Biochemical Properties of Recombinant Protein M28AP

The biochemical properties of recombinant protein M28AP, while using PBS buffer as the control and the activity of Asp, was assayed by the hydrolysis of L-leucine-7-amido-4-methylcoumarin hydrochloride (Leu-AMC) and l-arginine-AMC (Arg-AMC, Sigma-Aldrich, st. Louis, MO, USA). We added approximately 1 μg/mL the recombinant protein M28AP solution to 195 μL of assay buffer (50 mM Tris-HCl, pH 8.0) containing 10 μM Leu-AMC or Arg-AMC up to 200 μL, which was then incubated for 30 min at 37 °C. The release of fluorescence was measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm while using a FlexStation 3 Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA) [21 (link)].