Construction of a fully synthetic human scFv antibody library with CDR3 regions randomized by a split-mix-split method and its application.
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.
Yin
Chang-Cheng
CC
National Laboratory of Biomacromolecules, and CAS-UT joint laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
Ren
Li-Li
LL
Zhu
Lin-Lin
LL
Wang
Xiang-Bin
XB
Zhang
Zhong
Z
Huang
Hua-Liang
HL
Yan
Xi-Yun
XY
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
D000906:Antibodies; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library