title: A simple in vitro acylation assay based on optimized HlyA and HlyC purification.
abstract: HlyA is a toxin secreted by uropathogenic Escherichia coli strains. HlyA belongs to the repeats in the toxin protein family and needs (i) a posttranslational, fatty acylation at two internal lysines by the acyltransferase HlyC and (ii) extracellular ion binding to achieve its active conformation. Both processes are not fully understood and experiments are often limited due to the low amounts of protein available. Here, we present an optimized purification protocol for the proteins involved in HlyA activation as well as a quick and nonradioactive assay for in vitro HlyA acylation. These may simplify future experiments, e.g., activity scanning and characterization of HlyA or HlyC mutants as demonstrated with single and double HlyA lysine mutants.
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Thomas
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
authors.forename: Sander H J
authors.initials: SH
authors.lastname: Smits
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany. Electronic address: lutz.schmitt@hhu.de.
authors.forename: Lutz
authors.initials: L
authors.lastname: Schmitt
mesh_terms: D000215:Acylation; D000217:Acyltransferases / Q000302:isolation & purification*; D001483:Base Sequence; D017931:DNA Primers; D029968:Escherichia coli Proteins / Q000302:isolation & purification*; D006460:Hemolysin Proteins / Q000302:isolation & purification*; D066298:In Vitro Techniques
chemical_list: D017931:DNA Primers; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D000217:Acyltransferases; C092237:HlyC protein, E coli
keywords: Erythrocytes; Hemolytic activity; Ion binding; RTX toxin; UPEC