MMP activity was measured using the SensoLyte 520 generic MMP assay kit (AnaSpec), according to the instructions of the manufacturer. Briefly, purified proteins (30 ng), containing His-tagged CpGV-MMP, His-tagged CpGV-MMP-Mut, induced pET32a empty vector proteins, or control human MMP-9 (30 ng; AnaSpec) were diluted in MMP assay buffer (200 mM NaCl, 5 mM CaCl2, 20 µM ZnS04, 0.05% Brij 35, and 50 mM Tris–HCl, NS 6180 7.5) to a final volume (50 µl). Proteins were mixed with generic fluorescent MMP substrate (50 µl; 5-FAM/QXLTM520) in 96-well microtiter plates and incubated for 15 min at 37 °C. To test for MMP specific activity, GM6001 (400 µM; broad-spectrum MMP inhibitor; Millipore) and EDTA (metal chelator) were used to inhibit MMP activity. Fluorescence measurements were performed every 5 min for 2 h using a basidiomycetes Wallac Victor3TM (PerkinElmer) plate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
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