High-yield production of human big endothelin-1 by a combination of chemical modification and proteolysis of a fusion protein in Escherichia coli.

A protein modification method has been developed for the production of human big endothelin (ET)-1. Production of a large quantity of big ET-1 by the method described here is expected to facilitate future experiments such as X-ray crystallography and nuclear magnetic resonance studies, aimed at understanding the tertiary structure of big ET-1 and its dynamics. The plasmid pETB-50 used for the synthesis carries the gene for a fusion protein consisting of 34-amino acid (aa) residues of an N-terminal portion of beta-galactosidase and the 38-aa residues of big ET-1. The fusion protein ETB-50P contains an arginine residue in the big ET-1 portion at its second C-terminal site and three lysine residues including the C-terminal site in the beta-galactosidase portion, all of which are susceptible to trypsin. Tryptic digestion of the fusion protein quantitatively produced big ET-1 (1-37), which is depleted in the C-terminal serine. However, a treatment of the fusion protein with 1,2-cyclohexanedione prior to tryptic digestion gave full-length big ET-1 with N7, -N8-(1,2-dihydroxycyclohex-1,2- ylene)-arginine. This modification was reversed to the intact arginine residue when the modified big ET-1 was incubated in 0.5 M TRIS-HCl buffer, pH 8.0. Consequently, a combination of such a reversible protein modification and tryptic digestion gave 1.74 mg of recombinant big ET-1 from 2.01 of culture broth. The procedure described here may be applied to produce other arginine-containing peptides from fusion proteins.