title: Ligation-based assembly for constructing mouse synthetic scFv libraries by chain shuffling with in vivo-amplified VH and VL fragments.
abstract: In vitro assembly of two or three PCR fragments using primers is a common method of constructing scFv fragments for display on the surface of phage. However, mismatch annealing often occurs during in this step, leading to cloning and display of incomplete Fab or scFv fragments. To overcome this limitation, we developed a ligation-based two-fragment assembly (LTFA) protocol that involved separately cloning VH and Vκ fragments into the high-copy-number plasmid pUC18. The VH and Vκ fragments had randomized complementarity-determining region 3 (CDR3) and were joined with a peptidyl linker composed of (G4S)3. Using this approach, complete sequences of scFv fragments were successfully constructed, and the sequencing of 83 scFv clones revealed that none of the sequences, including the linker region, contained deletions or mutations. In contrast, linker sequences generated using a conventional two-fragment PCR assembly (TFPA) protocol often contained sequence anomalies, including large truncations. Using the LTFA protocol, a final library size of 1.0×10(8)cfu was achieved. Examination of the amino acid profiles of the generated scFv fragments within the randomized regions introduced using degenerate codons did not detect any bias from that expected based on stochastic distribution. After several cycles of panning with this library, antigen-specific scFvs against two reference antigens, hen egg lysozyme and streptavidin were detected. In addition, scFvs with specificity against peptidyl antigens in the loop region of the Medaka ortholog of human C6orf89, which encodes a histone deacetylase enhancer that interacts with the bombesin receptor, were also obtained. The LTFA protocol developed here is robust and allows for the easy construction of integral scFv fragments compared with conventional TFPA. Utilizing LTFA, other CDRs can be readily combined. This approach also allows for the in vitro maturation of scFv fragments by separately introducing randomization in CDRs or using error-prone PCR for the amplification of pre-selected sequences as a template scaffold.
authors.affiliation: Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho Nagaham, Shiga 526-0829, Japan.
authors.forename: Michiru
authors.initials: M
authors.lastname: Nishi
authors.affiliation: Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho Nagaham, Shiga 526-0829, Japan.
authors.forename: Nan
authors.initials: N
authors.lastname: Jian
authors.affiliation: Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho Nagaham, Shiga 526-0829, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho Nagaham, Shiga 526-0829, Japan.
authors.forename: Haruyo
authors.initials: H
authors.lastname: Seto
authors.affiliation: Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho Nagaham, Shiga 526-0829, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Nishida
authors.affiliation: Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho Nagaham, Shiga 526-0829, Japan; Advanced Research Center for Genome Super Power, Keio University, 2 Okubo, Tsukuba, Ibaraki 300-2611, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Tonoyama
authors.affiliation: Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho Nagaham, Shiga 526-0829, Japan; Advanced Research Center for Genome Super Power, Keio University, 2 Okubo, Tsukuba, Ibaraki 300-2611, Japan.
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Shimizu
authors.affiliation: Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho Nagaham, Shiga 526-0829, Japan. Electronic address: y_nishi@nagahama-i-bio.ac.jp.
authors.forename: Yoshisuke
authors.initials: Y
authors.lastname: Nishi
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D003001:Cloning, Molecular; D032621:DNA Shuffling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D009113:Muramidase / Q000276:immunology; D009990:Oryzias; D011506:Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics*; D019809:Streptavidin / Q000276:immunology
chemical_list: C519991:C6orf89 protein, human; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011506:Proteins; D057127:Single-Chain Antibodies; D019809:Streptavidin; C421864:hen egg lysozyme; D009113:Muramidase
keywords: Chain-shuffling; Ligation; Mouse scFv; Phage display; Randomization; Synthetic library