Development of new assays and improved procedures for the purification of recombinant human chymase.
Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin-angiotensin system. This enzyme is also involved in other physio-pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C-activated, newly raised anti-chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96-well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC-based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium-throughput screening (up to 10,000 points per day), about 800 microg x L(-1) of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.
Ferry
G
G
Division de Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Croissy sur Seine, France; Fukuoka University, Department of Cardiology, Fukuoka, Japan; Institut de Recherches Internationales Servier, Courbevoie, France.
Gillet
L
L
Bruneau
V
V
Banales
J M
JM
Beauverger
P
P
Cogé
F
F
Galizzi
J P
JP
Scalbert
E
E
Okamoto
T
T
Urata
H
H
Boutin
J A
JA
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D053818:Chymases; D006224:Cricetinae; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053818:Chymases