Cloning, expression, and characterization of ScMC2

In cells, programmed cell death (PCD) is a pathway important for the maintenance of homeostasis. Dysfunction of PCD can lead to various diseases including cancer. Programmed cell death can be classified into three categories: apoptosis, autophagy, and programmed necrosis. Many multicellular organisms use apoptosis for cell control, and recent studies have shown that apoptosis can also occur in unicellular organisms. Similar to other pathways in the cell, apoptosis and programmed cell death must be highly controlled. In mammals proteins called caspases have been identified as key players in apoptosis control, while metacaspases have been implicated in the cell death pathway in protozoa, fungi and plants. Metacaspases are cysteine-dependent proteases and are distantly related to the caspases found in mammals. Schizophyllum commune is a fungus that contains five metacaspases (ScMC1-5). The gene for ScMC2 has been successfully cloned using PCR to amplify and then Gibson Assembly to assemble a plasmid containing the ScMC2 gene, a His-tag for purification, and an additional sequence to aid in folding. After confirming the gene sequence ScMC2 protein was then successful expressed in E. coli and purified using nickel affinity chromatography. Purification quality was analyzed using SDS-PAGE and enzymatic assays with the peptide Gly-Arg-Arg as a substrate. Optimal pH and optimal concentrations of CaCl2 and NaCl for activity were determined. Kinetic assays were then performed to determine the kinetic constants Km and Vmax.