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Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system
Authors:Mira Song  Da-Young Park  Youngkwan Kim  Kyung-Jin Lee  Zhe Lu  Kinarm Ko  Young Kug Choo  Yeon Soo Han  Mi-Hyun Ahn  Doo-Byoung Oh  Kisung Ko
Affiliation:1 Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan, Jeonbuk 570-749, Korea;2 Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea;3 Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Mendelstrasse 7, 48149 Muenster, Germany;4 Department of Agricultural Biology, College of Agriculture and Life Science, Jeonnam University, Gwangju 500-757, Korea
Abstract:Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733. The heavy chain (HC) and light chain (LC) genes of mAb CO17-1A were cloned under the control of P10 and Polyhedrin promoters in the pFastBac™ dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. Confocal immunofluorescence and Western blot analyses confirmed expression of mAb CO17-1A in baculovirus-infected insect cells. The optimum conditions for mAb expression were evaluated at 24, 48, and 72 h after the virus infection at an optimum virus multiplicity of infection of 1. Expression of mAb CO17-1A in insect cells significantly increased at 72 h after infection. HPLC analysis of glycosylation status revealed that the insect-derived mAb (mAbI) CO17-1A had insect specific glycan structures. ELISA showed that the purified mAbI from cell culture supernatant specifically bound to SW948 human colorectal cancer cells. Fluorescence-activated cell sorting analysis showed that, although mAbI had insect specific glycan structures that differed from their mammalian counterparts, mAbI similarly interacted with CD64 (FcγRI) and Fc of IgG, compared to the interactions of mammalian-derived mAb. These results suggest that the baculovirus insect cell expression system is able to express, assemble, and secrete biofunctional full size mAb.
Keywords:Baculovirus  mAb CO17-1A  Insect cell  Glycosylation  Bac-to-Bac system
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