Biomedical Engineering Reference
In-Depth Information
Table 5.4
Peptide epitopes, and the
antibodies that recognize them, for use
in assaying fusion proteins.
Peptide sequence
Antibody recognition
-Glu-Gln-Lys-Leu-Ile Ser-GIu-Glu-Asp-Leu-
Anti-
myc
antibody
-His-His-His-His-His-His-COOH
Anti-His (C-terminal) antibody
-Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-
Anti-V5 antibody
Asp-Ser-Thr-
epitopes are given in Table 5.4. These antibodies can
be used to detect, by western blotting, fusion pro-
teins carrying the appropriate epitope. Note that a
polyhistidine tag at the C terminus can function for
both assay and purification.
Biotin is an essential cofactor for a number of
carboxylases important in cell metabolism. The
biotin in these enzyme complexes is covalently
attached at a specific lysine residue of the biotin
carboxylase carrier protein. Fusions made to a
segment of the carrier protein are recognized in
E.
coli
by biotin ligase, the product of the
bir
A gene, and
biotin is covalently attached in an ATP-dependent
reaction. The expressed fusion protein can be
purified using streptavidin affinity chromatography
(Fig. 5.14).
E. coli
expresses a single endogenous
biotinylated protein, but it does not bind to strepta-
vidin in its native configuration, making the affinity
purification highly specific for the recombinant
fusion protein. The presence of biotin on the fusion
protein has an additional advantage: its presence can
be detected with enzymes coupled to streptavidin.
The affinity purification systems described above
suffer from the disadvantage that a protease is
required to separate the target protein from the
affinity tag. Also, the protease has to be separated
from the protein of interest. Chong
et al.
(1997,
1998) have described a unique purification system
that has neither of these disadvantages. The system
utilizes a protein splicing element, an intein, from
the
Saccharomyces cerevisiae VMA1
gene (see Box 5.2).
The intein is modified such that it undergoes a
self-cleavage reaction at its N terminus at low
temperatures in the presence of thiols, such as
cysteine, dithiothreitol or
the N terminus of the gene encoding the intein. DNA
encoding a small (5 kDa) chitin-binding domain
from
Bacillus circulans
was added to the C terminus
of the intein for affinity purification (Fig. 5.15).
The above construct is placed under the control of
an IPTG-inducible T7 promoter. When crude extracts
from induced cells are passed through a chitin col-
umn, the fusion protein binds and all contaminating
proteins are washed through. The fusion is then
induced to undergo intein-mediated self-cleavage on
the column by incubation with a thiol. This releases
the target protein, while the intein chitin-binding
domain remains bound to the column.
Vectors to promote solubilization of
expressed proteins
One of the problems associated with the overproduc-
tion of proteins in
E. coli
is the sequestration of the
product into insoluble aggregates or 'inclusion bod-
ies' (Fig. 5.16). They were first reported in strains
overproducing insulin A and B chains (Williams
et al.
1982). At first, their formation was thought to
be restricted to the overexpression of heterologous
proteins in
E. coli
, but they can form in the presence
of high levels of normal
E. coli
proteins, e.g. subunits
of RNA polymerase (Gribskov & Burgess 1983).
Two parameters that can be manipulated to re-
duce inclusion-body formation are temperature and
growth rate. There are a number of reports which
show that lowering the temperature of growth
increases the yield of correctly folded, soluble protein
(Schein & Noteborn 1988, Takagi
et al
. 1988,
Schein 1991). Media compositions and pH values
that reduce the growth rate also reduce inclusion-
body formation. Renaturation of misfolded proteins
can sometimes be achieved following solubilization
in guanidinium hydrochloride (Lilie
et al.
1998).
-mercaptoethanol. The
gene encoding the target protein is inserted into a
multiple cloning site (MCS) of a vector to create a
fusion between the C terminus of the target gene and
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