusion for purification, the
gene of interest is first engineered into a vector in
which there is a polylinker downstream of six hist-
idine residues and a proteolytic cleavage site. In the
example shown in Fig. 5.13, the cleavage site is that
for enterokinase. After induction of synthesis of the
fusion protein, the cells are lysed and the viscosity
of the lysate is reduced by nuclease treatment.
The lysate is then applied to a column containing
immobilized divalent nickel, which selectively binds
the polyhistidine tag. After washing away any
contaminating proteins, the fusion protein is eluted
from the column and treated with enterokinase to
release the cloned gene product.
N
N
Transcription
C
C
CAP
I
1
I
2
pBAD
(b)
Fig. 5.12
Regulation of the pBAD promoter. (a) The
conformational changes that take place on addition of
arabinose. (b) Western blot showing the increase in synthesis
of a cloned gene product when different levels of arabinose are
added to a culture of the host cell.
negative regulator of this promoter,
ara
C. AraC is a
transcriptional regulator that forms a complex with
-arabinose. In the absence of arabinose, AraC binds
to the O
2
and I
1
half-sites of the
ara
BAD operon,
forming a 210 bp DNA loop and thereby blocking
transcription (Fig. 5.12). As arabinose is added to
the growth medium, it binds to AraC, thereby releas-
ing the O
2
site. This in turn causes AraC to bind to
the I
2
site adjacent to the I
1
site. This releases the
DNA loop and allows transcription to begin. Binding
of AraC to I
1
and I
2
is activated in the presence of
cAMP activator protein (CAP) plus cAMP. If glucose
is added to the growth medium, this will lead to a
repression of cAMP synthesis, thereby decreasing
expression from the
ara
BAD promoter. Thus one can
titrate the level of cloned gene product by varying
