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-pat hological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were repor ted in the literature. Most of these reports were not successful in obtaini ng 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, t o be cathepsin C-activated, newly raised anti-chymase Ig were used to follo w 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, o r the native hormone. Chymase was produced in CHO cells and appropriately m atured. The amount of enzyme obtained at the end of the process is compatib le with the medium-throughput screening (up to 10000 points per day), about 800 mug.L-1 of culture medium with a specific activity of 6.16 mmol of ang iotensin I cleaved per minute per mg of protein. All the biological and tec hnical tools are now available for the discovery of new classes of chymase inhibitors.