Shinji Takai1, Shin-ichiro Sumi2, Masaki Aoike2,
Yoshimi Itoh3, Denan Jin1, Eiko Matsumura3 and Mizuo Miyazaki1
1Department of Pharmacology, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan
2Institute for Biotechnology Research, Wakunaga Pharmaceutical Co., Ltd., Kodacho, Takatagun, Hiroshima 739-1195, Japan
3Department of Cell Biology, Osaka University of Pharmaceutical Sciences, Takatsuki City, Osaka 569-1041, Japan
Abstract: We compared recombinant human chymase expressed in Escherichia coli with human chymase purified from vascular tissues. The recombinant chymase, the structure of which was NH2-enterokinase cleavage site-chymase-COOH, was expressed in Escherichia coli and then was solubilized and renatured. The protein did not have a chymase activity, but gained this activity after the cleavage of the N-terminal site by enterokinase. The enzyme was purified by heparin affinity and gel filtration columns. The N-terminal sequence of the protein was identical to the sequence for human chymase. The molecular weights of the recombinant chymase and chymase purified from human vascular tissues were 26 and 30 kDa, respectively, and the 4 kDa difference was thought to be due to the presence or absence of glycan. The optimum pH of the recombinant enzyme activity was between 7.5 and 9.0. The activity of the recombinant enzyme was inhibited by chymostatin, soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, but not by ethylenediaminetetraacetic acid and aprotinin. This enzyme cleaved specifically the Phe8-His9 bond of angiotensin (Ang) I to form Ang II and that of big endothelin (ET)-1 to form ET-1-(1-31). These findings demonstrated that the enzymatic characteristics of the recombinant enzyme were identical to that of native human chymase.
Keywords: Chymase, Recombinant, Angiotensin II, Inhibitor