title: Enzymatic Assembly for scFv Library Construction.
abstract: Recombinant monoclonal antibodies can be established by displaying single-chain variable fragment (scFv) antibody libraries on phages and then biopanning against the target. For constructing superior scFv libraries, antibody light-chain variable region (VL) and heavy-chain variable region (VH) fragments must be assembled into scFvs without loss of diversity. A high-quality scFv library is a prerequisite for obtaining strong binders from the scFv library. However, the technical challenges associated with the construction of a diverse library have been the bottleneck in the establishment of recombinant antibodies through biopanning. Here, we describe a simple and efficient method for assembling VLand VHfragments through the concerted action of λ-exonuclease and Bst DNA polymerase. We successfully used this method to construct a diverse chicken scFv library.
authors.affiliation: Bio-Peak Co., Ltd., 584-70 Shimonojo, Takasaki, 370-0854, Japan.
authors.forename: Mieko
authors.initials: M
authors.lastname: Kato
authors.affiliation: Structure Physiology Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566, Japan. y.hanyu@aist.go.jp.
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Hanyu
mesh_terms: D000818:Animals; D002645:Chickens / Q000235:genetics / Q000276:immunology; D016172:DNA Fingerprinting; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D019151:Peptide Library; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics; D061905:Single-Domain Antibodies / Q000235:genetics* / Q000378:metabolism
chemical_list: D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D061905:Single-Domain Antibodies; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases
keywords: Antibody; Assembly; Phage display library; scFv; λ-exonuclease