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The impact of a His-tag on DNA binding by RNA polymerase alpha-C-terminal domain from Helicobacter pylori.
Paul, Navjit K; Baksh, Karina A; Arias, Joaquin F; Zamble, Deborah B.
Afiliação
  • Paul NK; Department of Chemistry, University of Toronto, Toronto, Ontario, M5S 3H6, Canada.
  • Baksh KA; Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
  • Arias JF; Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
  • Zamble DB; Department of Chemistry, University of Toronto, Toronto, Ontario, M5S 3H6, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada. Electronic address: dzamble@chem.utoronto.ca.
Protein Expr Purif ; 167: 105541, 2020 03.
Article em En | MEDLINE | ID: mdl-31756376
Polyhistidine tags (His-tags) are commonly employed in protein purification strategies due to the high affinity and specificity for metal-NTA columns, the relative simplicity of such protocols, and the assumption that His-tags do not affect the native activities of proteins. However, there is a growing body of evidence that such tags can modulate protein structure and function. In this study, we demonstrate that a His-tag impacts DNA complex formation by the C-terminal domain of the α-subunit (αCTD) of Helicobacter pylori RNA polymerase in a metal-dependent fashion. The αCTD was purified with a cleavable His-tag, and complex formation between αCTD, the nickel-responsive metalloregulator HpNikR, and DNA was investigated using electrophoretic mobility shift assays. An interaction between His-tagged αCTD (HisαCTD) and the HpNikR-DNA complex was observed; however, this interaction was not observed upon removal of the His-tag. Further analysis revealed that complex formation between HisαCTD and DNA is non-specific and dependent on the type of metal ions present. Overall, the results indicate that a histidine tag is able to modulate DNA-binding activity and suggests that the impact of metal affinity tags should be considered when analyzing the in vitro biomolecular interactions of metalloproteins.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: RNA Polimerase III / Helicobacter pylori / Etiquetas de Sequências Expressas / Proteínas de Ligação a DNA Idioma: En Revista: Protein Expr Purif Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Canadá País de publicação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: RNA Polimerase III / Helicobacter pylori / Etiquetas de Sequências Expressas / Proteínas de Ligação a DNA Idioma: En Revista: Protein Expr Purif Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Canadá País de publicação: Estados Unidos