Metacaspase are cysteine-dependent proteases that are found in fungi, plants, and protozoa and are dependent on calcium for activity. Proteases are defined as proteins that cleave other proteins as well as themselves (autoproteolysis). They play a critical role in programmed cell death and innate immune response in these organisms and do so by cleaving specifically after arginine or lysine residues. There are two types of metacaspases classified as type I and type II. Type II metacaspases lack the N-terminal prodomain that is found in Type I and contain a linker region between their two domains. Five different Type I metacaspases have been extensively studied in our lab. To start, we will study S. commune Type II metacaspases to determine if they are another form of Type I or if they are consistent with the literature definition of Type II enzymes. The goal of this project is to express and purify the possible Type II metacaspases and determine their activity. To purify the protein, nickel affinity chromatography was used to isolate the metacaspase after overexpression in E. coli. Analysis techniques consist of gel electrophoresis as well as activity assays to determine the activity and cleavage patterns due to autoproteolysis. Once purified, we will compare both calcium dependence and activity of the possible Type II in comparison with the Type I metacaspases.
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