Oligohis-tags: mechanisms of binding to Ni2+-NTA surfaces.

Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni(2+) immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C- or N-terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni(2+)-NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni(2+)-NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (K(D)) of 14 +/- 1 nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding.

Knecht S ，Ricklin D ，Eberle AN ，Ernst B ... -  《-》

Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on ni-nitrilotriacetic acid surfaces.

Khan F ，He M ，Taussig MJ 《ANALYTICAL CHEMISTRY》

Development and characterization of Ni-NTA-bearing microspheres.

Lauer SA ，Nolan JP 《-》

Immobilization of bacteriophage Qbeta on metal-derivatized surfaces via polyvalent display of hexahistidine tags.

Udit AK ，Brown S ，Baksh MM ，Finn MG ... -  《-》

BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip.

While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system. We show here that the histidine (His) tag, routinely used in protein purification and in detection is an ideal tag for immobilization, despite the intrinsically low affinity between an immobilized metal ion and the His tag. This is due to strong rebinding effects caused by the high surface density of immobilized Ni2+-nitrilotriacetic acid (NTA) on the chips used here. The immobilization of the ligand can be adjusted to a low level using the same chip, such that mass transport limitation and rebinding of the analyte to the immobilized ligand is minimal. Nine different proteins with different numbers of His tags were tested for stable binding to the Ni2+-NTA surface. Most proteins with one His tag dissociate very rapidly from the Ni2+-NTA surface, and the KD for the interaction between His tag and Ni2+-NTA was estimated to about 10(-6) m at neutral pH. In contrast, two His tags are usually found to be sufficient for stable binding. The kinetics of the chaperonin system of Escherichia coli GroEL and GroES were analyzed as a model using this system and found to be very similar to those obtained with covalently immobilized ligands. The sensor chip can be reused many times, because of the powerful regeneration methods. The ligand can be freshly immobilized after each cycle, thus eliminating potential denaturation upon regeneration as a source of error.

Nieba L ，Nieba-Axmann SE ，Persson A ，Hämäläinen M ，Edebratt F ，Hansson A ，Lidholm J ，Magnusson K ，Karlsson AF ，Plückthun A ... -  《ANALYTICAL BIOCHEMISTRY》