title: Lessons from an α-Helical Membrane Enzyme: Expression, Purification, and Detergent Optimization for Biophysical and Structural Characterization.
abstract: This chapter outlines the protocol developed in our lab to produce a multipass α-helical membrane protein. We present our work flow, from ortholog selection to protein purification, including molecular biology for plasmid construction, protein expression in E. coli, membrane isolation and detergent solubilization, protein purification and tag removal, biophysical assessment of protein stability in different detergents, and detergent concentration determination using thin-layer chromatography. We focus on results from our ongoing work with intramembrane aspartyl proteases from archaeal organisms.
authors.affiliation: School of Chemistry & Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA, 30332-0400, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Johnson
authors.affiliation: School of Chemistry & Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA, 30332-0400, USA.
authors.forename: Sibel
authors.initials: S
authors.lastname: Kalyoncu
authors.affiliation: School of Chemistry & Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA, 30332-0400, USA. raquel.lieberman@chemistry.gatech.edu.
authors.forename: Raquel L
authors.initials: RL
authors.lastname: Lieberman
mesh_terms: D001105:Archaea / Q000201:enzymology*; D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002855:Chromatography, Thin Layer; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003902:Detergents / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012995:Solubility
chemical_list: D003902:Detergents; D057055:Aspartic Acid Proteases
keywords: Circular dichroism; Cloning; Detergent screening; Expression; Intramembrane aspartyl protease; Membrane protein; Purification; Signal peptide peptidase; Thin-layer chromatography