Immobilization of His-tagged proteins on NiO foams for recyclable enzymatic reactors

Highlights

• NiO foams with high surface area were synthesised.

• His-tag proteins bind to the NiO foam and remain bound upon wash.

• Protein binding to the foam results in microporous cavities alterations.

• DFT suggests His-tags prefer stretched conformations and act as bidentate ligands.

• Immobilized His-tag enzyme remains active and the support is recyclable.

Abstract

We report for the first time the use of NiO foams for specific protein binding. These affordable porous materials, with high surface area, possess the features required as supports for enzymatic catalysis. A physicochemical and computational characterization of the interaction between this support and a model His-tagged sulfotransferase was performed as a first approach to the use of NiO foams for specific protein binding. Results confirm that the His-tag binds the NiO foam and remains bound when the support is washed. The foam microporous cavities undergo marked rearrangement upon protein binding and removal, in agreement with the successful immobilization and posterior removal of the protein on the foam. Computational results suggest that the His-tag prefers a stretched conformation, acting as a bidentate ligand. Enzymatic activity of the immobilized protein was confirmed, as well as the ability to recycle the support. These results demonstrate that NiO foams constitute an affordable and effective material for His-tag protein immobilization, with potential applications, namely protein isolation and enzymatic reactor applications. The ultimate aim of this work is to contribute to a better understanding of the kinetic activity at surface level, as well as the comparison between the experimental results and the computational output.

Introduction

Polyhistidine tags are a motif of usually 6 His residues added at the C or N terminus of a protein to assist in protein purification using an affinity approach [1]. The addition of this tag to either end of the coding sequence by recombinant DNA methods leads to the expression of a hybrid recombinant protein that can be easily purified by immobilized metal-affinity chromatography (IMAC) [2], [3], [4]. Purification of His-tagged proteins is based on the affinity of the imidazole moiety to transition metals – histidine displays a higher affinity to copper ions, and a decreasing affinity towards nickel, zinc and cobalt [2], [5]. Purification of proteins with His-tags is the most commonly used method due to its versatility and compatibility with most experimental conditions [3], [5]. His-tag protein purification by IMAC is accomplished using elution buffers with imidazole, at concentrations typically ranging from 20 to 500 mM, out-competing the proteins for the metal sites [3].

A large number of IMAC systems are also commercially available, from Ni-loaded resins to magnetic spheres decorated with Ni complexes, that can be applied to virtually all expression and purification strategies. The most conventional form for IMAC His-tag protein purification is the use of chromatography resins loaded with Ni ions, but a growing number of other supports have been developed in the last years. Nickel ferrite or gold nanoparticles [6], [7], agarose beads [8], graphene [9] and metal–organic frameworks (MOFs) [10] have been successfully used to bind and immobilize His-tag proteins, affording enzymatic reactors at different scales, including at a microfluidic scale [11].

Enzymatic reactions carried on with immobilized enzymes have a widespread application and can be efficiently prepared for use in various research and industrial applications, including drug metabolism. For example, the elusive cytochrome P450 (CYP) 2C9 has been successfully expressed and immobilized on a poly(methyl methacrylate) surface to generate a functional in vitro drug metabolism reactor [12]. CYP-functionalized CdTe quantum dots [13] and CYP immobilization on gold surfaces [14] have also led to functional CYP 2C9 reactors. Other drug-metabolizing enzymes, such as sulfotransferase 1A1, were also successfully immobilized on magnetic microparticles, affording a functional reactor [15].

3D porous NiO foams, due to their particularly high specific surface area, have been suggested as a promising material in several applications, among which as an electrode for supercapacitors [16], as a catalyst [17], in sensors [18], to support cleaning and separation applications [19], or for solar thermal water evaporation [20]. From the several techniques used to achieve these NiO 3D structures, like sol–gel, hydrothermal and chemical bath deposition, electrodeposition is highlighted due to clear advantages from cost and ecological viewpoints. This is a simple and straightforward route to achieve immobilized 3D porous structures. To prepare these 3D porous metal foams with macroporous ramified walls, hydrogen bubbles act as a dynamic template during the fast metal deposition attained by the high cathodic current densities applied [21].

In this work, we analysed whether NiO foams can be used as a solid support for protein immobilization. A combined scanning electron microscopy (SEM), X-ray diffraction (XRD), attenuated total reflectance (ATR), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry, Brunauer-Emmett-Teller (BET) approach, together with a DFT-based computational study, was used to characterize the foams before and after immobilization of a recombinant His-tagged human sulfotransferase. The enzymatic activity of the immobilized proteins was also confirmed using model substrates.