Tetra-nickel substituted polyoxotungsate as an efficient sorbent for the isolation of His6-tagged proteins from cell lysate
Graphical abstract
Introduction
With the development of proteomics in disease diagnosis and treatment, efficient methods for the separation and purification of recombinant proteins are urgently demanded [1], [2], [3], [4]. His-tagged recombinant proteins have been widely utilized for various purposes in molecular biology such as the production of antibodies or investigation of the mechanism of protein-protein interactions. As His-tagged proteins usually coexist with other components, the separation of His-tagged proteins from complicated matrices is of great importance for ensuing investigations. Among the techniques for the separation of histidine protein, immobilized metal affinity chromatography (IMAC) is the extensively adopted one by virtue of the interactions between immobilized metal ions and the electron donor groups such as histidine residues of protein [5], and now various IMAC-based commercial kits are available everywhere. However, the practical application of IMAC techniques is usually annoyed by the long separation time, high pressure as well as low surface metal ion density on the metal affinity columns [6], [7]. Great efforts have been taken for the design and development of novel functional materials to replace the conventional metal-affinity columns, and the reported Au-Ni-Au nanorods [8], Ni/NiO core/shell nanoparticles [9], [10], Fe3O4@NiO heteronanoparticles [11] and polymer brush-modified magnetic nanoparticles [12] have been demonstrated to be potential materials in the separation/purification of protein species.
Polyoxometalates (POMs) are a class of metal-oxygen clusters containing transition elements such as Mo、W、V、Nb and Ta [13]. The intriguing structures and unique physic-chemical properties have gaining POMs considerable interest in the fields of catalysis [14], [15], medicine [16] and material sciences [17]. POMs of lacunary structure could be easily achieved by removing metal “octahedra” from the original POMs [18] or by simple one-step synthetic approach [19], and the lacunary structures provide available sites for the incorporation of transition metal ions to produce high-nuclear transition-metal-substituted POMs (TMSPs) [20], [21], [22], which are merited with the properties of both original POMs and the incorporated metal ions. For instance, the tetra-nickel-containing polyoxotungstate Na6K4-Ni4P2 reported by Lv et al. [23] has been demonstrated to be an efficient and robust molecular catalyst for hydrogen production due to the rich redox potential and high stability of POMs, and the excellent ability of nickel as hydride donor [24]. A series of high nuclear cobalt-incorporated POMs such as [{Co4(OH)3(PO4)}4(PW9O34)4]28- have been synthesized by Han et al. via sandwiching the [Co4] cluster among lacunary POM units [25]. The cobalt-incorporated POMs show highly efficiency in the photocatalysis of water oxidation because of the highest oxidated W/Mo centers in lacunary POMs and the ability of self-assemble upon the oxidation of Co2+ [26].
The low cytotoxicity and favorable biocompatibility are also gaining POMs increasing popularity in anticancer, antibacterial and antiviral fields [27], [28], [29]. For better understanding the POMs’ biological activity, some efforts have been taken to investigate the interactions between POMs and protein species of interest [30]. Zhang et al. find that phosphomolybdate acid (PMo12) can spontaneously bind on the surface of BHb through hydrophobic interaction using fluorescence quenching technique, which provides a promising tool to study the interactions of inorganic drugs and proteins [31]. Sap et al. demonstrate that the ZrIV-substituted Keggin POM can be used as efficient artificial protease for the selective hydrolysis of lysozyme, resulting from the fact that the negatively charged Zr-Keggin POM can co-crystallized with positively charged protein via electrostatic interaction [32].
In present study, a trivacant lacunary tungstophosphate ligand [PW9O34]9- is firstly prepared by using Na2WO4·2H2O and Na2HPO4·12H2O as source materials via a one-step route. Then, Ni2+ is introduced into the trivacant lacunary structure of [PW9O34]9- by forming tetra-nickel cluster [Ni4] via the metal-oxo coordinating interactions between Ni2+ and [PW9O34]9-, giving rise to the product of tetra-nickel substituted polyoxotungsate K6Na4[Ni4(H2O)2(PW9O34)2] (Ni4P2). The Ni4P2 is then immobilized onto the surface of SiO2 nanoparticles (NH2-SiO2) by self-assembly to achieve the solidification of POMs, and offers the final product of Ni4P2@SiO2 composites (Scheme 1). Due to the substituted nickel ions on the surface of composites, the obtained Ni4P2@SiO2 exhibits excellent adsorption selectivity towards histidine protein. His6-tagged proteins are successfully isolated from cell lysate with the obtained Ni4P2@SiO2 composite as sorbent, and improved performance such as stability and recyclability are obtained when compared with commercial Ni2+-based column.
Section snippets
Materials and reagents
Sodium tungstate dehydrate (Na2WO4·2H2O), nickel (Ⅱ) acetate tetrahydrate (Ni(OOCCH3)2·4H2O), sodium hydroxide (NaOH), sodium chloride (NaCl), ethanol absolute (C2H6O), ammonium hydroxide (NH3·H2O), tetraethyl orthosilicate (TEOS, Si(OC2H5)4), calcium hydride (CaH2), glycine (C2H5NO2), acrylamide (C3H5NO), methylene-bis-acrylamide (C7H10N2O2), sodium dodecyl sulfate (SDS, C12H25SO4Na), imidazole (C3H4N2), Coomassie Brilliant Blue G-250 and R-250 are purchased from Sinopharm Chemical Reagent
Preparation and characterizations of Ni4P2@SiO2 composites
Polyoxometalate of lacunary structure is a significant class of POMs because of their diversity of structure and abundant oxygen-coordinating sites. Trivacant [PW9O34]9- (PW9) is a kind of lacunary POMs with seven exposed oxygen atoms, and these oxygen atoms on the surface of lacunary PW9 are high nucleophilic and prone to coordinate with metal atoms.
In present work, PW9 is firstly obtained by one-step route, then Ni2+ is encapsulated into trivacant ligands PW9 by the formation of tetranuclear
Conclusions
A nickel substituted Keggin type polyoxotungsate Ni4P2@SiO2 composite has been prepared. The lacunary structures of POMs provide available sites for the introduction of metal ions, and the encapsulation of Ni2+ into POMs promotes the selective adsorption of histidine protein onto the surface under the affinity of Ni towards histidine protein. The Ni4P2@SiO2 is successfully applied to isolate His6-tagged proteins from crude cell lysate, with favorable selectivity and recyclability. Considering
Acknowledgments
The authors appreciate financial supports from the Natural Science Foundation of China (21475017, 21275027, 21235010), and Fundamental Research Funds for the Central Universities (N150502001, N141008001).
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