title: [Affinity maturation of a single chain antibody against VEGFR2 by hydrophilic shuffling].
abstract: Abstract: This study is to improve the affinity of scFv-AK404R against VEGFR2. The secondary mutational library was constructed by hydrophilic shuffling in CDR3 region of the heavy chain. VEGFR2-specific screening was performed by phage display technology and the protein of mutants was expressed in periplasm of E.coli HB2151 and purified by affinity chromatography. The affinity constant of scFvs was measured by competitive ELISA, and the structure of scFvs was analyzed by bioinformatics. The result showed that a library with 6.4x10(5) scFv members was established by electro-transformation. Two mutated clones with high absorbance value were isolated after screening. After purification by affinity chromatography, electrophoretically pure scFv proteins were obtained. The competitive ELISA showed that the affinities of WZ01 and WZ02 were three times higher than that of the parental AK404R, and bioinformatics analysis showed that the enlarged contact surface and fitted closely with KDR3 surface may be the reasons for improved affinity. These results suggest that introducing hydrophilic amino acids to the heavy chain CDR3 region is an effective approach to improve the affinity of scFv.
authors.affiliation: China Pharmaceutical University, Nanjing, China.
authors.forename: Hai-di
authors.initials: HD
authors.lastname: Qi
authors.forename: Xiao-niu
authors.initials: XN
authors.lastname: Miao
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.forename: Kai
authors.initials: K
authors.lastname: Gu
authors.forename: Si-wei
authors.initials: SW
authors.lastname: Zhang
authors.forename: Min
authors.initials: M
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D002846:Chromatography, Affinity; D019295:Computational Biology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D019151:Peptide Library; D057127:Single-Chain Antibodies; D040301:Vascular Endothelial Growth Factor Receptor-2