MW = 20.1 kDa calculated. Recombinant Matrix Metalloproteinase-14 (MMP-14, Membrane-Type Matrix Metalloproteinase1, MT1- MMP) cloned from human cDNA, expressed in E. coli. The enzyme consists of the catalytic domain of human MMP-14 (residues 114-290, UniProtKB accession P50281). The protein has the mutation C127S to increase its stability. The catalytic activity rates are not affected by the mutation.
120 130 140 150 160 IQGLKWQ HNEITFSIQN YTPKVGEYAT YEAIRKAFRV WESATPLRFR 170 180 190 200 210 EVPYAYIREG HEKQADIMIF FAEGFHGDST PFDGEGGFLA HAYFPGPNIG 220 230 240 250 260 GDTHFDSAEP WTVRNEDLNG NDIFLVAVHE LGHALGLEHS SDPSAIMAPF 270 280 290 YQWMDTENFV LPDDDRRGIQ QLYGGESGFP
> 95% by SDS-PAGE. The protein is observed, in denaturing conditions, as a single band migrating at a molecular weight between 18.8 and 25.0 kDa.
0.2 mg/mL solution in Tris 20 mM pH 7.2, CaCl2 10 mM, ZnCl2 0.1 mM, NaCl 0.3 M, acetohydroxamic acid (AHA) 0.5 M. The concentration is calculated by the analysis of the absorbance at 280 nm (ε280 = 33920 M-1cm-1 calculated).
> 150 U/μg. Activity described as U=100 pmol/min at 25°C using a colorimetric assay with thiopeptide Ac-Pro-Leu-Gly-[2- mercapto-4-methyl-pentanoyl]-Leu-Gly-OC2H5 (Biomol) as substrate.
-80°C. After initial defrost, aliquot the product into individual tubes and refreeze at -80°C.
Avoid repeated freeze/thaw cycles.
Enzyme kinetic studies, cleavage of target substrates and screening of inhibitors.
K. Lehti et al. J. Biol. Chem. 2000, 275:15006-13.
G. Murphy and V. Knäuper Matrix Biol. 1997, 15, 511.
W. Bode et al. Cell Mol Life Sci 1999, 55:639-52.