Expression, Purification, and Enzymatic Characterization of Intramembrane Proteases.

Intramembrane proteases catalyze peptide bond hydrolysis in the lipid bilayer and play a key role in numerous cellular processes. These integral membrane enzymes consist of four classes: site-2 protease (S2P), rhomboid serine protease, Rce1-type glutamyl protease, and aspartyl protease exemplified by presenilin and signal peptide peptidase (SPP). Structural elucidation of these enzymes is important for mechanistic understanding of their functions, particularly their roles in cell signaling and debilitating diseases such as Parkinson's disease and Alzheimer's disease. In the past decade, rigorous effort has led to determination of the crystal structures of S2P from archaebacterium, rhomboid serine protease from E. coli (GlpG), and presenilin/SPP from archaebacterium (PSH). A novel method has been developed to express well-behaved human γ-secretase, which facilitated its structure determination by cryoelectron microscopy (cryo-EM). In this chapter, we will discuss the expression and purification of intramembrane proteases including human γ-secretase and describe the enzymatic activity assays for these intramembrane proteases.