title: Expression and Purification of Haemophilus influenzae Rhomboid Intramembrane Protease GlpG for Structural Studies.
abstract: Rhomboid proteases are membrane-embedded proteases that cleave peptide bonds of transmembrane proteins. They play a variety of roles in cell signaling events. The rhomboid protease GlpG from Haemophilus influenzae (hiGlpG) is a canonical form of rhomboid protease having six transmembrane segments. In this unit, detailed protocols are presented for optimization of hiGlpG expression using the araBAD promotor system in the pBAD vector. The parameters for optimization include concentration of inducing agent, induction temperature, and time. Optimization of these key factors led to the development of a protocol yielding 1.6 to 2.5 mg/liter protein purified after ion metal affinity chromatography (IMAC). Further purification can include size exclusion chromatography (SEC).
authors.affiliation: Department of Biochemistry, Membrane Protein Disease Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Pankaj
authors.initials: P
authors.lastname: Panwar
authors.affiliation: Department of Biochemistry, Membrane Protein Disease Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: M Joanne
authors.initials: MJ
authors.lastname: Lemieux
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010450:Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D005822:Genetic Vectors; D006193:Haemophilus influenzae / Q000201:enzymology / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases
keywords: IMAC; SEC; expression study; pBAD; rhomboid protease