Production of Antibody Fragments Using the Baculovirus–Insect Cell System

Furuta, Takanori; Ogawa, Takafumi; Yamaji, Hideki

doi:10.1007/978-1-61779-974-7_22

Cited by 6 publications

(1 citation statement)

References 13 publications

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“…Trichoplusia ni BTI-TN-5B1-4 (High Five; Thermo Fisher Scientific, Waltham, MA, USA) cells were cultured in a serum-free medium Express Five SFM (Thermo Fisher Scientific) supplemented with 2.6 g/L Lglutamine (Nacalai Tesque, Kyoto, Japan) and 10 mg/L gentamycin sulfate (Thermo Fisher Scientific). The cells were maintained at 27°C in T-flasks in a non-humidified incubator (25). Cell density was determined by microscopically counting the number of cells using the Countess II automated cell counter (Thermo Fisher Scientific), while cell viability was judged by the exclusion of trypan blue dye.…”

Section: Insect Cells and The Culture Mediummentioning

confidence: 99%

Production of an antibody Fab fragment using 2A peptide in insect cells

Mizote

Masumi-Koizumi

Katsuda

et al. 2020

Journal of Bioscience and Bioengineering

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Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence.Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells.

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Section: Insect Cells and The Culture Mediummentioning

confidence: 99%

Production of an antibody Fab fragment using 2A peptide in insect cells

Mizote

Masumi-Koizumi

Katsuda

et al. 2020

Journal of Bioscience and Bioengineering

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Production of Japanese Encephalitis Virus-Like Particles Using Insect Cell Expression Systems

Yamaji

Konishi

2016

Vaccine Design

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Virus-like particles (VLPs) can be produced via the expression of virus surface proteins that self-assemble into particulate structures in recombinant protein expression systems. Expression of the DNA fragment encoding the Japanese encephalitis (JE) virus prM signal peptide, the precursor (prM) of the viral membrane protein (M), and the envelope glycoprotein (E) allows the production of a secretory form of VLPs. Expression systems that use lepidopteran insect cells, such as the baculovirus-insect cell system and stably transformed insect cells, can be used for the efficient production of JE VLPs. This chapter describes the production of JE VLPs from stably transformed lepidopteran insect cells.

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Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) inEscherichia coli

Akbari

Sadeghi

Jafrian-Dehkordi

et al. 2014

Journal of Industrial Microbiology and Biotechnology

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The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand-HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.

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Production of Antibody Fragments Using the Baculovirus–Insect Cell System

Cited by 6 publications

References 13 publications

Production of an antibody Fab fragment using 2A peptide in insect cells

Production of an antibody Fab fragment using 2A peptide in insect cells

Production of Japanese Encephalitis Virus-Like Particles Using Insect Cell Expression Systems

Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) inEscherichia coli

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