Mutagenesis to Improve Protein Expression and Study Proteolytic Sites in Metacaspases

In multicellular organisms, apoptosis is an important process in maintaining the health of the organism. Apoptosis is the process of systematic cell death, in which damaged, old, or dysfunctional cells are eliminated in a controlled manner. In this process, metacaspases, which are cysteine-dependent proteases found in plants, fungi, and protozoa, play a critical role in the function of apoptosis. Metacaspases depend on calcium for their function and they cleave after arginine and lysine residues. The structure and therefore function of the metacaspase is dependent on the chain of amino acids, and how the protein folds into its tertiary structure. In the Fox Lab, we study mutations in the DNA code and how those mutations affect the function of the metacaspase. My project is twofold, first I created a single-point mutation in which I changed lysine to glutamine at a predicted proteolysis site. Secondly, I inserted the missing lac repressor binding sequence into the plasmid containing the metacaspase DNA sequence. The lac operon plays an important role in allowing transcription to occur in the presence of lactose or lactose analogs. Both mutations were successfully produced and verified by DNA sequencing.