title: Design and construction of small perturbation mutagenesis libraries for antibody affinity maturation using massive microchip-synthesized oligonucleotides.
abstract: We report a rational strategy to design and construct multiple small perturbation mutagenesis (SPM) libraries using massively parallel synthesis of oligonucleotides on a microchip for affinity maturation of an engineered anti-ErbB2 antibody chA21. On the basis of a comprehensive analysis of the sequence and structural relationships of six complementary determination regions (CDRs) in the Kabatman database, a computational algorithm was developed to introduce single-site and double-site mutations into variable CDR positions using ambiguous nucleotides. The six SPM libraries were composed of 419 degenerate oligonucleotides that can be expanded into 161,832 unique CDR sequences with a high coverage ratio of 95% natural amino acid diversity. We used Illumina next-generation sequencing to demonstrate that the synthetic CDR library sequences, as well as relative quantities per sequence, can be controlled precisely by adjusting reaction chamber assignment and input nucleoside composition. The microchip-synthesized oligonucleotides were used for construction of single-chain antibody fragment (scFv) phage libraries through one-step mutagenic PCR of double-stranded plasmids with >10(6)E. coli transformants. A variant with combinatorial mutations from four individual CDRs achieved more than 19-fold affinity increase. The strategy described herein should be broadly applicable to affinity and selectivity studies of antibodies and other proteins.
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China.
authors.forename: Man
authors.initials: M
authors.lastname: Xu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China.
authors.forename: Siyi
authors.initials: S
authors.lastname: Hu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Ding
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China.
authors.forename: Caiyi
authors.initials: C
authors.lastname: Fei
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Wan
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Hu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China.
authors.forename: Ruikai
authors.initials: R
authors.lastname: Du
authors.affiliation: LC Sciences, 2575 West Bellfort St. STE 270, Houston, TX 77054, USA.
authors.forename: Xiaochuan
authors.initials: X
authors.lastname: Zhou
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China.
authors.forename: Jiong
authors.initials: J
authors.lastname: Hong
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Liu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China; Department of Biology and Biochemistry, University of Houston, 4800 Calhoun Rd, Houston, TX 77004, USA. Electronic address: xgao@uh.edu.
authors.forename: Xiaolian
authors.initials: X
authors.lastname: Gao
authors.affiliation: School of Life Sciences, University of Science and Technology of China, 443 Huangshan Rd, Hefei, Anhui 230027, China. Electronic address: jliu@ustc.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
mesh_terms: D000915:Antibody Affinity; D015723:Gene Library; D016296:Mutagenesis; D009841:Oligonucleotides / Q000737:chemistry*
chemical_list: D009841:Oligonucleotides
keywords: Antibody affinity maturation; Microchip oligonucleotide synthesis; Next-generation sequencing; Phage display; Small perturbation mutagenesis