Cytoplasmic production of soluble and functional single-chain Fv-Fc fusion protein in Escherichia coli

▶ Soluble and functional scFv-Fc is expressed in E. coli trxB/gor double mutant. ▶ Solubility of scFv-Fc in the cytoplasm is improved by co-expression of GroELS. ▶ Productivity of more than 10mg/L is achieved in shake-flask culture. We describe the soluble production of a mouse anti-bovine ribonucle...

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Bibliographic Details
Published in:Biochemical engineering journal 2011-02, Vol.53 (3), p.253-259
Main Authors: Sonoda, Hiroyuki, Kumada, Yoichi, Katsuda, Tomohisa, Yamaji, Hideki
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Escherichia coli
Fc fusion protein
Fundamental and applied biological sciences. Psychology
GroELS
Molecular chaperone
Single-chain variable fragment
Soluble production
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Summary:▶ Soluble and functional scFv-Fc is expressed in E. coli trxB/gor double mutant. ▶ Solubility of scFv-Fc in the cytoplasm is improved by co-expression of GroELS. ▶ Productivity of more than 10mg/L is achieved in shake-flask culture. We describe the soluble production of a mouse anti-bovine ribonuclease A single-chain variable fragment (scFv) fused with the Fc region of human IgG1 in Escherichia coli. Two production systems, secretory production using a pelB signal peptide and cytoplasmic production in a trxB/gor double mutant strain with an oxidizing cytoplasm, were investigated for efficient production of soluble and functional scFv-Fc fusion protein. Antigen-binding activity was observed in both systems but almost all of the scFv-Fc that was expressed formed insoluble aggregates. Hence, the co-expression of molecular chaperones was examined. Co-expression of GroEL/GroES showed a 4.6-fold increase in antigen-binding activity in the cytoplasmic production system but not in the secretory system. By contrast, the other two chaperones, DnaK/DnaJ/GrpE and trigger factor, had no effect in either production system. The protein solubility was also improved markedly by the co-expression of GroEL/GroES and approximately 70% of the 3A21 scFv-Fc protein was soluble. A practical productivity of more than 10mg/L was achieved with a simple batch shake-flask culture. These results indicate that the E. coli cytoplasmic production system with oxidizing cytoplasm and molecular chaperones might be one of the choices for the soluble production of scFv-Fcs and other Fc fusion proteins.
ISSN:1369-703X
1873-295X
DOI:10.1016/j.bej.2010.11.003