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Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of K M and V max to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein., (© 2017 Elsevier Inc. All rights reserved.)

Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases., (© 2017 Elsevier Inc. All rights reserved.)