Protein Engineering, Vol 11, 825-832, Copyright © 1998 by Oxford University Press
PS Daugherty, G Chen, MJ Olsen, BL Iverson and G Georgiou
A quantitative system for screening combinatorial single-chain Fv (scFv)
antibody libraries was developed utilizing surface display on Escherichia
coli and fluorescence-activated cell sorting (FACS). This system was
employed to isolate clones with high-affinity to a fluorescently-labeled
hapten from libraries constructed by randomizing heavy and light-chain
residues in the anti-digoxin 26-10 derived antibody, scFv(dig). The use of
flow cytometry enabled the detection of rare library members directly in
heterogeneous populations and the optimization of selection conditions
prior to sorting. A heavy-chain mutant having wild-type affinity (KD =
0.91+/-0.22 nM) and an expected representation frequency of less than 1 x
10(6), was selected to homogeneity after three rounds utilizing
increasingly stringent selection conditions. The isolated clone possessed
two distinct point mutations relative to the wild-type DNA sequence, yet
still coded for the wild-type amino acid sequence, suggesting that the
wild-type residues may be optimal at the randomized positions. An affinity
improved clone (KD = 0.30+/-0.05 nM), having a dissociation constant
approximately threefold lower than the wild-type antibody, was isolated
from a smaller light-chain library in a single sorting step. Flow cytometry
was shown to be a simple and rapid method for the determination of the
relative hapten dissociation rate constants of selected clones without
requiring subcloning. The relative rate constants estimated by FACS were
confirmed by producing the scFv antibodies in soluble form and measuring
hapten binding kinetics by surface plasmon resonance (SPR). These results
demonstrate that E.coli surface display, coupled with quantitative
selection and analysis using FACS, has the potential to become a powerful
tool for rapid isolation and characterization of desirable mutants from
large polypeptide libraries.
ARTICLES
Antibody affinity maturation using bacterial surface display
Department of Chemical Engineering, University of Texas, Austin 78712, USA.
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