80
Combinatorial Phage Antibody Libraries
dimer called a
diabody
or a trimer called a
triabody
.
Both Fab and scFv formats are com-
monly used, and there can be both ad-
vantages and disadvantages of an scFv
form. The use of splicing overlap extension
PCR in scFv library construction reduces
the number of restriction enzymes and
cloning steps, which may lead to better
library diversity. In addition, scFvs are
typically produced in much higher yields
in
Escherichia coli
than their Fab counter-
parts. ScFvs may be advantageous when
a smaller molecule is desirable, such as
for probing narrow cavities. Finally, scFvs,
with their tendency to oligomerize, may
be exploited for multivalent interactions,
although this feature is often a disadvan-
tage when oligomerization is unwanted.
Hence, Fabs may be preferable to scFvs if
stability is an issue, or if the intention
is eventually to generate a whole anti-
body molecule.
Structurally, the Ff (includes f1, fd,
and M13) ±lamentous phage particle is
composed of a closed circular single-
stranded DNA genome encapsulated by
a long thin protein coat. The majority
of the phage particle is composed of
approximately 2700 copies of major coat
protein
8
(g8p
or
pVIII),
which
are
distributed along the length of the virion.
About ±ve copies of a minor coat protein
3 (g3p or pIII) are arranged at the tail of
the phage. The pIII has two ‘‘N-terminal
domains’’ (N1 and N2) and one C-terminal
domain (CT) that are separated by glycine-
rich linkers. The N2 domain binds to F
0
pili, facilitating infection of male
E. coli
,
and N1N2 additionally serves to confer
immunity from superinfection by other Ff
phages. The membrane-bound CT domain
serves to cap the protein to the trailing end
of the phage ±lament. Of the other minor
coat proteins, pVI is also present in about
±ve copies with pIII at the tail of the phage,
with about ±ve copies each of pVII and pIX
deployed at the head.
All the coat proteins on the ±lamentous
phage have been used as fusions for
displaying polypeptides on the phage coat,
and, to date, all but pVI have been
used to display antibody fragments (pVI
displays polypeptides via its C-terminus
and has been used primarily for cDNA
libraries). Although antibody display by
pVIII is possible, it has not gained wide-
spread use (see below). An Fab library
has been described in which the heavy
and light chains are fused to pVII and
pIX respectively, and an scFv library based
on pIX has been successfully screened
to yield novel antibodies. Display by pIX
may have the advantage that because the
antibody is displayed at the opposite end
of the phage from pIII, infectivity is less
likely to be affected. Nevertheless, the most
common choice by far for antibody display
is pIII, and pIII display has proven to be
extremely robust.
Filamentous phage are nonlytic and, un-
like their lytic counterparts, new virus
particles are not formed in the cytoplasm
of the host cell for release by lysis. In-
stead, virions are extruded through the
outer membrane, leaving the bacterial cell
intact. The extrusion process begins as
the ±lamentous phage particle assembles
on the inner bacterial membrane. Then, a
pool of pIII molecules is secreted into the
inner membrane for incorporation into
an emerging phage particle. These pIII
molecules are anchored in the membrane
via their CT domains, with the remain-
der of the sequence extending into the
periplasmic space. Of the coat proteins,
only pIII and pVIII have leader sequences
at their N-termini, which are cleaved off
in the periplasm during phage assembly.
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