title: Expression, Purification, and Enzymatic Characterization of Intramembrane Proteases.
abstract: 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.
authors.affiliation: Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China. Electronic address: zhour13@mails.tsinghua.edu.cn.
authors.forename: R
authors.initials: R
authors.lastname: Zhou
authors.affiliation: Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China. Electronic address: shil-lab@tsinghua.edu.cn.
authors.forename: Y
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China. Electronic address: ygh12@mails.tsinghua.edu.cn.
authors.forename: G
authors.initials: G
authors.lastname: Yang
mesh_terms: D000544:Alzheimer Disease / Q000201:enzymology; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology*; D020285:Cryoelectron Microscopy / Q000379:methods*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008967:Molecular Biology / Q000379:methods*; D010300:Parkinson Disease / Q000201:enzymology; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; C491326:RCE1 protein, human; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase; D008666:Metalloendopeptidases; C120654:MBTPS2 protein, human
keywords: Enzymatic activity assay; Expression; I-CLiPs; Intramembrane protease; Purification; Structure; γ-Secretase