Date of Award

6-2020

Document Type

Restricted (Opt-Out)

Degree Name

Bachelor of Science

Department

Biochemistry

First Advisor

Kristin Fox

Keywords

metacaspase, purification, mutation, autoproteolysis, PCR, SDS-PAGE

Abstract

Metacaspases are cysteine-dependent proteolytic enzymes that cleave after arginine and lysine amino acid residues. These enzymes are found in plants, fungi, and protozoa, but not animals, giving them potential as targets for anti-fungal treatments. Metacaspases are found to exhibit optimal activity on small peptides in the presence of calcium, and some require autoproteolytic cleavage of their N-terminal domains for full activity. Schizophyllum commune is a mushroom-forming fungus that has been found to contain the genes of five Type I metacaspases, named ScMCA-Ia-e. These metacaspases have also been expressed without their N-terminal prodomains, and consequently named ScMCA-Ia-e∆pro. All five metacaspases have been characterized, with their optimal calcium and pH levels having been previously determined. The desire to prevent autoproteolysis from occurring in vitro in these metacaspases has led to extensive research into the cleavage of ScMCA-Ia∆pro. While it has been previously proposed that ScMCA-Ia∆pro undergoes cleavage after R389 and R447, the study of deletion mutants after R389 and R447 have shown that this is not the case. Interestingly, time course studies have indicated that R447 does still hold an important role in the autoproteolytic process. N-terminal protein sequencing of a lower molecular weight cleavage fragment has pointed to two lysine amino acids, K368 and K375, as potential sites of cleavage in the protein. To determine if either of these lysine residues form the cleavage site of ScMCA-Ia∆pro, further work needs to be done to determine the effect created on the protein by mutating these residues.

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In Copyright - Educational Use Permitted.