||Recombinant Human matrix metalloproteinase-3 (MMP-3, stromelysin-1, transin) cloned from human cDNA was expressed inE. coli. The enzyme consists of the catalytic domain of human MMP-3 (residues 105-265 swissprot accession P08254). The protein has been mutated to increase its stability, as the mutation drastically reduces the enzyme's rate of autoproteolysis. The catalytic activity rates are not affected by the mutation. MW=18 kDa.
||Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP"s are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. This gene encodes an enzyme which degrades fibronectin, laminin, collagens III, IV, IX, and X, and cartilage proteoglycans. The enzyme is thought to be involved in wound repair, progression of atherosclerosis, and tumor initiation. The gene is part of a cluster of MMP genes which localize to chromosome 11q22.3.
||> 95% by SDS-PAGE. The enzyme was observed as a single band migrating at a molecular weight of < 20 kDa.
||>30U/μg. Activity described as U=100 pmol/min at 37ºC using a colorimetric assay with thiopeptolide Ac-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gly-OC2H5(Biomol) as substrate.
||Enzyme kinetic studies, cleavage of target substrates and screening of inhibitors.
||0.2mg /ml in 20mM Tris, pH 7.2, 10mM CaCl2, 0.1mM ZnCl2, 0.3M NaCl, 0.2M Acetohydroxamic Acid (AHA). The concentration is calculated from the absorbance at 280nm, (e280 = 27310 M-1 cm-1).
||Under the above described conditions, to avoid precipitation or protein dimerization, the product can be concentrated to a maximum of 0.5mM.
||-80ºC. The enzyme is stable at -20ºC for at least 1 week. After initial defrost, aliquot enzyme into individual tubes and refreeze at -80ºC. Avoid repeated freeze/defrost cycles.