An improved method for large-scale purification of recombinant human glucagon.
Glucagon was expressed in Escherichia coli as a fusion protein including the glucagon sequence [Ishizaki et al. (1992), Appl. Microbiol. Biotechnol. 36, 483-486]. The high-level expression of a protein in E. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report [Yoshikawa et 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.
Okamoto
H
H
Shionogi Research Laboratories, Shionogi & Co., Osaka, Japan.
Iwamoto
H
H
Tsuzuki
H
H
Teraoka
H
H
Yoshida
N
N
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism; D005934:Glucagon / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D012995:Solubility
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005934:Glucagon; D010450:Endopeptidases; D012697:Serine Endopeptidases; C078180:blaSE protein, Bacillus licheniformis