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Journal of Biochemistry Advance Access published online on August 19, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn103
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© 2008 The Japanese Biochemical Society

Construction of a fully synthetic human scFv antibody library with CDR3 regions randomized by split-mix-split method and its application

Chang-Cheng Yin1,2, Li-Li Ren2, Lin-Lin Zhu2, Xiang-Bin Wang2, Zhong Zhang2, Hua-Liang Huang2,* and Xi-Yun Yan1,*

1National Laboratory of Biomacromolecules, and CAS-UT joint laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
2Beijing ABT Genetic Engineering Technology Co., Ltd, Beijing, 102206, China

*Corresponding author: Xi-Yun Yan: Tel, 86-10-64888583; Fax, 86-10-64888584, E-mail, yanxy{at}sun5.ibp.ac.cn. Hua-Liang Huang: Tel & Fax, 86-10-80726906, E-mail, hlhuang{at}genetics.ac.cn

Received May 1, 2008; Accepted August 13, 2008


  Abstract

The randomsation scheme of hypervariable region takes crucial roles in construction of a synthetic antibody library. The codon bias and inevitable ‘stop’ codon of conventional "NNK" and "NNS" codons limit their applications. Here we report a split-mix-split DNA synthesis method that can control over the amino acid composition and distribution of randomized sequences effectually. A fully synthetic human antibody library with a diversity of 1.56x109 was successfully generated with complementarity determining region 3 (CDR3) randomized by this strategy. Sequencing analysis indicated that more than 60% of colonies had completely correct scFv genes and the amino acid composition and distribution were accordance with design well. The utility was demonstrated by screening of scFv clones against BHL (anti-CD3xanti-carcinoma bispecific antibody). These results proved the feasibility of the split-mix-split DNA randomsation strategy in library construction and site-directed mutagenesis.

Key Words: antibody library, biopanning, phage display, randomization, single-chain antibody


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