Recombinant human glucagon: large-scale purification and biochemical characterization.
Recombinant glucagon was expressed in Escherichia coli as a fusion protein including the glucagon sequence therein as previously reported [Ishizaki et al. (1992). Appl. Microbiol. Biotechnol. 36, 483-486]. We developed a large-scale method for the isolation and purification of recombinant glucagon. After cell disruption, the resultant pellets were solubilized with 2 M guanidine-HCl, to which Staphylococcus aureus V8 protease had been added, and were digested into intermediates composed of 53- and 60-residue peptides containing the glucagon moiety. After the digestion came to an end, the solution was desalted, and the remaining V8 protease was allowed to resume digestion of the intermediates into glucagon, followed by partial purification by S-Sepharose and Sephacryl S-100 chromatographies. The glucagon obtained was found to be not less than 99.5% pure by analytical HPLC. One liter of culture produced about 180 mg of pure glucagon. The amino acid composition and the sequence agreed well with the theoretical values. Radioreceptor assay gave an affinity constant similar to that of pancreatic glucagon, and similar activities in cAMP production and glycogenolysis were also observed. Thus, the recombinant glucagon was confirmed to be biochemically identical with pancreatic glucagon.
Yoshikawa
K
K
Shionogi Research Laboratories, Shionogi and Co., Ltd., Osaka, Japan.
Tsuzuki
H
H
Fujimoto
M
M
Tohkin
M
M
Matsubara
T
T
Yonezawa
H
H
Okamoto
H
H
Teraoka
H
H
Yoshida
N
N
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D000242:Cyclic AMP / Q000096:biosynthesis; D005934:Glucagon / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism; D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism
D010446:Peptide Fragments; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005934:Glucagon; D000242:Cyclic AMP; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase