Synthesis of human big endothelin-1 by sequence-specific proteolysis of a fusion protein in Escherichia coli.
Three DNA constructs, pETB-40, 41, and 42, encoding human big endothelin-1 (ET-1) preceded by the specific recognition sequence (Ile-Glu-Gly-Arg) for the activated blood coagulation factor Xa (FXa), fused in frame to the N-terminal portion of beta Gal, were expressed in Escherichia coli. The fusion proteins, pETB-40P, 41P, or 42P, consisted of the 55-, 51-, or 42-aa N-terminal peptide of beta Gal and the 38-aa of big ET-1, and had 1, 0, or 0 Cys residues and 5, 5, or 1 Arg residues in the N-terminal peptide of beta Gal, respectively. Enzymatic cleavage of the purified fusion proteins by FXa or trypsin allowed the recovery of authentic human big ET-1. The rates of conversion of pETB-40P, 41P, and 42P to big ET-1 by FXa digestion were 5.6, 11.2, and 30.0%, respectively. pETB-40P with a deletion of one Cys residue and four Arg residues in the N-terminal part was a better substrate than the other two for FXa or trypsin in the production of big ET-1.
Ohashi
H
H
Biochemistry, Central Research Laboratories, Banyu Pharmaceutical Co., Ltd., Tokyo.
Katsuta-Enomoto
Y
Y
Yasufuku
K
K
Okada
K
K
Yano
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010920:Placenta / Q000502:physiology; D011247:Pregnancy; D011498:Protein Precursors / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping
D019332:Endothelin-1; D016232:Endothelins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA; D008666:Metalloendopeptidases