title: Large-scale preparation of recombinant human calcitonin from a multimeric fusion protein produced in Escherichia coli.
abstract: In order to develop a large-scale, high-yield production process for human calcitonin (hCT) in Escherichia coli, a stable expression plasmid was constructed and the expressed protein was modified for efficient cleavage by protease. Multiple copies of a synthetic gene encoding hCT-Leu-Arg, a substrate for C-terminal amidation by carboxypeptidase Y (CPY), were inserted into the stable expression plasmid. Using this plasmid, the expression of a multimeric fusion protein was induced by shifting the temperature from 34 to 38 degrees C. The multimeric fusion protein was accumulated as inclusion bodies. After the fermentation, the cells were harvested and disrupted by a homogenizer. The insoluble multimeric fusion protein was suspended in 6.6 M urea solution. At this time, however, the protein could not be solubilized and thus the efficiency of cleavage by protease was low. To solubilize the protein and protect Lys residues against digestion by trypsin, the protein was citraconilated with citraconic anhydride. Following S-sulfonation with Na2SO3-CuSO4, almost all the protein was solubilized. The solubilized, citraconilated, and S-sulfonated protein was digested with trypsin efficiently. By treatment with trypsin, the multimeric fusion protein was cleaved into monomers of citraconilated and S-sulfonated hCT-Leu-Arg (S-hCT-Leu-Arg). The subsequent decitraconilation was performed at low pH. S-hCT-Leu-Arg, isolated by preparative HPLC, was directly converted into S-hCT-HN2 by CPY without removal of the Arg residue. Finally, S-hCT-NH2 was desulfonated and converted into mature hCT. In this way, a large amount of recombinant mature hCT was obtained in a tank fermentation. To our knowledge, this is the first report of industrial-scale, human calcitonin production using a multimeric fusion protein expression system.
authors.affiliation: Pharmacology and Molecular Biology Research Laboratories, Sankyo Co. Ltd., 2-58 Hiromachi 1-chome, Tokyo 140-8710, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Ishikawa
authors.forename: J
authors.initials: J
authors.lastname: Kawaguchi
authors.forename: Y
authors.initials: Y
authors.lastname: Yao
authors.forename: H
authors.initials: H
authors.lastname: Tamaoki
authors.forename: T
authors.initials: T
authors.lastname: Ono
authors.forename: F
authors.initials: F
authors.lastname: Fukui
authors.forename: H
authors.initials: H
authors.lastname: Yoshikawa