Expression and characterization of phospholipase Cβ and ε enzymes

Phospholipase C enzymes play a key role in regulating intracellular calcium release downstream of G protein-coupled receptors and receptor tyrosine kinases. PLCβ and PLCε play key roles in maximal cardiac contractility, cardiac hypertrophy, opioid analgesia, and oncogenesis. Structural and biochemical studies of PLCs have been essential in furthering our understanding of their basal regulation and activation by small G proteins and heterotrimeric G protein subunits. Here, we detail purification protocols for PLCβ and PLCε variants. The resulting proteins have been successfully used for high-resolution structural studies as well as detailed functional studies in vitro. We also describe a receptor-independent in vitro assay, allowing studies of PLC regulation and activation to be carried out using purified proteins. Finally, we describe also detail the methodology for a radioactivity-based assay that can be used to characterize these lipases in cellulo.