Oligohis‐tags: mechanisms of binding to Ni²⁺‐NTA surfaces

Abstract

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.

Extracellular vesicles as a platform for membrane‐associated therapeutic protein delivery

“…A related approach in which liposomes containing NTA-Ni lipids were used to display histidine-tagged proteins failed due to low dissociation constants associated with unstable immobilization [66,67]. …”

Reversible Major Histocompatibility Complex I-Peptide Multimers Containing Ni2+-Nitrilotriacetic Acid Peptides and Histidine Tags Improve Analysis and Sorting of CD8+ T Cells

“…For kinetic experiments A2-His 6 Flu peptide complexes were diluted in running buffer in 1:2 serial dilutions starting from 22 M, and A2-His 12 or A2-2ϫHis 6 peptide complexes in 1:3 serial dilutions, starting from 7.4 M and passed over the Ni 2ϩ -NTA-biotin-SA-coated and control flow cells at 50 l/min. As described previously (16,17), the steps were as follows: (i) rinsing the flow cells with running buffer; (ii) activation of peptide-NTA with Ni 2ϩ (500 M NiCl 2ϩ in running buffer); (iii) rinsing in running buffer; (iv) injection of His-tagged A2/Flu complexes at different concentrations and measuring changes in RU measured over 60 s and then over an uninterrupted dissociation period of 6 min; (v) His-tagged protein removal by washing with imidazole solution (500 mM in water); (vi) NiCl 2ϩ removal with regeneration buffer (10 mM HEPES, 150 mM NaCl, 0.005% polysorbate 20, 350 mM EDTA, pH 7.4); and (vii) rinse in dispenser buffer (10 mM HEPES, 150 mM NaCl, 0.005% polysorbate 20, 3 mM EDTA, pH 7.4). The k on and k off values were calculated assuming 1:1 Langmuir binding, and data were analyzed using BIAevaluation 4.1 software and a global fit algorithm.…”

“…Mono-Ni 2ϩ -NTA compounds form reversible coordination complexes with oligohistidines, which for a His 6 have a K D of ϳ10 Ϫ6 M (13)(14)(15)(16)(17). Although this is sufficient for purification of His-tagged recombinant proteins from culture supernatants (3), it is not sufficient for the preparation of staining reagents, which must be stable for months.…”

Immobilization of His-tagged kinase JAK2 onto the surface of a plasmon resonance gold disc modified with different copper (II) complexes

“…The His-tag typically consists of five or six consecutive His residues added to the C-or N-terminus of the protein [20]. The most popular systems for oriented immobilization of His-tagged proteins are complexes of nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) with transition metal ions (Ni 2+ , Cu 2+ ) [20][21][22][23][24][25][26][27][28]. Nitrilotriacetic acid forms a tetradentatechelate with Ni 2+ and Cu 2+ , which is able to bind His-tagged proteins via coordinated bonds [29].…”

“…A coordinative complex with a binding affinity of K D equal to 1 mM is simply formed between one molecule of NTA and two imidazole rings on His-tagged protein [20,27]. Dextran-based SPR chips modified with covalently linked NTA molecules are commercially available and have been used for the detection of the complex between Me 2+ -NTA and His-tagged proteins [20,24,26,30].…”