title: Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.
abstract: For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6°C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol.
authors.affiliation: Institute of Genetics, Technische Universität Dresden, Helmholtzstr. 10, 01062 Dresden, Germany.
authors.forename: Susan
authors.initials: S
authors.lastname: Ibe
authors.affiliation: Institute of Genetics, Technische Universität Dresden, Helmholtzstr. 10, 01062 Dresden, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Schirrmeister
authors.affiliation: Institute of Genetics, Technische Universität Dresden, Helmholtzstr. 10, 01062 Dresden, Germany. Electronic address: susanne.zehner@tu-dresden.de.
authors.forename: Susanne
authors.initials: S
authors.lastname: Zehner
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002118:Calcium / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008345:Manganese / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014733:Vibrio / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D008345:Manganese; D002118:Calcium
keywords: MIIA domain; Metal ion-inducible autocleavage; Protein linker; Single step purification; Tag removal