Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli(BIOCHEMICAL ENGINEERING)
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概要
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The effects of cytoplasmic and periplasmic chaperones on the secretory production of an anti-bovine ribonudease A single-chain variable fragment (scFv) 3A21 in Escherichia coli were investigated. Co-expression of a cytoplasmic chaperone, GroEL/ES, DnaK/DnaJ/GrpE, trigger factor, or SecB with 3A21 scFv affected the proportions of antigen-binding activity in the cytoplasmic soluble fraction, the periplasmic fraction, and the extracellular medium, but there was no significant difference in the total activity compared to the control without chaperone co-expression. On the other hand, co-expression of a periplasmic chaperone, Skp or FkpA, with the exception of DsbC, greatly increased the binding activity in all the soluble fractions. Co-expression of both Skp and FkpA had no synergistic effect. Combinations of cytoplasmic and periplasmic chaperones decreased the productivity. In shake-flask cultures of cells co-expressing Skp or FkpA, considerable amounts of 3A21 scFv were detected in the extracellular medium by enzyme-linked immunosorbent assay (ELISA) and Western blot, and the extracellular production level of 3A21 scFv was calculated to be around 40mg/l. The binding activity of 3A21 scFv co-expressed with Skp was slightly higher than that with FkpA. These results indicate that the co-expression of periplasmic chaperones Skp and FkpA is extremely useful for the secretory production of scFvs in a culture medium using E. coli, but cytoplasmic chaperones and multiple-chaperone combinations may not be effective.
著者
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Kumada Yoichi
Department of Chemistry and Materials Technology, Kyoto Institute of Technology
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Kumada Yoichi
Department Of Bioscience And Biotechnology Okayama University
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Katsuda Tomohisa
Department Of Chemical Science And Engineering Graduate School Of Engineering Kobe University
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Yamaji Hideki
Department Of Chemical Science And Engineering Faculty Of Engineering Kobe University
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Sonoda Hiroyuki
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University
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Sonoda Hiroyuki
Department Of Chemical Science And Engineering Graduate School Of Engineering Kobe University:resear
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Sonoda Hiroyuki
Department Of Biological Science Graduate School Of Science Hiroshima University
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