Expression in yeast of the metacaspase scp3 from the fungus Schizophyllum commune (1010.8)

Caspase homologs, metacaspases, are found in fungi, protists, and protozoa and are involved in programmed cell death‐like behavior. There is also evidence that metacaspases are additionally involved in proteostasis and cell cycle regulation. Metacaspases contain a cysteine histidine catalytic diad and cleave after lysine and arginine. Some metacaspases are activated by calcium and autocatalytic processing occurs in several, but is not necessary for activity. Five metacaspases, named scp1‐5, have been identified from the fungus Schizophyllum commune with the goal of expressing and characterizing them. Previous attempts to express these metacaspases in E. coli have resulted in either low amounts of protein or protein that forms inclusion bodies. The yeast Saccharomyces cerevisiae is often used to express eukaryotic proteins and should allow the production of large amounts of soluble protein. Therefore, the expression of these metacaspases in yeast is being investigated as an alternative to expression in E. coli. The scp3 gene has been successfully cloned from S. commune and inserted into the vector pYES2.1/V5‐His‐TOPO (Life Technologies). The vector containing scp3 was transformed into S. cerevisiae. Expression has been attempted by induction with galactose through the GAL1 promoter in the vector. Scp3 will be detected by western blotting using a V5 antibody (Invitrogen).