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J Biosci Bioeng. 2017 Aug;124(2):221-226. doi: 10.1016/j.jbiosc.2017.03.007. Epub 2017 Apr 11.

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

Author information

1
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
2
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; Institute of Pathology, Kyodo Byori, Inc., 2-7-12 Otsuwa, Nishi-ku, Kobe 651-2112, Japan.
3
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; Manufacturing Technology Association of Biologics, c/o Integrated Research Center of Kobe University, 7-1-49 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
4
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan; Manufacturing Technology Association of Biologics, c/o Integrated Research Center of Kobe University, 7-1-49 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan. Electronic address: yamaji@kobe-u.ac.jp.

Abstract

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins.

KEYWORDS:

Fab fragment; High Five cell; Insect cell; Recombinant protein production; Transient gene expression

PMID:
28410897
DOI:
10.1016/j.jbiosc.2017.03.007
[Indexed for MEDLINE]

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