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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

Paper ID Volume ID Publish Year Pages File Format Full-Text
21496 43225 2010 6 PDF Available
Title
Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system
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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Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 110, Issue 2, August 2010, Pages 135–140
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us