title: Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase.
abstract: Escherichia coli tyrosine kinase (Etk) is a membrane bound kinase in gram-negative bacteria that regulates the export of capsular polysaccharides (CPS). The molecular mechanism behind CPS regulation remains unclear, despite access to a crystal structure of the cytoplasmic kinase domain of Etk. In this study, an efficient protocol to produce full length Etk solubilized in n-dodecyl-β-d-maltoside has been established with high yield. We have determined that detergent solubilized Etk retains kinase activity, but the protein is prone to aggregation, degradation, and has been unsuccessful in protein crystallization trials. In response, we designed and characterized truncations of Etk that do not aggregate and have led to successful crystallization experiments. In this article, we discuss our optimized expression and purification protocol for Etk, the design of Etk protein truncations, and the behavior of Etk during purification in a range of stabilizing detergents. These efforts have successfully produced protein suitable for crystallization. Our results will be a useful guide for future structural and functional studies of the bacterial tyrosine kinase family.
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Chelsy
authors.initials: C
authors.lastname: Chesterman
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, Ontario K7L 3N6, Canada. Electronic address: jia@queensu.ca.
authors.forename: Zongchao
authors.initials: Z
authors.lastname: Jia
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003460:Crystallization; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010766:Phosphorylation; D066329:Protein Aggregates; D011485:Protein Binding; D000072417:Protein Domains; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D029968:Escherichia coli Proteins; D066329:Protein Aggregates; D011505:Protein-Tyrosine Kinases
keywords: Crystallization; Detergent; E. coli tyrosine kinase; Membrane protein purification