Optimization of expression conditions for production of anti-colorectal cancer monoclonal antibody CO17-1A in baculovirus-insect cell system.
The baculovirus-insect cell system is considered a feasible expression system for recombinant glycoprotein production due to its several advantages, including high capacity, flexibility, and glycosylation capability. However, accurate titering of the recombinant baculovirus is required to ensure high expression in insect cells using a commercial and expensive immunoassay titer kit in which the envelope glycoprotein of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-type baculovirus is detected by anti-envelope glycoprotein antibody and a secondary antibody conjugated to horseradish peroxidase (HRP). In this study, conditions for the expression of the CO17-1A immunotherapeutic monoclonal antibody (MAb) against colorectal cancer cells in a baculovirus system were optimized without using a commercial titering kit. Several variables were investigated to optimize antibody expression in a baculovirus-insect cell system, including baculovirus passage, volume of the infecting baculovirus inoculum (100, 200, 400, and 800 μL), and the harvest time of insect cells or cell supernatants after virus infection (24, 48, and 72 h). Two different pFastBac vectors carrying the CO17-1A MAb genes with or without the KDEL endoplasmic reticulum (ER) retention motif (Lys-Asp-Glu-Leu) fused to the HC (MAb CO17-1A K and MAb CO17-1A, respectively) were constructed and used to generate baculoviruses. Immunoblot analysis was conducted to confirm expression of MAb CO17-1A K and MAb CO17-1A in baculovirus-infected insect cells. Densitometry analysis of the protein bands was used to quantify the relative expression under different conditions. The highest expression was observed in lysed cells infected with 400 μL of passage 3 baculovirus (P(3) BV) carrying the gene encoding the CO17-1A MAb without KDEL at 72 h after virus infection. These results suggest that the infection conditions, the number of virus passages, baculovirus inoculum volume, and the harvest time can be modified to optimize MAb expression without using a BaculoELISA titer kit in a baculovirus-insect cell system.
Department of Biological Science, Biotechnology Institute, College of Natural Sciences, Wonkwang University, Iksan, Korea.
Da-Young
DY
Park
Jung-Hwan
JH
Lee
Yang-Kang
YK
So
Young-Kwan
YK
Kim
Kinarm
K
Ko
Sang-Won
SW
Park
Yong Seok
YS
Lee
Yeon Soo
YS
Han
Kisung
K
Ko
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000276:immunology; D000970:Antineoplastic Agents / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms; D004721:Endoplasmic Reticulum; D005822:Genetic Vectors; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology; D014162:Transfection
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C050928:carcinoma-associated antigen 17-1A