Hexahistidine (His6)-tag dependent protein dimerization: a cautionary tale.
Nickel nitrilotriacetic acid (Ni2+-NTA) immobilization of hexahistidine (His6) tagged proteins has become one of the most commonly used methods of affinity chromatography. Perhaps the greatest utility of this protein purification method stems from the general belief that His-tagged proteins (comprised of His6) are little affected in their activities or efficiencies, while alterations in specificity are unexpected. Although this is certainly true in many instances, we present a case in which the biochemical properties of proteins being studied were fundamentally altered due to the presence of His-tags. We carried out these studies using variants of the pi(30.5) protein of plasmid R6K, a DNA binding protein which negatively regulates plasmid replication. Pi(30.5) can bind DNA containing a target sequence (TGAGR) arranged either asymmetrically (direct repeats) in the gamma origin, or symmetrically in inverted half-repeats (IR's) in the operator of its own gene, pir. Importantly, dimers of pi protein bind to an IR; this property allows researchers to quickly assess whether different regulatory variants of pi proteins exhibit altered dimerization properties. For example, pi(30.5) containing a single amino-acid substitution, F107S (pi200(30.5)), has been shown to be monomeric in solution and dimers were not observed bound to IR's. Here we demonstrate that the presence of a His-tag partially restores the ability of pi200(30.5) to dimerize in solution and bind to an IR in dimeric form. This report sends an important message that (other) proteins containing His-tags may differ from their wild type counterparts in dimerization/oligomerization properties.
Wu
J
J
Department of Bacteriology, University of Wisconsin, Madison 53706, USA.
Filutowicz
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins; D019281:Dimerization; D006639:Histidine / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009842:Oligopeptides; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D006639:Histidine; D004265:DNA Helicases