Characterization of recombinant human chymase expressed in Escherichia coli

Citation
S. Takai et al., Characterization of recombinant human chymase expressed in Escherichia coli, JPN J PHARM, 82(2), 2000, pp. 144-149
Citations number
33
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pharmacology & Toxicology
Journal title
JAPANESE JOURNAL OF PHARMACOLOGY
ISSN journal
0021-5198 → ACNP
Volume
82
Issue
2
Year of publication
2000
Pages
144 - 149
Database
ISI
SICI code
0021-5198(200002)82:2<144:CORHCE>2.0.ZU;2-#
Abstract
We compared recombinant human chymase expressed in Escherichia coli with hu man chymase purified from vascular tissues. The recombinant chymase, the st ructure of which was NH2-enterokinase cleavage site-chymase-COOH, was expre ssed in Escherichia coli and then was solubilized and renatured. The protei n did not have a chymase activity, but gained this activity after the cleav age of the N-terminal site by enterokinase. The enzyme was purified by hepa rin affinity and gel filtration columns. The N-terminal sequence of the pro tein was identical to the sequence for human chymase. The molecular weights of the recombinant chymase and chymase purified from human vascular tissue s 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 recombi nant enzyme activity was between 7.5 and 9.0. The activity of the recombina nt enzyme was inhibited by chymostatin, soybean trypsin inhibitor and pheny lmethylsulfonyl fluoride, but not by ethylenediaminetetraacetic acid and ap rotinin. This enzyme cleaved specifically the Phe(8)-His(9) bond of angiote nsin (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 t he recombinant enzyme were identical to that of native human chymase.