title: Construction of a fully synthetic human scFv antibody library with CDR3 regions randomized by a split-mix-split method and its application.
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 10(9) 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.
authors: National Laboratory of Biomacromolecules, and CAS-UT joint laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors: Chang-Cheng
authors: CC
authors: Yin
authors: Li-Li
authors: LL
authors: Ren
authors: Lin-Lin
authors: LL
authors: Zhu
authors: Xiang-Bin
authors: XB
authors: Wang
authors: Zhong
authors: Z
authors: Zhang
authors: Hua-Liang
authors: HL
authors: Huang
authors: Xi-Yun
authors: XY
authors: Yan
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies; D022801:Complementarity Determining Regions / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D000906:Antibodies; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library