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

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 BT1-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. (C) 2012 Elsevier B.V. All rights reserved.