High-yield production of recombinant endothelin-1.
A fusion protein (pETB-42P), which encodes the 42-amino acid leader peptide and the 38-amino acid peptide of human big endothelin (ET)-1, was synthesized in Escherichia coli, isolated as inclusion bodies, and purified by DEAE-chromatography. Trypsin digestion of the purified pETB-42P gave big ET-1(1-37) in a yield of 70%; then pepsin digestion of the purified big ET-1(1-37) gave ET-1(1-21) in a yield of 74% (overall yield: 52%). Sequential trypsin and pepsin digestions of the purified fusion protein in the same reaction vessel also allowed recovery of ET-1 in a yield of 60%. One milligram of ET-1 or 2.0 mg of big ET-1(1-37) was obtained from 1.8 liters of culture broth. Recombinant ET-1 thus obtained was identical to authentic ET-1 in terms of amino acid sequence and vasoconstrictor potency, and recombinant big ET-1(1-37) had almost the same in vitro and in vivo biological activities as big ET-1(1-38).
Yasufuku
K
K
New Drug Discovery Research Laboratories, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd.
Ohashi
H
H
Katsuta-Enomoto
Y
Y
Fukuroda
T
T
Noguchi
K
K
Yano
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology; D003001:Cloning, Molecular; D016232:Endothelins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010434:Pepsin A; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014357:Trypsin; D014661:Vasoconstriction / Q000187:drug effects
D016232:Endothelins; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D010434:Pepsin A