The Effect of Varying Temperatures on Metacaspase Yield in Escherichia coli

Metacaspases are cysteine-dependent proteases (analogous to caspases in animal systems) which play a critical role in programmed cell death and stress responses in non-animal organisms. Despite their biological significance, the impact of environmental conditions on metacaspase yield remains underexplored. This study investigates the effect of temperature on metacaspase production in Escherichia coli (E. coli), with the hypothesis that lowering the temperature at which the cells are grown will result in both better expression and a greater amount of metacaspase per weight of cells. This hypothesis lays the foundation for potentially increasing metacaspase expression and yield by lowering the temperature at which the cells are grown, thereby increasing protein concentration. E. coli cultures were grown at 27°C, 20°C, and 15°C, and metacaspase production was assessed through unpurified cell pellet mass, Bradford assays with both purified and unpurified metacaspase samples, and SDS-PAGE analysis of only purified metacaspase samples. Results demonstrated a significant decline in total cell pellet and protein concentration as well as metacaspase-specific concentration at lower temperatures. At 27°C, cell pellet mass and protein yield were highest, whereas at 15°C, both were significantly reduced. SDS-PAGE analysis further confirmed the temperature-dependent decrease in metacaspase expression. These findings highlight the sensitivity of metacaspase production to temperature variation, providing insights into optimal conditions for studying metacaspases and their potential biochemical and medical applications.