title: False positive RNA binding activities after Ni-affinity purification from Escherichia coli.
abstract: A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Tetyana
authors.initials: T
authors.lastname: Milojevic
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Sonnleitner
authors.forename: Alessandra
authors.initials: A
authors.lastname: Romeo
authors.forename: Kristina
authors.initials: K
authors.lastname: Djinović-Carugo
authors.forename: Udo
authors.initials: U
authors.lastname: Bläsi
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D035001:Host Factor 1 Protein / Q000302:isolation & purification / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D006639:Histidine; D012313:RNA
keywords: Crc; EMSA; Hfq; IMAC; Ni-affinity purification; RNA binding proteins