Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification.

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads. Our examination involved FTIR, EDS, XRD, SDS-PAGE, and Western blotting methods. FTIR spectra revealed notable changes in the stretching patterns and intensities of hydroxyl, amine, carbonyl, imine and amide chemical groups, when Mpro protein was present in the Ni-M/A sample. XRD spectra demonstrated the occurrence of two Nickel peaks at 35-40 deg and 40-45 deg in Ni-M/A, but only one nickel peak at 35-40 deg in Ni-M/A-Mpro, indicating the binding of Mpro on the Nickel ions. EDS analysis reported a decrease in the concentration of Nickel on the surface of Ni-M/A from 16% to 7% when Mpro protein was loaded into the stationary phase. Importantly, our data indicated that the purity of the His-tagged protein Mpro after purification reached 97% after just one-step purification using the Ni-M/A stationary phase. Moreover, the binding capacity of Ni-M/A for Mpro was approximately 5.2 mg/g with recovery efficiency of 40%. Our results suggested Ni-M/A as a highly potential solid phase for affinity chromatography in the purification of His-tagged protein.

Selective binding, magnetic separation and purification of histidine-tagged protein using biopolymer magnetic core-shell nanoparticles.

In previous studies, we synthesized the magnetic core-shell structured Fe3O4/PMG/IDA-Ni2+ nanoparticles. The Ni2+ on the surface of nanoparticles provides abundant docking sites for histidine, and the composite nanoparticles showed potential applications in the separation and purification of histidine-tagged (His-tagged) proteins. Meanwhile, the presence of the superparamagnetic core (Fe3O4) in the nanoparticles allows them to be quickly separated and purified by an external magnetic field. Herein, the ability of magnetic nanoparticles to purify His-tagged human superoxide dismutase 1 (hSOD1) was verified. SDS-PAGE and activity data showed His-tagged hSOD1 specifically bound to Fe3O4/PMG/IDA-Ni2+, and there was no significant competition for binding between final and three intermediate products. The binding capacity of nanoparticles can reach to 62.0 mg/g (dry weight of hSOD1/nanoparticles). The nanoparticle-bound hSOD1 exhibited better thermal and storage stability compared to free hSOD1. Furthermore, the purification efficiency of the magnetic nanoparticles in the separation and purification of His-tagged proteins was comparable to the other two commercial materials (High Affinity Ni-NTA Resin, HisPur Ni-NTA Magnetic Beads). Finally, the magnetic nanoparticles can be reused in the binding of His-tagged protein for multiple times. In conclusion, the nanoparticles are ready to be applied in the separation and purification of His-tagged protein.

Layer by layer coating of NH2-silicate/polycarboxylic acid polymer saturated by Ni2+ onto the super magnetic NiFe2O4 nanoparticles for sensitive and bio-valuable separation of His-tagged proteins.

Magnetic nanoparticles NiFe2O4 was synthesized and covered in the silicate lattice of (3-Aminopropyl) triethoxysilane (APS) by the sol-gel process. Subsequently, the EDTA-dianhydride was attached to the amino surface of magnetic nanoparticles (MNPs) during the nucleophilic attack. This polycarboxylic layer trapped the high level of nickel ions for selective bonding to the His-tagged recombinant protein. The surface of MNPs was investigated by TEM, XRD, SEM (EDSA), VSM, BET, FT-IR and zeta potential analysis which characterized the size, chemical lattice, morphology, magnetic strength, specific surface area, functional groups and charge of the surface of nanoparticles. The performance and validity of the nanoparticles were studied by the purification of His-tagged green fluorescence protein (His-GFP). Also, the safety of proposed Ni-MNPs in the purification procedure of His-tagged proteins for pharmaceutical applications was proved by the determination of the nickel leakage level in the purified final protein using atomic absorption spectroscopy. In vitro cytotoxicity of Ni-MNPs and trace metal ions was investigated by the MTS assay technique. In addition, the comparison of biological activity in purified protein (GM-CSF) and commercial sample did not show any toxic effect.

Rapid labeling of intracellular His-tagged proteins in living cells.

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Chitosan/cellulose-based beads for the affinity purification of histidine-tagged proteins.

Chitosan/cellulose-based beads (CCBs) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as a solvent, and their structures were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli. The results showed that Zn2+-CCB exhibited more specific adsorption capacity toward the target protein compared with Ni2+-CCB and Cu2+-CCB. The maximum adsorption of EGFP was 1.84 mg/g of Zn2+-CCB, with 90% purity under the optimized conditions (ionic strength (1.0 M NaCl), pH (7.2) and imidazole concentration (500 mM)). In addition, a regeneration method for the sorbent was further developed by washing with ethylenediaminetetraacetic acid disodium and then reimmobilizing with metal ions. This technique is an alternative method for the purification of his-tagged proteins, making the process more economical, fast, stable, and large batch.

Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

Carboligation reactions mediated by benzoylformate decarboxylase immobilized on a magnetic solid support.

In this study, magnetic nanoparticles (Fe3 O4 , magnetite) with immobilized metal affinity ligands (MSS) were prepared and characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), and vibrating-sample magnetometer (VSM) methods for purification and immobilization of the histidine-tagged recombinant benzoylformate decarboxylase (BFD). The MSS support was shown to be eligible for selective binding of HIS-tagged BFD by SDS-page analysis. Loading capacity of the MSS support was determined as 43.6 ± 1.1 mg/g. The regeneration ability for protein binding was also studied. An immobilized BFD was tested to catalyze benzoin condensation and representative cross acyloin reaction. Conversion and enantiomeric excess values were comparable with that of free enzyme catalyzed reactions.

Metal organic frameworks-in-nanochannels: A tailorable chromatography membrane for isolation of target protein.

Metal-organic frameworks (MOFs) demonstrate strong potential in biosample separation. However, the obtained MOFs powders are unsuitable for recovery techniques in an aqueous solution, especially the challenges of withdrawing MOFs particles and expanding their functions for specific applications. Herein, a general strategy is designed utilizing metal oxide-nanochannel arrays as precursors and templates for in-situ selective growth of MOFs structures. The exemplary MOFs (Ni-bipy) with tailored composition are selectively grown in NiO/TiO2 nanochannel membrane (NM) using NiO as the sacrificial precursor, which enables one to achieve a ∼262 times concentration of histidine-tagged proteins within 100 min. The significantly improved adsorption efficiency in a wide pH range and the effective enrichment from a complex matrix as a nanofilter illustrate the great potential of MOFs in nanochannels membranes for the high-efficiency recovery of essential proteins in complex biological samples. The porous self-aligned Ni-MOFs/TiO2 NM exhibits biocompatibility and flexible functionalities, which is desirable for the generation of multifunctional nanofilter devices and developing biomacromolecule delivery vehicles.

Synthesis of Ni2+-functionalized polydopamine magnetic beads for facilitated purification of histidine-tagged proteins.

Facilitated purification of proteins, at a low cost and a short time, is one of the key steps in the industrial production of recombinant proteins. In the current study, polydopamine nanoparticles (PDA-NPs) are considered in the synthesis of magnetic beads for purifying recombinant proteins due to advantages such as biocompatibility/ biodegradability, easy synthesis, as well as the ability to directly chelate metal ions. They were synthesized in Tris buffer (pH: 8:5), then chelated with Fe3+(20 mg) and Ni2+ ions at concentrations of 2, 3, 5, and 7 mg/ml. Prepared nanoparticles were characterized through scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), Inductively Coupled Plasma (ICP), and vibrating sample magnetometer (VSM). The size distribution of the particles was reported in the narrow range of 120-140 nm and 200 to 220 nm by the SEM image and DLS analysis, respectively. The chelation of ions on the surface of the nanoparticle was confirmed by the ICP technique with a magnetization of 35.42 emu/g. The highest adsorption rate of Ni2+ ions to polydopamine was obtained at a ratio of 1.4. The SDS-PAGE and western blot analysis confirmed the purification of eGFP and Hsp40 by PDA/Fe3+/Ni2+ at 26 and 40 kDa compared to the commercial nickel column. Moreover, the concentration of purified eGFP by PDA/Fe3+/Ni2+ was reported 138.83 µg/ml by the fluorescent signals, which is almost equal to or more than the protein purified by commercial Ni-NTA column (108.28 µg/ ml). The stability of PDA/Fe3+/Ni2+ has also been evaluated by ICP-OES for 10 days, and the result suggested that PDA magnetic beads were stable. Therefore, it can be concluded that PDA/Fe3+/Ni2+ have the ability to purify recombinant proteins in one less step and shorter time.

Ni(II) functionalized polyhedral oligomeric silsesquioxane based capillary monolith for purification of histidine-tagged proteins by immobilized metal affinity micro-chromatography.

A new capillary monolithic stationary phase was synthesized for the purification of histidine tagged proteins by immobilized metal affinity micro-chromatography (µ-IMAC). For this purpose, mercaptosuccinic acid (MSA) linked-polyhedral oligomeric silsesquioxane [MSA@poly(POSS-MA)] monolith 300 µm in diameter was obtained by thiol-methacrylate polymerization using methacryl substituted-polyhedral oligomeric silsesquioxane (POSS-MA) and MSA as the thiol functionalized agent in a fused silica capillary tubing. Ni(II) cations were immobilized onto the porous monolith via metal-chelate complex formation with double carboxyl functionality of bound MSA segments. µ-IMAC separations aiming the purification of histidine tagged-green fluorescent protein (His-GFP) from Escherichia coli extract were carried out on Ni(II)@MSA functionalized-poly(POSS-MA) [Ni(II)@MSA@poly(POSS-MA)] capillary monolith. His-GFP was succesfully isolated by µ-IMAC on Ni(II)@MSA@poly(POSS-MA) capillary monolith with the isolation yield of 85 % and the purity of 92 % from E. coli extract. Higher His-GFP isolation yields were obtained with lower His-GFP feed concentrations and lower feed flow rates. The monolith was used for consecutive His-GFP purifications with a tolerable decrease in equilibrium His-GFP adsorption over five runs.

Synthesis of Highly Monodisperse Nickel and Nickel Phosphide Nanoparticles.

Nickel and nickel phosphide nanoparticles are highly useful in various fields, owing to their catalytic and magnetic properties. Although several synthetic protocols to produce nickel and nickel phosphide nanoparticles have been previously proposed, controllable synthesis of nanoparticles using these methods is challenging. Herein, we synthesized highly monodisperse nickel and nickel phosphide nanoparticles via thermal decomposition of nickel-oleylamine-phosphine complexes in organic solvents. The size and composition of the nickel and nickel phosphide nanoparticles were easily controlled by changing the aging temperature, precursor concentration, and phosphine surfactant type. Large-sized monodisperse nickel nanoparticles obtained using our method were successfully applied for the purification of histidine-tagged proteins.

Supramolecular Assembly of His-Tagged Fluorescent Protein Guests within Coiled-Coil Peptide Crystal Hosts: Three-Dimensional Ordering and Protein Thermal Stability.

The use of biomaterials for the inclusion and stabilization of biopolymers is an ongoing challenge. Herein, we disclose three-dimensional (3D) coiled-coil peptide crystals with metal ions that include and overgrow His-tagged fluorescent proteins within the crystal. The protein guests are found within two symmetry-related growth sectors of the crystalline host that are associated with faces of the growing crystal that display ligands for metal ions. The fluorescent proteins are included within this "hourglass" region of the crystals at a notably high level, display order within the crystal hosts, and demonstrate sufficiently tight packing to enable energy transfer between a donor-acceptor pair. His-tagged fluorescent proteins display remarkable thermal stability to denaturation over extended periods of time (days) at high temperatures when within the crystals. Ultimately, this strategy may prove useful for the prolonged storage of thermally sensitive biopolymer guests within a 3D crystalline matrix.

Fabrication of copolymer brushes grafted superporous agarose gels: Towards the ultimate ideal particles for efficient affinity chromatography.

A composite immobilized-metal affinity agarose particle was designed for the selective separation and purification of histidine-tagged proteins from complicated biological samples. The composite particle was constructed using superporous agarose particles as supporting matrix, flexible copolymer brushes as scaffolds to render higher ligand densities, and Ni2+-chelated iminodiacetic acids as recognition elements. Superporous agarose composite particles endow high permeability and interfering substance tolerance. The copolymer brush was prepared by surface-initiated atom transfer radical polymerization of N-isopropylacrylamide and glycidyl methacrylate, followed by iminodiacetic acids and Ni2+ ions. The physical and chemical properities of the composite particle were thoroughly investigated. The composite particles were shown to be able to selectively separate histidine-tagged recombinant proteins in the presence of high quantities of interfering chemicals in a model protein-binding experiment. By altering the temperature, the protein binding of the composite particles can be modulated. The superporous agarose particles supported polymer brush enables fast and efficient separation and purification of target proteins with high permeability, low backpressure, and high interfering matrix tolerance, which pave the path for bioseparation through designing and fabrication of novel agarose particles-based functional materials.

Highly Selective Entrapment of His-Tagged Enzymes on Superparamagnetic Zirconium-Based MOFs with Robust Renewability to Enhance pH and Thermal Stability.

Metal-organic frameworks (MOFs), as a kind of poriferous nanoparticle, are promising candidates for enzyme immobilization to enhance their stability and reusability. However, most MOFs could not specifically immobilize enzymes and regenerate easily, which inevitably leads to serious high consumption and environmental pollution. In this study, renewable and magnetic MOFs were first constructed to specially immobilize His-tagged enzymes from the cell lysates without purification. The immobilized ß-glucuronidase exhibited wider pH adaptability and temperature stability. The relative activity of immobilized ß-glucuronidase was still maintained at ∼80% after eight cycles. Importantly, after simple treatment, the immobilization capacity of regenerated MOFs after simple treatment was restored to more than 90% in the first three times. The specific magnetic MOFs were proven to be an efficient and renewable platform for one-step immobilization and purification of His-tagged enzymes, showing great potential in industrial applications of nanotechnology and biocatalysis.

Rapid and regenerable surface plasmon resonance determinations of biomarker concentration and biomolecular interaction based on tris-nitrilotriacetic acid chips.

The tris-nitrilotriacetic acid (tris-NTA) chip has been used for surface plasmon resonance (SPR) kinetic studies involving histidine (His)-tagged proteins. However, its full potential, especially for analyte quantification in complex biological media, has not been realized due to a lack of systematic studies on the factors governing ligand immobilization, surface regeneration, and data analysis. We demonstrate that the tris-NTA chip not only retains His-tagged proteins more strongly than its mono-NTA counterpart, but also orients them more uniformly than protein molecules coupled to carboxymethylated dextran films. We accurately and rapidly quantified immunoglobulin (IgG) molecules in sera by using the initial association phase of their conjugation with His-tagged protein G densely immobilized onto the tris-NTA chip, and established criteria for selecting the optimal time for constructing the calibration curve. The method is highly reproducible (less than 2% RSD) and three orders of magnitude more sensitive than immunoturbidimetry. In addition, we found that the amount of His-protein immobilized is highly dependent on the protein isoelectric point (pI). Reliable kinetic data in a multi-channel SPR instrument can also be rapidly obtained by using a low density of immobilized His-tagged protein. The experimental parameters and procedures outlined in this study help expand the range of SPR applications involving His-tagged proteins.

Green Fluorescent Probe for Imaging His6-Tagged Proteins Inside Living Cells.

Small molecule-based fluorescent probes offer great opportunities for specifically tracking proteins in living systems with minimal perturbation on the protein function and localization. Herein, we report a small green fluorescent probe (Ni2+- NTA-AF) consisting of a Ni2+-NTA moiety, a fluorescein, and an arylazide group, that binds specifically to His6-tagged proteins with fluorescence enhancement in vitro upon photoactivation of the arylazide group. Importantly, the probe can cross the cell membranes and stoichiometrically label His6-tagged proteins rapidly (∼15 min) in living prokaryotic and eukaryotic cells exemplified by a DNA repair protein Xeroderma pigmentosum group A (XPA). Using the probe, we successfully visualized Sirtuin 5, which is localized to the mitochondria. This probe exhibits high quantum yields and improved solubility, offering a new opportunity for imaging intracellular His6-tagged proteins inside living cells with better contrast.

Nanoparticle-supported temperature responsive polymer brushes for affinity separation of histidine-tagged recombinant proteins.

We developed a modular approach for the preparation of nanoparticle-supported polymer brushes carrying repeating iminodiacetate units for affinity separation of histidine-tagged recombinant proteins. The nanoparticle-supported polymer brushes were prepared via the combination of surface-initiated atom transfer radical polymerization with Cu(I)-catalyzed azide-alkyne cycloaddition reaction. The nanocomposite materials were characterized to determine the particle size, morphology, organic content, densities of polymer chains and the affinity ligand. Protein binding assay illustrated that the iminodiacetate-rich polymer brushes enable to selectively bind histidine-tagged recombinant proteins in the presence of abundant interfering proteins. More importantly, the protein binding capacity can be tuned by adjusting the environmental temperature. STATEMENT OF SIGNIFICANCE: The nanoparticle core-polymer brush structure enables selective binding of histidine-tagged recombinant proteins via multiple metal-coordination interactions. The soft and flexible structure of the polymer brushes was found beneficial for lowering the steric hindrance in protein binding. Taking advantage of the conformational changes of the polymer brushes at different temperatures, it is possible to modulate the protein binding on the nanocomposite by adjusting the environmental temperature. In general, the iminodiacetate-rich core-brush nano adsorbents are attractive for purifying histidine-tagged recombinant proteins practically. The synthetic approach reported here may be expanded to develop other advanced functional materials for applications in various biomedical fields ranging from biosensors to drug delivery.

Facile fabrication of nickel immobilized on magnetic nanoparticles as an efficient affinity adsorbent for purification of his-tagged protein.

In the present research, an efficient, convenient, and inexpensive method for the one-pot synthesis of Fe3O4@Histidine is developed. Histidine is readily loaded on magnetic nanoparticles by one step and simple method without any supplemental linkers. In the structure of Fe3O4@Histidine, histidine covalently immobilized on the surface of Fe3O4, magnetic nanoparticles are able to trap Ni2+ ions through a strong interaction between nickel and histidines in protein tag. Two coordination sites of nickel are occupied with ligand on the surface of magnetic nanoparticles and four coordination sites have been remained that these sites will be occupied with histidine tag of recombinant protein A. The functionalized nanoparticles were spherical and well separated with an average diameter around 30nm. The obtained magnetic nanoparticles have a saturation magnetization of about 54emu/g. Fe3O4@Histidine-Ni was used to enrich and purify 6×histidine-tagged recombinant protein-A directly from the mixture of lysed cells. It has been found that Ni(II)-immobilized Fe3O4@Histidine magnetic nanoparticles present negligible nonspecific protein adsorption and high His-tag protein binding capacity The average binding capacity (MW 42k Da), is 700±25µg·mg-1 (protein/Fe3O4@Histidine-Ni).

Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC).

Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography.

Selective binding and magnetic separation of histidine-tagged proteins using Fe3O4/Cu-apatite nanoparticles.

Hierarchical Fe3O4@Cu-apatite nanoparticles (NPs) were synthesized via a facile hydrothermal method. The Fe3O4 cores present spherical shape and have a mean diameter of 300nm, and the Cu-apatite shell with thickness of about 50nm is composed of a large number of sheets. Using the high affinity of Cu(2+) on the surface toward histidine tags, the Fe3O4@Cu-apatite NPs can be applied to enrich and magnetically separate histidine tagged (His-tagged) proteins directly from the mixture of lysed cells. Research results indicated that the Fe3O4@Cu-apatite NPs presented negligible nonspecific protein adsorption and high protein binding ability.