Expression and purification of a human anti-cyclin D1 single-chain variable fragment antibody AD5 and its characterization

Wu,Yan; Zou,Desheng; Cao,Yuhua; Yao,Nannan; Wang,Junye; Wang,Wenhan; Jiang,Hongyu; Li,Guiying

doi:10.3892/ijmm.2013.1523

Expression and purification of a human anti-cyclin D1 single-chain variable fragment antibody AD5 and its characterization

Authors:
- Yan Wu
- Desheng Zou
- Yuhua Cao
- Nannan Yao
- Junye Wang
- Wenhan Wang
- Hongyu Jiang
- Guiying Li
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Affiliations: Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, College of Life Science, Jilin University, Changchun, Jilin 130012, P.R. China, Department of General Internal Medicine, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
Published online on: October 11, 2013 https://doi.org/10.3892/ijmm.2013.1523
Pages: 1451-1457

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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.

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