title: Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein.
abstract: Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure.
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary.
authors.forename: Ria K
authors.initials: RK
authors.lastname: Balogh
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary.
authors.forename: Béla
authors.initials: B
authors.lastname: Gyurcsik
authors.affiliation: Laboratory of Proteomics, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, Hungary.
authors.forename: Éva
authors.initials: É
authors.lastname: Hunyadi-Gulyás
authors.affiliation: Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 207, 2800 Kgs. Lyngby, Denmark. Electronic address: hemc@kemi.dtu.dk.
authors.forename: Hans E M
authors.initials: HE
authors.lastname: Christensen
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary. Electronic address: jancso@chem.u-szeged.hu.
authors.forename: Attila
authors.initials: A
authors.lastname: Jancsó
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D010957:Plasmids / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C051124:MerR protein, Bacteria; C545158:cysteine thiolate; D003300:Copper; D003545:Cysteine
keywords: Copper(I); Cysteine; DNA binding; Four step purification; Metalloregulatory proteins