Chymases are a group of chymotrypsin-like serine proteases secreted by mast cells (1). They are synthesized as inactive precursors containing a 2-residue propeptide, which needs to be removed by dipeptidyl peptidase I/cathepsin C for the enzymatic activity (2). Human chymase encoded by the CMA1 gene is known to be involved in hypertention and heart failure through its ability to convert angiotensin I (Ang I) to angiotensin II (Ang II), which plays a key role in the regulation of arterial pressure (3). In addition, it is also important in physiological and pathological conditions including inflammation, fibrosis and processing of cytokines (4). Therefore, designing a specific inhibitor for chymase activity has been a pharmacologic strategy to develop therapeutic agents. The recombinant human chymase encoded by CMA1 gene (rhChymase, residues 1-247, P23946) was expressed with a C-terminal 10X His tag in a mouse myeloma cell line, NS0. Molecular Mass: The secreted, purified rhChymase has an N-terminal sequence of G20 EIIGGTE, which corresponds to the pro form. The 238 amino acid His-tagged protein predicts a molecular mass of 27kD and migrates with an apparent molecular mass of approximately 38kD in SDS-PAGE under reducing conditions. Activity: Measured by its ability to cleave a fluorogenic peptide substrate, SUV-Ala-Ala-Pro-Phe-AMC. The specific activity, measured with 100uM substrate and 100ng activated enzyme in 100ul of 20mM Tris, 2.0M KCl, 0.02% Triton X-100, pH 9.0 at room temperature, is > 80 pmoles/min/ug. Storage and Stability: Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
