title: Escherichia coli-based expression system for the heterologous expression and purification of the elicitin β-cinnamomin from Phytophthora cinnamomi.
abstract: Elicitins are sterol carrier proteins from the Oomycete genera Phytophthora and Phytium and elicit a hypersensitive response in many economically important plants, in some cases causing a systemic acquired resistance. Their recombinant expression in bacteria is complicated by the presence of three disulfide bonds in the elicitin structure. In consequence, elicitins have so far only been produced in soluble form by isolation from native Phytophthora or Phytium strains or by recombinant expression in the yeast Pichia pastoris. Here, for the first time, we report the soluble expression of the elicitin β-cinnamomin from Phytophthora cinnamomi in Escherichia coli by secretion of the protein into the periplasm. β-Cinnamomin yields have been significantly improved after careful selection of the optimum secretion signal sequence. In total, 17.6 mg β-cinnamomin per liter cell culture have been obtained in shake flasks with the secretion signal sequence of the maltose-binding protein MalE from E. coli. Furthermore, by making use of a C-terminal His-tag, β-cinnamomin purification has been significantly simplified with only one step of immobilized metal ion affinity chromatography yielding protein of high purity (>90%). The established protocol has further been successfully applied to the soluble expression of another elicitin.
authors.affiliation: Institute of Biotechnology, RWTH Aachen University, Aachen, Germany. s.hofzumahaus@biotec.rwth-aachen.de
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hofzumahaus
authors.forename: Anett
authors.initials: A
authors.lastname: Schallmey
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010838:Phytophthora / Q000737:chemistry / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054790:Ribosome Inactivating Proteins, Type 2 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; C061930:cinnamomin, Phytophthora cinnamomi
keywords: Affinity chromatography; Disulfide bonds; Periplasmic expression; Secretion signal peptide; Sterol carrier protein