title: Expression and purification of a human anti-cyclin D1 single-chain variable fragment antibody AD5 and its characterization.
abstract: Cyclin D1 plays an important role in cell cycle progression. Increasing evidence indicates that cyclin D1 is overexpressed in the majority of tumor cells and has become a potential target for tumor therapy. However, little research has been done on the specific inhibition of cyclin D1 for cancer therapy. With the rapid development of the phage display antibody library technique, single-chain variable fragment (scFv) antibodies have emerged, which have tremendous application prospects in cancer therapy and diagonosis. In this study, a human scFv binding specifically to cyclin D1 (AD5) that was derived from a human semi-synthetic scFv phage library was expressed in the soluble form in Escherichia coli (E. coli) HB2151 cells. To characterize AD5, soluble AD5 was purified successfully through ammonium sulfate precipitation and affinity chromatography from the culture supernatant of AD5/HB2151. ELISA assay revealed that purified soluble AD5 could specifically bind to human recombinant cyclin D1 with approximately (1.19±0.056) x 107 M-1 affinity constant and showed approximately 52% competitive inhibition with the anti-cyclin D1 polyclonal antibody for binding to cyclin D1 in vitro. These results suggest that the scFv antibody against cyclin D1 may be a novel potential tool for targeting cyclin D1 in cancer therapy and diagnosis.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, College of Life Science, Jilin University, Changchun, Jilin 130012, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.forename: Desheng
authors.initials: D
authors.lastname: Zou
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Cao
authors.forename: Nannan
authors.initials: N
authors.lastname: Yao
authors.forename: Junye
authors.initials: J
authors.lastname: Wang
authors.forename: Wenhan
authors.initials: W
authors.lastname: Wang
authors.forename: Hongyu
authors.initials: H
authors.lastname: Jiang
authors.forename: Guiying
authors.initials: G
authors.lastname: Li
mesh_terms: D001667:Binding, Competitive; D019938:Cyclin D1 / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D061986:MCF-7 Cells; D019151:Peptide Library; D011485:Protein Binding; D057127:Single-Chain Antibodies / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D019151:Peptide Library; D057127:Single-Chain Antibodies; D019938:Cyclin D1