Cobalt(III)‐Mediated Permanent and Stable Immobilization of Histidine‐Tagged Proteins on NTA‐Functionalized Surfaces

Abstract

We present the cobalt(III)-mediated interaction between polyhistidine (His)-tagged proteins and nitrilotriacetic acid (NTA)-modified surfaces as a general approach for a permanent, oriented, and specific protein immobilization. In this approach, we first form the well-established Co(2+) -mediated interaction between NTA and His-tagged proteins and subsequently oxidize the Co(2+) center in the complex to Co(3+) . Unlike conventionally used Ni(2+) - or Co(2+) -mediated immobilization, the resulting Co(3+) -mediated immobilization is resistant toward strong ligands, such as imidazole and ethylenediaminetetraacetic acid (EDTA), and washing off over time because of the high thermodynamic and kinetic stability of the Co(3+) complex. This immobilization method is compatible with a wide variety of surface coatings, including silane self-assembled monolayers (SAMs) on glass, thiol SAMs on gold surfaces, and supported lipid bilayers. Furthermore, once the cobalt center has been oxidized, it becomes inert toward reducing agents, specific and unspecific interactions, so that it can be used to orthogonally functionalize surfaces with multiple proteins. Overall, the large number of available His-tagged proteins and materials with NTA groups make the Co(3+) -mediated interaction an attractive and widely applicable platform for protein immobilization.

Formation of supramolecular protein structures on gold surfaces

“…The inclusion of a tag (which locates to the center of the dodecameric ring) also provides a convenient method for metal binding through chelation to the six histidine residues. 10,29,30 This study reports the first findings of the surface assembly of HsPrx3-6his. We investigated the interaction of HsPrx3-6his with gold surfaces, with the goal of forming surface tethered supramolecular protein structures that could later be used for the formation of ordered nanoscale assemblies.…”

Activated αIIbβ3 on platelets mediates flow-dependent NETosis via SLC44A2

“…To each channel, 20l of 3.75M VWF A1 or A1* were applied at room temperature for 20 minutes in a humidified chamber. Channels were then incubated with H 2 O 2 for 30 min to oxidize Co 2+ to Co 3+ , which stabilizes the binding of His-tagged A1/A1* (Wegner et al, 2016).…”

Superparamagnetic Gold Nanoparticles Synthesized on Protein Particle Scaffolds for Cancer Theragnosis

“…The engineered HBV capsids were mixed with chloro(trimethyl phosphine) gold(I) in Tris buffer (50 × 10 −3 m Tris‐HCl, 500 × 10 −3 m NaCl, pH 7.0) to chemisorb the gold ions [(CH 3 ) 3 PAu + ] through the coordination bond with the surface‐localized His 6, followed by reducing the chemisorbed gold ions using a reducing agent (NaBH 4 ) to synthesize the raspberry‐like cluster of AuNPs on the engineered HBV capsid. Regarding metal–polyhistidine coordination, other approaches such as cobalt‐mediated immobilization of histidine‐rich proteins and attachment of polyhistidine‐tagged antigen on cobalt‐coated liposomes have been recently reported . Depending on the reducing condition (see Supporting Information), the two different types of AuNP clusters were synthesized: superparamagnetic AuNP clusters (SPAuNCs) (38.7 ± 3.4 nm) composed of smaller AuNPs (1.4 ± 0.2 nm) and diamagnetic AuNP clusters (DAuNCs) (41.6 ± 3.3 nm) composed of larger AuNPs (4.5 ± 0.9 nm), as confirmed through transmission electron microscopy (TEM), dynamic light scattering (DLS), and energy dispersive X‐ray spectroscopy analyses (Figure b and Figure S3, Supporting Information).…”