Skip Navigation


Journal of Biochemistry Advance Access originally published online on August 19, 2008
Journal of Biochemistry 2008 144(5):591-598; doi:10.1093/jb/mvn103
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
144/5/591    most recent
mvn103v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Yin, C.-C.
Right arrow Articles by Yan, X.-Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yin, C.-C.
Right arrow Articles by Yan, X.-Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2008 The Japanese Biochemical Society

Construction of a Fully Synthetic Human scFv Antibody Library with CDR3 Regions Randomized by a 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,{dagger}

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

*To whom correspondence should be addressed. Tel/Fax: +86-10-80726906, E-mail: hlhuang{at}genetics.ac.cn

{dagger}Correspondence may also be addressed: Tel: +86-10-64888583, Fax: +86-10-64888584, E-mail: yanxy{at}sun5.ibp.ac.cn

Received May 1, 2008; Accepted August 13, 2008


  Abstract

The randomization scheme of hypervariable region takes crucial role 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 1.56 x 109 was successfully generated with complementarity determining region 3 (CDR3) randomized by this strategy. Sequencing analysis indicated that >60% of colonies had completely correct scFv genes and the amino acid composition and distribution were designed well in accordance. The utility was demonstrated by screening of scFv clones against BHL (anti-CD3 x anti-ovarian carcinoma bispecific antibody). These results proved the feasibility of the split–mix–split DNA randomization strategy in library construction and site-directed mutagenesis.

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

Abbreviations: BHL, anti-CD3 x anti-ovarian carcinoma bispecific single-chain antibody; CDR, complementarity determining region; HuCAL, fully gene-synthetic human combinatoryial antibody library; scFv, single-chain variable fragment; SMS, split–mix–split DNA synthesis; SOE PCR, splice overlap extension PCR; TRIM, trinucleotide-mutagenesis; VH, variable region of heavy chain; VL, variable region of light chain; V{kappa}, variable regionof {kappa} light chain; V{lambda}, variable region of {lambda} light chain


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.