82
Combinatorial Phage Antibody Libraries
lac
Z promoter. The heavy chain possesses
a
pel
B leader sequence, whereas the light
chain has an
omp
A leader sequence to di-
rect secretion into the periplasmic space
in
E. coli
.Th
eF
dch
a
ini
sfu
s
e
dw
i
th
a sequence encoding the CT domain of
pIII. A ±ve–amino acid tether (GGGGS)
is positioned between the Fd and the
pIII to minimize interaction. The Fd frag-
ment is thus cotranslated with the pIII
CT domain and, following translocation to
the periplasm, the fusion protein is an-
chored to the inner membrane via the
pIII domain, with the antibody project-
ing into the periplasm (Fig. 2). The light
ch
a
ini
sth
enab
l
e
,g
i
v
enth
er
edo
xpo
-
tential of the periplasmic environment, to
assemble on the heavy chain to yield a
functional Fab fragment. The inclusion of
an f1 intergenic region in the vector per-
mits superinfection with an M13 helper
phage to be followed by the packaging of
single-stranded, phagemid-carrying genes
encoding heavy- and light-chain sequences
(Fig. 3). Competition between native pIII,
comprising the N-terminal domain essen-
tial for virion infectivity, and the Fab–pIII
fusion yields a phage particle carrying na-
tive pIII and monomerically displaying
Fab at the head of the ±lamentous virion.
The pComb3X vector is nearly identical
to pComb3H except that a hexahistidine
and a hemagglutinin (HA) tag are fused
to the heavy chain, followed by an am-
ber stop codon and the
gene3
sequence,
such that soluble Fab or scFv can be pro-
duced readily in nonsuppressor strains
of
E. coli
without the need for excising
Innermembran
e
O
utermembrane
C
L
N
ompA
L
LacZ
pComb3H
Hc
Amp
R
f1
Lc
pIII
His6-HA-
amber stop
pComb3X only
V
H
V
L
C
L
C
C
H1
+
+
+
+
N
pelB
V
H
C
H1
Coat
protein
III
+
+
+
+
Fig. 2
The proposed pathway of Fab
assembly in the bacterial periplasm.
Polycistronic expression of cloned light
chain (Lc) and Fd (Hc, heavy chain)/pIII
fusion is controlled by a single
lac
promoter/operator sequence in
pComb3H and pComb3X. Both vectors
are improved versions of the original
pComb3 vector and have enhanced
stability. The pComb3X vector is nearly
identical to pComb3H except that a
hexahistidine and HA tag followed by an
amber stop codon have been inserted
in-frame between Fd and
gene3,
allowing for facile detection of the
cloned single chain Fvs. The translated
antibody is directed to the periplasmic
space by
omp
A(Lc)and
pel
B(Hc)
leader sequences. Here, the light chain
assembles onto the heavy-chain
template, which is anchored to the inner
membrane by the pIII fusion. (From
Barbas et al. (1991)
Methods: A
Companion to Methods in Enzymology
,
Vol. 2, Academic Press, Orlando, FL,
pp. 119–124.)
previous page 1402 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1404 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off