Combinatorial Phage Antibody Libraries
83
f1
d.s.
s.s.
pComb3H
phagemid
Proteins
Fab
pIII
Phage DNA
Helper
phage
Phage
Fab
Infection
Phagemid DNA
Phage DNA
Phagemid DNA
Fig. 3
Helper phage rescue of pComb3H phagemid yielding a phage
display library. Incorporation of the f1 origin of replication into the
pComb3H phagemid means that upon coinfection with an M13 helper
phage, single-stranded pComb3H DNA will be packaged and extruded
from the bacterial cell as an assembled phage. As the assembling phage is
extruded from the cell, it is ‘‘capped’’ by both the native pIII (provided by
the helper phage) and the pIII–Fab complex (derived from pComb3H).
The resulting phage has Fab displayed primarily monovalently on its
surface and contains the corresponding antibody genes inside. [Adapted
from Burton, D.R. and Barbas, C.F. (1993)
Immunomethods
,
3
, 155–163.]
gene3
.Weno
tetha
tinsomephaged
i
s
-
play systems, the entire
gene3
sequence is
incorporated rather than just the CT do-
main, in the phagemid fusion. Since one
function of the amino-terminal half of pIII
is to prevent superinfection of
E. coli
by
other Flamentous phages, in this approach
the expression of the phagemid must be
halted for helper phage infection and in-
duced for phage display, thus complicating
the selection procedure.
Some antibody fragments are poorly
displayed on phages. To increase the
display of such antibodies on the sur-
face of the phage, some creative strate-
gies have been devised. ±or example,
overexpressing certain
chaperones
has
facilitated increased display and stability
of poorly folding antibody fragments. In
another example, a random-mutagenesis-
selection approach resulted in increased
display of antibodies and other polypep-
tides, the success of which could either
be traced to beneFcial mutations in the
antibody itself or to the leader peptide
sequence. ±inally, a general strategy for in-
creasing antibody display on pIII has been
simply to use a specially designed helper
phage that has a defective
gene3
,suchthat
oligovalent display of phagemid-encoded
antibody is enforced.
Some alternatives to phage display have
been developed in recent years, and two of
them deserve particular mention. Yeast
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