Biomedical Engineering Reference
In-Depth Information
Promoter
Signal
Random
peptide
DNA
M13 gene
III
Phage
vector
f1ori
Infect
male
E. coli
Phage
particle
Gene
III
protein
DNA
Random
protein
Test binding to antibody
or receptor
Fig. 7.13
The principle of phage display
of random peptides.
carrying a single copy of the P3 or P8 gene from
M13 plus the M13
ori
sequence (i.e. a phagemid,
see p. 70). As before, the random DNA sequence is
inserted into the P3 or P8 gene downstream from
the signal peptide-cleavage site and the construct
transformed into
E. coli
. Phage particles displaying
the amino acid sequences encoded by the DNA insert
are obtained by superinfecting the transformed cells
with helper phage. The resulting phage particles are
phenotypically mixed and their surfaces are a
mosaic of normal coat protein and fusion protein.
Specialized phagemid display vectors have been
developed for particular purposes. For example,
phagemids have been constructed that have an
amber (chain-terminating) codon immediately down-
stream from the foreign DNA insert and upstream
from the body of P3 or P8. When the recombinant
phagemid is transformed into non-suppressing
strains of
E. coli
, the protein encoded by the foreign
DNA terminates at the amber codon and is secreted
into the medium. However, if the phagemid is
transformed into cells carrying an amber suppres-
sor, the entire fusion protein is synthesized and
displayed on the surface of the secreted phage par-
ticles (Winter
et al.
1994). Other studies (Jespers
et al.
1995, Fuh & Sidhu 2000, Fuh
et al.
2000) have
shown that proteins can be displayed as fusions to
the carboxy terminus of P3, P6 and P8. Although
amino-terminal display formats are likely to domin-
ate established applications, carboxy-terminal display
permits constructs that are unsuited to amino-
terminal display.
