An improved method for large-scale purification of recombinant human glucagon |
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Authors: | Hiroyuki Okamoto Hideo Iwamoto Hiroshige Tsuzuki Hiroshi Teraoka and Nobuo Yoshida |
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Institution: | (1) Shionogi Research Laboratories, Shionogi & Co., 5-12-4, Sagisu, Fukushima-ku, 553 Osaka, Japan |
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Abstract: | Glucagon was expressed inEscherichia coli as a fusion protein including the glucagon sequence Ishizakiet al. (1992),Appl. Microbiol. Biotechnol.36, 483–486]. The high-level expression of a protein inE. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report Yoshikawaet al. (1992),J. Protein Chem.
11, 517–525], we solubilized this inclusion body by using guanidinium chloride. However, the existence of denaturant caused problems such as a low proteolytic activity for transforming the fusion protein into glucagon and complicated purification methods. We tried to improve the method to enable large-scale purification. At alkaline pH, the inclusion body could be solubilized to a high concentration and cleaved by amino acid-specific endopeptidases. By utilizing isoelectric precipitations as a new economical purification method for glucagon from intermediates, the glucagon obtained was shown to be over 99.5% pure by analytical RP-HPLC. The yield was almost equal that of our previous method, and the glucagon produced was chemically and biochemically equivalent to natural glucagon. |
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Keywords: | BLase V8 protease isoelectric precipitation inclusion body |
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