ABSTRACT
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.
