title: Parameters for effective in vitro production of zinc finger nucleic acid-binding proteins.
abstract: Immobilized metal affinity chromatography (IMAC) is widely used for the production of recombinant proteins for a variety of applications; however, a number of challenges are typically encountered by researchers depending on the properties of the specific proteins in question. Here, we describe technical issues we have encountered in production of recombinant zinc finger nucleic acid-binding proteins by IMAC intended for detailed and accurate in vitro analysis. The process encountered leading to a modified IMAC protocol for effective production of high-purity, native zinc finger nucleic acid-binding proteins is described in detail. The parameters with respect to solubility, lysis and redox conditions, removal of residual metal ions with chelating agents, and renaturation in the presence of divalent metal cations are described. These procedures have been extended to production of a wide array of RNA-binding proteins in our laboratory and would be relevant to a number of protein purification applications.
authors.affiliation: Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, Saskatoon, Canada.
authors.forename: Zachery R
authors.initials: ZR
authors.lastname: Belak
authors.forename: Manoj
authors.initials: M
authors.lastname: Nair
authors.forename: Nick
authors.initials: N
authors.lastname: Ovsenek
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008670:Metals / Q000737:chemistry; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D016335:Zinc Fingers
chemical_list: D002614:Chelating Agents; D004268:DNA-Binding Proteins; D008670:Metals; D011994:Recombinant Proteins