title: Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli.
abstract: The effects of cytoplasmic and periplasmic chaperones on the secretory production of an anti-bovine ribonuclease 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.
authors.affiliation: Department of Chemical Science and Engineering, Kobe University, Kobe 657-8501, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sonoda
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kumada
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Katsuda
authors.forename: Hideki
authors.initials: H
authors.lastname: Yamaji
mesh_terms: D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis*
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D057127:Single-Chain Antibodies; C072721:Skp protein, E coli; D012259:Ribonuclease, Pancreatic; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli