title: Construction of an scFv library by enzymatic assembly of V(L) and V(H) genes.
abstract: The single-chain Fv fragment (scFv) is the most frequently used form of recombinant antibody. It is possible to establish clones specific to a certain target by displaying the scFv library on phages followed by biopanning against the target. For the construction of superior scFv libraries, the light-chain variable region (VL) and the heavy-chain variable region (VH) fragments should be assembled into the scFv without loss of diversity. We have provided an efficient method for constructing scFvs by enzymatic assembly of the VL and VH domains using the concerted action of λ-exonuclease and Bst DNA polymerase. First, we amplified the chicken VL and VH fragments using a phosphorylated primer with a 21-nucleotide overlap in the linker region. Then we recessed the overlapping parts of the VL and VH fragments with λ-exonuclease, which yielded single-stranded overhangs that specifically annealed between the VL and VH fragments; the complete double-stranded scFv was formed using Bst DNA polymerase. Complete scFvs were obtained using this method, whereby a library of scFvs was constructed from the immune library of chicken IgG. The diversity of this scFv library was analyzed by DNA fingerprinting method. The scFvs specific to the antigen could be isolated from this library after 5 rounds of panning.
authors.affiliation: Bio-Peak Co., Ltd., 584-70 Shimonojo, Takasaki, 370-0854 Japan; Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566 Japan.
authors.forename: Mieko
authors.initials: M
authors.lastname: Kato
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Hanyu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000235:genetics / Q000276:immunology; D018076:DNA, Complementary; D005260:Female; D050438:Genes, Immunoglobulin Heavy Chain; D050439:Genes, Immunoglobulin Light Chain; D007074:Immunoglobulin G / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics* / Q000378:metabolism; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: Antibody engineering; Assembly; CDR; Library; Phage display; Single-chain Fv fragment; V(H); V(L); complementarity-determining region.; heavy-chain variable region; light-chain variable region; scFv; single-chain Fv fragment; λ-Exonuclease