MW = 18.6 kDa calculated. Recombinant Matrix Metalloproteinase-10 (MMP-10, Stromelysin-2, Transin 2) cloned from human cDNA, expressed in E. coli. The enzyme consists of the catalytic domain of human MMP-10 (residues 99-263 UniProtKB accession P09238) with the mutation F170N. 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.
100 110 120 130 140 M-FS SFPGMPKWRK THLTYRIVNY TPDLPRDAVD SAIEKALKVW 150 160 170 180 190 EEVTPLTFSR LYEGEADIMI SFAVKEHGDN YSFDGPGHSL AHAYPPGPGL 200 210 220 230 240 YGDIHFDDDE KWTEDASGTN LFLVAAHELG HSLGLFHSAN TEALMYPLYN 250 260 SFTELAQFRL SQDDVNGIQS LYG
> 95% by SDS-PAGE. The protein is observed, in denaturing conditions, as a single band migrating at a molecular weight between 18.4 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) 0.2 M. The concentration is calculated by the analysis of the absorbance at 280 nm (ε280 = 29910 M-1cm-1 calculated).
> 10U/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.
Bode, W. et al. Cell. Mol. Life Sci. 55 (4), 639-652 (1999).
Murphy, G. & Knäuper, V. Matrix Biol. 15 (8-9), 511-518 (1997).