Enzymatic Assembly for scFv Library Construction

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1575)

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 VL and VH fragments through the concerted action of λ-exonuclease and Bst DNA polymerase. We successfully used this method to construct a diverse chicken scFv library.

Key words

scFv Phage display library λ-exonuclease Assembly Antibody 

Abbreviations

scFv

Single-chain variable fragment

VL

Light-chain variable region

VH

Heavy-chain variable region

CDR

Complementarity-determining region

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Bio-Peak Co., Ltd.TakasakiJapan
  2. 2.Structure Physiology Research Group, Biomedical Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan

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