title: Stable and functional immobilization of histidine-tagged proteins via multivalent chelator headgroups on a molecular poly(ethylene glycol) brush.
abstract: We present a generic approach for immobilizing oligohistidine-tagged proteins with high stability and homogeneous functionality onto glass-type surfaces. Multivalent chelator heads (MCH) carrying two and three nitrilotriacetic acid (NTA) moieties were coupled with controlled surface concentration to glass surfaces premodified with an ultrathin two-dimensional polymer brush of a bifunctional poly(ethylene glycol). Low roughness and lateral homogeneity of these surfaces were confirmed by AFM and fluorescence microscopy, respectively. Protein immobilization and interactions at these interfaces were studied by label-free and fluorescence detection. Oligohistidine-tagged proteins bound specifically to NTA loaded with nickel(II) ions and could be eluted with imidazole. More than 90% of the immobilized protein preserved its activity. In contrast to mono-NTA, immobilized multivalent chelator heads bound oligohistidine-tagged proteins stoichiometrically and with high stability, even at very low chelator surface concentrations. Thus, an excess of the metal chelator sites was not necessary, and excessive binding sites could be quantitatively blocked with an indifferent protein. As a consequence, increased functional stability of the immobilized protein and a substantial reduction in nonspecific adsorption were achieved. Binding of histidine-tagged proteins to the MCH-modified surface was efficiently blocked by stoichiometric amounts of soluble MCH, and biomolecular interaction unbiased by the interaction of the histidine tag to the surface-bound MCH was observed. These excellent features and the compatibility with many solid-phase analytical techniques make this surface chemistry beneficial for functional protein analysis.
authors.affiliation: Institute of Biochemistry, Johann Wolfgang Goethe-University, Marie-Curie-Strasse 9, 60439 Frankfurt am Main, Germany.
authors.forename: Suman
authors.initials: S
authors.lastname: Lata
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
mesh_terms: D002614:Chelating Agents / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity
chemical_list: D002614:Chelating Agents; D009942:Organometallic Compounds; D011506:Proteins; C088321:nickel nitrilotriacetic acid; D011092:Polyethylene Glycols; D006639:Histidine; D009571:Nitrilotriacetic Acid