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Production of single-chain Fv–Fc fusion protein in stably transformed insect cells

Paper ID Volume ID Publish Year Pages File Format Full-Text
3506 173 2012 6 PDF Available
Title
Production of single-chain Fv–Fc fusion protein in stably transformed insect cells
Abstract

We describe the secretory production of a mouse anti-bovine ribonuclease A single-chain variable fragment (scFv) fused with the Fc region of human IgG1 in stably transformed lepidopteran insect cells. Use of the insect-derived Bip and melittin signal peptides resulted in higher yields of the secreted scFv–Fc fusion protein than use of the baculovirus gp64 signal peptide. After cotransfection with an expression vector that contains the Bip signal sequence upstream of the DNA encoding the scFv–Fc and a selection vector that carries a neomycin resistance gene, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418. Colonies of resistant cells were obtained around 2 weeks after adding G418, and clonal cells were screened by enzyme-linked immunosorbent assay (ELISA) of the culture supernatant. The yield of the scFv–Fc protein secreted from the most productive clone was around 60 mg/l in a shake-flask culture. To improve the productivity, we investigated the effect of medium supplements of sodium butyrate (NaBu), dimethyl sulfoxide (DMSO), and sericin. Supplementing culture medium with sericin increased the scFv–Fc protein yield to 82 mg/l, but productivity was not increased by either NaBu or DMSO. These results indicate that the stably transformed insect cells may allow for the efficient production of scFv–Fc and other Fc fusion proteins.

► Stably transformed lepidopteran insect cells secreting scFv–Fc fusion protein. ► Use of a Drosophila Bip secretion signal resulted in a high production level. ► Yield of 60 mg/l scFv–Fc was achieved in a shake-flask culture. ► Supplementing the medium with sericin improved culture viability and product yield.

Keywords
Insect cell culture; Recombinant protein production; Stable expression; Single-chain variable fragment; Fc fusion protein; Medium supplement
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Production of single-chain Fv–Fc fusion protein in stably transformed insect cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 67, 15 August 2012, Pages 77–82
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