title: Production of Recombinant Rhomboid Proteases.
abstract: 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.
authors.affiliation: Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Arutyunova
authors.affiliation: Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Panigrahi
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Electronic address: kvido.strisovsky@uochb.cas.cz.
authors.forename: K
authors.initials: K
authors.lastname: Strisovsky
authors.affiliation: Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada. Electronic address: mlemieux@ualberta.ca.
authors.forename: M J
authors.initials: MJ
authors.lastname: Lemieux
mesh_terms: D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010450:Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006193:Haemophilus influenzae / Q000201:enzymology; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008967:Molecular Biology / Q000379:methods*; D011532:Providencia / Q000201:enzymology; D013329:Structure-Activity Relationship
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010450:Endopeptidases
keywords: AarA; Expression study; IMAC; Rhomboid protease; SEC; ecGlpG; hiGlpG; pBAD