Fast and highly efficient purification of 6×histidine-tagged recombinant proteins by Ni-decorated MnFe₂O₄@SiO₂@NH₂@2AB as novel and efficient affinity adsorbent magnetic nanoparticles

Abstract

The present study is aimed at the synthesis of MnFe₂O₄@SiO₂@NH₂@2AB-Ni, as a highly efficient and novel affinity adsorbent, for specific purification of 6×histidine-tagged recombinant proteins. The new immobilized metal ion affinity adsorbent was fabricated following co-precipitation synthesis of superparamagnetic manganese ferrite nanoparticles. Subsequently, tetraethyl orthosilicate (TEOS) was added in weak basic conditions (pH ∼ 9) to prevent oxidation and increase the density of –OH groups on the surface of MnFe₂O₄. Synthesized MnFe₂O₄@SiO₂ was then properly NH₂-functionalized with 3-aminopropyl-trimethoxylsilane (APTMS) as anchor molecules. Manganese ferrite nanoparticles were converted to bidentate ligands through a reaction between isatoic anhydride and amino-functionalized MnFe₂O₄@SiO₂. The stable surface functionalized nanoparticles were further linked with Ni²⁺ and used in powder form for efficient purification of 6×His-tagged proteins from the mixture of lysed cells. MnFe₂O₄@SiO₂@NH₂@2AB-Ni nanoparticles exhibited excellent performance in the separation of 6×histidine-tagged recombinant protein-A from cell lysate, with a binding capacity of about 220 mg g⁻¹. Indeed, the synthesized magnetic nanoparticles presented negligible nonspecific protein adsorption.

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Introduction

Proteins, the body's building blocks, play many critical roles in the body. They do most of the work in cells and are required for the structure, function, and regulation of tissues and organs. Thus separation and analysis of different kinds of proteins are fundamentally important to understand biological systems.¹ Protein purification is a series of processes for isolation of one protein from a complex mixture, cells, or tissues.^2,3 However, conventional protein separation techniques are multipart and wasteful, and the establishment of novel and efficient methods for enrichment and purification of proteins is currently the most cited topic.^4–6

Nowadays, many target proteins are usually expressed with a tag to facilitate the purification of proteins of interest from crude extracts,^7–9 for example histidine tag (His-tag) is widely used for affinity purification of recombinant proteins by immobilized metal-ion affinity chromatography (IMAC). This technique is based on the affinity of histidine residues and chelated transition metal ions (such as Ni²⁺, Co²⁺, and Cu²⁺) immobilized on a chelating resin. Common metal-chelating agents used in IMAC include iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), carboxymethylated aspartic acid (CM-Asp), and tris-carboxymethyl ethylene diamine (TED).^10–12

The most commonly used solid supports for IMAC are agarose and sepharos. While it is easy to handle, but the column format for the agarose beads is of low throughput with the demanding of large scale or high throughput purification.^13,14

Magnetic nanoparticles open a new window in biotechnology and biomedicine, such as drug delivery,¹⁵ hyperthermia,¹⁶ magnetic resonance imaging (MRI),¹⁷ cell and stem cell separation,^18–20 enzyme immobilization,²¹ and etc. In the past few years, many efforts have been made to develop functionalized magnetic particles and use of them in protein purifications due to their unique advantages in simple handling, fast purification, high throughput, and ease in automation. Also, they can be readily isolated from solutions using a strong external magnet.^22,23 There have been some reports of preparation of different types of magnetic particles for this aim.^24–26 For instance Zou and co-workers synthesized ferroferric oxide/L-cysteine (Fe₃O₄/Cys) nanospheres via a facile solvothermal route for capturing histidine-tagged proteins from the mixed-protein solutions.²⁷ Kim et al. synthesized new bis-nitrilotriacetic acid (NTA) chelated with catechol anchor and immobilized superparamagnetic iron oxide nanoparticles as affinity probes for enrichment of polyhistidine (His×6-tagged) fusion proteins.²⁸ Liao and co-workers reported the preparation of nitrilotriacetic acid/Co²⁺-linked, silica/boron-coated magnetite nanoparticles for purification of two model 6×histidine-tagged proteins.²⁹

In the present study, we reported preparation of novel MnFe₂O₄@SiO₂@NH₂@2AB-Ni magnetite nanoparticles, and investigated their application for highly efficient and selective purification of 6×His-tagged proteins from cell lysate in powdery form. MnFe₂O₄@SiO₂@NH₂@2AB-Ni nanoparticles exhibited excellent performance in terms of purity and capacity for purification of 6×histidine-tagged recombinant proteins from cell lysate.

Experimental

The chemicals used in this work were obtained from Fluka and Merck and were used without purification. FT-IR spectra were recorded as KBr pellets on a Perkin-Elmer 781 spectrophotometer and an Impact 400 Nicolet FT-IR spectrophotometer. X-ray diffraction (XRD) pattern of the as-synthesized material was obtained using a Holland Philips Xpert X-ray powder diffraction (XRD) diffractometer (CuK, radiation, λ = 0.154056 nm), at a scanning speed of 2° min⁻¹ from 10° to 100° (2θ). The content of nickel was determined by VISTA-PRO, CCD simultaneous ICP analyzer. Thermogravimetric/differential thermal analyses (TG/DTA) was performed on a Thermal Analyzer with a heating rate of 20 °C min⁻¹ over a temperature range of 25–1100 °C under flowing compressed N₂.

Synthesis of MnFe₂O₄@SiO₂@NH₂@2AB-Ni

Synthesis of MnFe₂O₄ nanoparticles. MnFe₂O₄ nanoparticles have been prepared following the reported standard protocol by co-precipitation of MnCl₂ and FeCl₃ in water in the presence of sodium hydroxide.³⁰ Briefly, MnCl₂·4H₂O and FeCl₃·6H₂O were taken in molar ratio of Mn²⁺ : Fe³⁺ = 1 : 2 to prepare 0.3 mol L⁻¹ metal ion solution of 100 mL containing 0.1 mol L⁻¹ Mn²⁺ and 0.2 mol L⁻¹ Fe³⁺, then this solution was slowly dropped into 100 mL 3 mol L⁻¹ of NaOH solution at the preheated temperature to 95 °C. After aging for 2 h with continuous stirring, the mixture was filtered, washed and dried at 60 °C for 12 h.

Silica coating over magnetite nanoparticles. For silica coating on the surface of MnFe₂O₄ nanoparticles, 0.5 g of MnFe₂O₄ nanoparticles were first ultrasonically treated with 50 mL of 0.1 M HCl aqueous solution for 10 min. The magnetic nanoparticles were then separated and washed with deionized water. Then the nanoparticles were homogeneously dispersed in a mixture of 80 mL ethanol, 20 mL deionized water and 5 mL concentrated ammonia aqueous solution (28%) for 30 min. Then 150 μL TEOS was added drop wise to the above mentioned mixture. After magnetically stirring at room temperature for 6 h, the product was separated and washed with ethanol and deionized water.³¹

Synthesis of amino-functionalized silica coated magnetic nanoparticles. The MnFe₂O₄@SiO₂ (0.5 g) was dispersed in a mixture of ethanol and water (100 mL, 1 : 1 by volume) and sonicated for 30 min at room temperature and then loaded into a round-bottomed flask. APTMS (0.87 mL, 5 mmol) was then added slowly, and the solution was heated at 40 °C with vigorous stirring for 24 h. The final product was separated by magnetic decantation and washed with deionized water and ethanol, then dried under vacuum at room temperature overnight to yield amino functionalized magnetic nanoparticles.

Grafting of 2-amino benzamide on the amino-functionalized magnetic nanoparticles. The amino-functionalized magnetic nanoparticles (0.5 g) were suspended in ethanol (99.5%, 100 mL) with sonication for 30 min to form a uniform dispersion. To this mixture isatoic anhydride (0.32 g, 2 mmol) was added and the resulted mixture was refluxed for 12 h. The prepared functionalized magnetic nanoparticles were separated by magnetic decantation and then washed with ethanol several times to remove the unreacted isatoic anhydride and dried under vacuum at 50 °C.

Immobilization of nickel on modified magnetic nanoparticles. For preparation of MnFe₂O₄@SiO₂@NH₂@2AB-Ni, firstly, 2-amino benzamide immobilized magnetic nanoparticles (0.5 g) were ultrasonically dispersed in 100 mL of ethanol to form a homogeneous dispersion, and then loaded into a round-bottomed flask. Ni(OAc)₂·4H₂O (0.248 g, 1 mmol) was added to this solution. The mixture was refluxed for 12 h. After stirring, the catalyst was harvested by aid of magnet, washed several times with ethanol to remove unreacted Ni(OAc)₂ and dried under vacuum at 50 °C, for 6 h.

Purification of 6×histidine-tagged recombinant protein-A in E. coli by MnFe₂O₄@SiO₂@NH₂@2AB-Ni

Recombinant protein to be purified. Protein-A with 6×histidine residues (His-tag) in their C-terminus, with molecular weights of about 42 KD was chosen to be separated using MnFe₂O₄@SiO₂@NH₂@2AB-Ni magnetic nanoparticles. Protein-A was expressed in to the bacterial cytosol.

Growth of bacteria and induction of gene expression. The expression plasmid, pET21a was prepared and transformed into E. coli BL21 (DE3) as host strain. A single transformed colony was inoculated into 3 mL of Luria–Bertani (LB, 10 g L⁻¹ tryptone, 5 g L⁻¹ yeast extract, and 10 g L⁻¹ NaCl) medium containing 50 μg.·m L⁻¹ kanamycin and grown overnight at 37 °C with shaking at 225 rpm. Next day, the overnight culture was inoculated 1 : 100 mL into fresh LB medium containing 50 μg mL⁻¹ kanamycin and grown at 37 °C until the optical density at 600 nm (OD600) reached 0.4. At this point, protein expression was induced by 100 μL of 1 M isopropyl-β Dthiogalactopyranoside (IPTG) to give a final concentration of 1 mM. The induced culture was continued for 4 hours and then processed for protein extraction.

Cell lysis and protein extraction. Bacterial cells were harvested by centrifugation of cell culture at 4000 rpm, 4 °C for 10 min. Supernatant was aspirated off and cells were washed three times with cold binding-wash solution (20 mM Na₂HPO₄, pH = 7.0). Cells were then resuspended in 2 mL cold lysis buffer (20 mM Na₂HPO₄, 10 mM imidazole, pH = 7.0). Cell lysis was further continued by sonication on ice (15 s at 70% power, four times, 1 min intervals with a M73 probe). The lysate was centrifuged at 12 000 rpm, 4 °C for 10 min to remove the crude precipitates. After centrifugation, supernatant was filtered through a 0.2 μm cellulose acetate filter (Millipore, USA) as Soluble Cell Extract (SCE).

Protein purification by MnFe₂O₄@SiO₂@NH₂@2AB-Ni magnetic nanoparticles. Firstly, 5 mg of MnFe₂O₄@SiO₂@NH₂@2AB-Ni magnetic nanoparticles in stable powdery form were transferred in to Eppendorf tubes, washed, and equilibrated three times with 500 μL of cold lysis buffer. Each time after pipetting and mixing, tube was placed on a magnet until the beads migrated to the side of the tube and the clarified liquids were discarded. Meantime, 800 μL soluble cell extracts were diluted with 200 μL cold lysis buffer before mixing with beads. The mixture mixed well by gentle pipetting and incubated for 30 minutes on a roller mixer for protein binding. After the binding process, tubes were placed in the magnetic separator, and except a small volume (30 μL) of the clarified supernatant which was collected and frozen for further analysis as flowthrough samples (FT); the rest was removed and discard. Wash steps were performed four times by adding 500 μL of wash buffer (50 mM NaH₂PO₄,300 mM NaCl, 10 mM imidazole, pH = 8.0) to remove nonspecifically adsorbed lysates, at each washing steps, a small portion of supernatant was collected (W1–4) and the rest was discarded. After four washing steps, the entrapped His-tagged proteins were eluted with 200 μL of elution buffers (50 mM NaH₂PO₄, 300 mM NaCl containing different concentrations of imidazole 250 mM, 500 mM, and 1 M pH 8.0). Supernatant from each elution steps (E1–4) was then collected and stored at –20 °C. All collected (SCE), flowthrough (FT), washes (W1–4) and elutions (E1–5) were prepared by mixing 30 μL aliquots of each preparation with 7 μL of 1 × SDS-PAGE loading buffer (50 mM Tris–HCl pH 6.8, 10% glycerol, 2.5% SDS, 0.1% bromophenol blue, 25 mM dithiothreitol). The samples were boiled for three minutes and verified in dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) 12% using Coomassie brilliant blue stain. In some settings and in order to verify the purity of the expressed protein, SDS-PAGE gels were stained with silver stain according to the protocol published elsewhere.^25b

Western blotting

After separation on SDS-PAGE gel, proteins were transferred to nitrocellulose membranes (Hybond-ECL; Amersham, Sweden) in 100 mM Tris–HCl pH 7.5, containing 2.5 M NaCl, 0.5% Tween 20, 2% Triton X-100 and 20% (v/v) methanol, for 1 h at 1 mA cm⁻², using a Bio-Rad apparatus (Berkeley, California, USA). To detect proteins, membranes were blocked in bovine serum albumin (BSA) 5% for 16 h followed by incubation for 1 h at 4 °C with a horseradish peroxidase (HRP)-conjugated monoclonal anti-His antibody at a 1 : 100 000 dilution.³² Membranes were developed with ECL detection kit according to the manufacturer's instruction.

Enzyme-linked immunosorbent assay (ELISA)

Direct ELISA was set up for determining capacity of purified protein A to bind Fc region of antibodies using HRP-conjugated Sheep immunoglobulin (Ig) (Sina Biotech, Tehran, Iran). In brief, microtiter plates were coated with titrating amounts of recombinant protein A for 1.5 h at 37 °C. Wells without coating served as negative reagent control. Plates were then washed with phosphate buffered saline (PBS)-Tween 0.5% and blocked with PBS-BSA 2.5% overnight. 1 : 2000 dilution of HRP-conjugated Sheep Ig was added to the wells and incubation was continued for 1.5 h. Signals were developed after addition of TMB (3,3′,5,5;-tetramethylbenzidine) as chromogen. Optical density (OD) of the wells was measured at 450 nm.

Results and discussion

The preparation of MnFe₂O₄@SiO₂@NH₂@2AB-Ni is illustrated in Scheme 1. In this protocol, firstly, superparamagnetic manganese ferrite nanoparticles were synthesized following a co-precipitation method in basic aqueous media.³⁰ The coating process was performed by suspending the magnetic nanoparticle in an ethanol–water solution and mixing with TEOS to form a silica shell under basic conditions.³¹ The SiO₂ shell was formed on the surface of the MnFe₂O₄ in order to prevent oxidation and the high density of –OH groups on the silica also allows further modification of the particles through silanization. The resulting nanoparticles were functionalized with APTMS to create amino groups on surface; the amino-functionalized magnetic nanoparticles were allowed to react with isatoic anhydride to form immobilized bidentate ligand, finally the Ni²⁺ ions loaded on the surface of magnetic nanoparticles through the reaction of MnFe₂O₄@SiO₂@NH₂@2AB with nickel acetate. Characterization of the prepared magnetic nanoparticles was performed with different physicochemical methods such as Fourier Transform Infrared (FT-IR), X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Vibrating Sample Magnetometry (VSM), thermo gravimetric analysis (TGA), and Energy Dispersive X-rays (EDX) analysis.

Scheme 1 Steps for fabricating of MnFe₂O₄@SiO₂@NH₂@2AB-Ni.

Characterization of MnFe₂O₄@SiO₂@NH₂@2AB-Ni

FT-IR spectra of MnFe₂O₄, MnFe₂O₄@SiO₂, MnFe₂O₄@SiO₂@NH₂, MnFe₂O₄@SiO₂@NH₂@2AB and MnFe₂O₄@SiO₂@NH₂@2AB-Ni were shown in Fig. 1. FT-IR spectra of pure manganese ferrite nanoparticles showed characteristic peaks at 3427 cm⁻¹ and 1630 cm⁻¹, were assigned to the vibration of hydroxyl groups,³³ in addition, an obvious peak at 584 cm⁻¹ attributed to Fe–O bond vibration (Fig. 1a). Compared with the curve a, in the curve b, the band at 1082 cm⁻¹ was assigned to the vibration of the Si–O bonds, demonstrating the existence of SiO₂ shell on the surface of MnFe₂O₄.³⁴ In the curve c, the peak at 3423 cm⁻¹ was probably attributed to the free amino groups, which is overlapped by the O–H stretching vibration. Two obvious peaks at 2950 cm⁻¹ and 2860 cm⁻¹ were observed, which were assigned to C–H stretching bands. These results provided the evidences that the amino groups were successfully attached to the surface of MnFe₂O₄ nanoparticles (Fig. 1c). For the immobilized ligand in order to the formation MnFe₂O₄@SiO₂@NH₂@2AB, the C=O stretch of the amide appeared at 1600 cm⁻¹ which was overlapped with the O–H vibration and, some weak peaks at 1400–1500 cm⁻¹ assigned to stretching vibrations of C=C aromatic rings (Fig. 1d). While in the complex, this band shifted to higher frequency and appears at 1702 cm⁻¹ because of coordination of the nitrogen with Ni(II) that indicating complexation of amide group of supported ligand with nickel (Fig. 1e).

Fig. 1 The comparative FT-IR spectra for (a) MnFe₂O₄, (b) MnFe₂O₄@SiO₂, (c) MnFe₂O₄@SiO₂@NH₂, (d) MnFe₂O₄@SiO₂@NH₂@2AB, and (e) MnFe₂O₄@SiO₂@NH₂@2AB-Ni.

The XRD patterns of MnFe₂O₄, MnFe₂O₄@SiO₂, MnFe₂O₄@SiO₂@NH₂, MnFe₂O₄@SiO₂@NH₂@2AB, and MnFe₂O₄@SiO₂@NH₂@2AB-Ni were shown in Fig. 2. It could be seen that the strong characteristic diffraction peaks at 2θ of 29.94°, 35.24°, 42.75°, 52.94°, 56.42°, and 61.86° corresponding to the signals of (220), (311), (400), (331), (422), and (333) of the MnFe₂O₄ (JCPDS 1964-73) were found in all samples. These results mean that the MnFe₂O₄@SiO₂@NH₂@2AB-Ni have been synthesized successfully without damaging the crystalline cubic spinel structure of MnFe₂O₄ core. According to the result calculated by Scherrer equation, it was found that the diameter of produced MnFe₂O₄ nanoparticles was about 35 nm and MnFe₂O₄@SiO₂@NH₂@2AB-Ni was obtained with a diameter of about 75 nm.

Fig. 2 XRD patterns of (a) MnFe₂O₄, (b) MnFe₂O₄@SiO₂, (c) MnFe₂O₄@SiO₂@NH₂, (d) MnFe₂O₄@SiO₂@NH₂@2AB, and (e) MnFe₂O₄@SiO₂@NH₂@2AB-Ni.

The size and morphology of MnFe₂O₄ and MnFe₂O₄@SiO₂@NH₂@2AB-Ni were determined by scanning electron microscopy (SEM). It can be seen from Fig. 3A that MnFe₂O₄ nanoparticles have a mean diameter of about 30–35 nm and a nearly spherical shape. The SEM image shown in Fig. 3B demonstrates that the particles after immobilization with the organic groups and complexation with nickel, revealed agglomerates of spherical particles with larger average size 70–85 nm. The nature of the complex does not have significant impact on the morphology of all the MNPs.

Fig. 3 The SEM image of (A) MnFe₂O₄ and (B) MnFe₂O₄@SiO₂@NH₂@2AB-Ni.

Morphology and particle size of MnFe₂O₄ were characterized using TEM technique. The TEM image shows that the these nanoparticles have a mean diameter of about 25–35 nm (Fig. 4).

Fig. 4 The TEM image of MnFe₂O₄.

The magnetic property of MnFe₂O₄ and MnFe₂O₄@SiO₂@NH₂@2AB-Ni was investigated at room temperature using vibrating sample magnetometry (VSM) (Fig. 5). The saturation magnetization of samples changed from 54 to 36 emu g⁻¹, because of the modification. Moreover, magnetization curves of the magnetic nanoparticle, before and after functionalization, exhibit no hysteresis which demonstrates its superparamagnetic characteristics. The strong magnetization of the nanoparticles was also revealed by simple attraction with an external magnet.

Fig. 5 Magnetization curves for the prepared MnFe₂O₄ (a) and MnFe₂O₄@SiO₂@NH₂@2AB-Ni (b) at 40 °C.

N₂ adsorption–desorption isotherms and the Barrett–Joyner–Halenda (BJH) pore-size distribution curves of MnFe₂O₄@SiO₂@NH₂@2-AB-Ni are depicted in Fig. 6. The BET analysis showed a high specific surface area for the prepared magnetic nanoparticles which was approximately 538.02 m² g⁻¹. The pore volume and pore size were also calculated from the N₂ adsorption result; approximately 0.15 cm² g⁻¹ and, 11 nm, respectively. As can be seen in Fig. 6, the samples displayed type II isotherms³⁵ and capillary condensation at relative pressures of 0.3 < P/P₀ < 0.5, which was the characteristic of mesoporous materials.

Fig. 6 N₂ adsorption–desorption isotherms of MnFe₂O₄@SiO₂@NH₂@2-AB-Ni.

The dynamic laser scattering (DLS) measurement of MnFe₂O₄@SiO₂@NH₂@2-AB-Ni magnetic nanoparticles was shown in Fig. 7. As can be seen in Fig. 6, the particle diameters range from 50 to 78 nm and there is a good agreement between this result and results in SEM image.

Fig. 7 Characterization of MnFe₂O₄@SiO₂@NH₂@2-AB-Ni using a dynamic light backscattering method.

Fig. 8a presents the TGA curves of MnFe₂O₄@SiO₂@NH₂ (a), MnFe₂O₄@SiO₂@NH₂@2AB (b), and MnFe₂O₄@SiO₂@NH₂@2AB-Ni (c). The lossed weight below 200 °C in all samples is attributed to the release of adsorbed water. TGA analysis of the amino-functionalized magnetic nanoparticles indicated that 0.81 mmol g⁻¹ of the amine groups was immobilized on the particles. TGA analysis of the immobilized 2-aminobenzamide ligand exhibited loading 0.52 mmol g⁻¹. Also, TGA analysis of the MnFe₂O₄@SiO₂@NH₂@2AB-Ni indicated that 0.32 mmol g⁻¹ of he complex was immobilized on the magnetic nanoparticles.

Fig. 8 TGA curves of (a) MnFe₂O₄@SiO₂@NH₂, (b) MnFe₂O₄@SiO₂@NH₂@2AB, and (c) MnFe₂O₄@SiO₂@NH₂@2AB-Ni.

Furthermore, to support the above mentioned observation, the catalyst was subjected to energy dispersive X-rays. EDX analysis of nickel confirms its immobilization on the magnetic nanoparticles (Fig. 9).

Fig. 9 EDX spectrum of MnFe₂O₄@SiO₂@NH₂@2AB-Ni.

Purification of 6×His-tagged proteins

Purification of recombinant proteins is routinely performed in various fields of biotechnology. In this regard, choosing a suitable purification method is a crucial step. One of the powerful techniques for protein purification is IMAC technique which is based on the coordination bonds between metal toward histidine. In this study, 6×histidine-tag recombinant protein-A, as a model protein, was purified using synthesized magnetic nanoparticles. This protein is a cell wall component of the bacterium Staphylococcus aureus that binds specifically to many mammalian immunoglobulins, most markedly IgG and is utilized for oriented immobilization of antibodies from different sources.^36–38

Here, Ni²⁺ was modified on the surface of MnFe₂O₄@SiO₂@NH₂@2AB and the application of these magnetic nanoparticles was investigated in purification of 6×histidine-tagged recombinant protein-A from crude E. coli lysate as depicted in Scheme 2. MnFe₂O₄@SiO₂@NH₂@2AB-Ni magnetic nanoparticles were added to the Soluble Cell Extract (SCE) and the mixture was incubated for 30 min with continuous shaking. The non-specific proteins were removed by washing of the magnetic nanoparticles in an external magnetic field. Subsequently, the captured 6×His-tagged protein-A was eluted with increasing imidazole concentration. All collected fractions were analyzed for protein-A using SDS-PAGE analysis.

Scheme 2 Procedure for purification of 6×His-tagged Protein-A by MnFe₂O₄@SiO₂@NH₂@2AB-Ni magnetic nanoparticles.

Fig. 10A shows the representative results of recombinant 6×His-tagged protein-A purification. Lane 1 was loaded with SCE containing overexpressed 6×histidine-tagged recombinant protein-A (42 kDa) and unrelated bacterial proteins. Following adsorption to the magnetic metrics, nonspecific proteins were passed (flow through) (lane 2). Only, a tiny amount of 6×histidine-tagged recombinant protein-A was appeared in flow through (lane 2) or after washing steps (lane 3–4) indicating very high capacity of the synthesized metrics for recombinant 6×His-tagged protein. The protein-A anchored to MnFe₂O₄@SiO₂@NH₂@2AB-Ni were purely and with very high recovery rate eluted with increasing concentrations of imidazole (lanes 5–8). Western blot analysis with anti-His tag antibody also confirmed the identity of the recombinant protein A (Fig. 10B). The reactivity of purified protein A was also checked using ELISA. As depicted in Fig. 10C, protein A exhibited excellent reactivity with Fc part of Sheep immunoglobuling indicating functionality of the expressed recombinant protein.

Fig. 10 Characterization of purified 6×His-tagged recombinant protein A. 6×His-tagged recombinant protein A was purified using MnFe₂O₄@SiO₂@NH₂@2AB-Ni magnetic nanoparticles and characterized by SDS-PAGE (A), western blotting (B) and silver stain (C). Upper right panel shows molecular weight marker. (A & C) Lane 1: SCE, lane 2: flow through fraction, lane 3: W₁, lane 4: W₄, lane 5: E₁₀₀, lane 6: E₁₀₀, lane 7: E₂₅₀, lane 8: E₂₅₀. Lane 9: E₅₀₀.

Also, reactivity of purified recombinant protein A with Sheep immunoglobulin was investigated and showed in Fig. 11. Titrating amounts of protein A was coated in wells of a microtiter plate followed by incubation with HRP-conjugated sheep immunoglobulin. Signals were read after addition of chromogen at 450 nm.

Fig. 11 Reactivity of purified recombinant protein A with Sheep immunoglobulin.

Notably, our MnFe₂O₄@SiO₂@NH₂@2AB-Ni magnetic nanoparticles displayed excellent capacity of 220 mg g⁻¹ for His-tagged protein-A which was much higher than the capacity reported for commercial micro beads (10–20 mg g⁻¹).

Other synthetic schemes for metal-chelating magnetic nanoparticles have been reported, for example Wang and co-workers reported the synthesis of nanostructured Ni-NTA functionalized magnetic nanoparticle for adsorption of his-tagged enzyme with binding capacity of 146 mg protein/mg MNPs.³⁹ Zhang et al. investigated the synthesis of uniform magnetic core/shell microspheres functionalized with Ni²⁺–iminodiacetic acid for one step purification and immobilization of his-tagged enzymes. It was determined the binding capacity of prepared magnetic nanoparticles to be around 103 mg g⁻¹ (protein/beads).⁴⁰ Feng and co-workers reported the synthesis of Fe₃O₄/SiO₂-GPTMS-Asp-Co for purification of histidine-tagged proteins. The binding capacity for per gram of Fe₃O₄/SiO₂-GPTMS-Asp-Co nanoparticles is more than 9.45 mg 6-histidine-tagged gp 41 proteins.⁴¹ Zhang and co-workers synthesized Fe₃O₄/Au–ANTA–Co²⁺ nanoparticles to separate His-tagged proteins from the mixed-protein solution. The binding capacity for His-tagged SSA1 is 74 μg mg⁻¹.⁴²

Conclusions

In summary, we presented here a facile and efficient synthesis of a novel MnFe₂O₄@SiO₂@NH₂@2AB-Ni, magnetic nano particles for purification of His-tagged recombinant proteins. The synthesis procedure was a simple and non-expensive approach leading to a production of a very stable powdery form of the magnetic nanoparticle matrix. The functionalized nanoparticles were spherical and well separated with an average diameter around 75 nm and they have a saturation magnetization of about 36 emu g⁻¹ and show superparamagnetic property. These nano particles exhibited high adsorption capacity and excellent specificity (220 μg mg⁻¹) towards 6×histidine-tagged recombinant protein-A. We tested the applicability of MnFe₂O₄@SiO₂@NH₂@2AB-Ni MNPs for purification of recombinant protein-A as a model protein. The silver stain result shows that the separated protein is pure and single band with remarkable purity. We think that our proposed MNPs could be potentially utilized for purification of different array of His-tagged recombinant proteins.

Acknowledgements

We are thankful from Iran National Science Foundation (INSF) for supporting this project numbered 92027471, University of Kashan for supporting this work by grant number 159148/34, and also gratefully acknowledge financial support from the Avicenna Research Institute.

References

1. B. Dennls, Nature, 1995, 376, 307–312.
2. C. Charcosset, J. Chem. Technol. Biotechnol., 1998, 71, 95–110.
3. S. Gräslund, P. Nordlund and J. Weigelt, et al., Nat. Methods, 2008, 5, 135–146.
4. C. Xu, K. Xu, H. Gu, X. Zhong, Z. Guo, R. Zheng, X. Zhang and B. Xu, J. Am. Chem. Soc., 2004, 126, 3392–3393.
5. M. Benelmekkia, E. Xuriguera, C. Caparros, E. Rodríguez-Carmona, R. Mendoza, J. L. Corchero, S. Lanceros-Mendez and L. l. M. Martinez, J. Colloid Interface Sci., 2012, 365, 156–162.
6. H. Y. Xie, R. Zhen, B. Wang, Y. J. Feng, P. Chen and J. Hao, J. Phys. Chem. C, 2010, 114, 4825–4830.
7. J. E. Gautrot, W. T. S. Huck, M. Welch and M. Ramstedt, ACS Appl. Mater. Interfaces, 2010, 2, 193–202.
8. G. Kaur-Atwal, D. J. Weston, P. L. R. Bonner, S. Crosland, P. S. Green and C. S. Creaser, Curr. Anal. Chem., 2008, 4, 127–135.
9. R. Gutierrez, E. M. Martin del Valle and M. A. Galan, Sep. Purif. Rev., 2007, 36, 71–111.
10. D. B. Shieh, C. H. Su, F. Y. Chang, Y. N. Wu, W. C. Su, J. R. Hwu, j. H. Chen and C. S. Yeh, Nanotechnology, 2006, 17, 4174–4182.
11. K. Yang and W. Su, Anal. Biochem., 2011, 408, 175–177.
12. H. Block, B. Maertens, A. Spriestersbach, N. Brinker, J. Kubicek, R. Fabis, J. Labahn and F. Schafer, Methods Enzymol., 2009, 463, 439–473.
13. (a) S. K. Sahu, A. Chakrabarty, D. Bhattacharya, S. K. Ghosh and P. Pramanik, J. Nanopart. Res., 2011, 13, 2475–2484; (b) E. Hochuli, H. Döbeli and A. Schacher, J. Chromatogr. A, 1987, 411, 177–184.
14. (a) K. Kracalıkova, G. Tishchenko and M. Bleha, J. Biochem. Biophys. Methods, 2006, 67, 7–25; (b) Y. Liao, Y. Cheng and Q. Li, J. Chromatogr. A, 2007, 1143, 65–71.
15. (a) B. Luo, S. Xu, A. Luo, W. R. Wang, S. L. Wang, J. Guo, Y. Lin, D. Y. Zhao and C. C. Wang, ACS Nano, 2011, 5, 1428–1435; (b) H. S. Cho, Z. Y. Dong, G. M. Pauletti, J. M. Zhang, H. Xu, H. C. Gu, L. M. Wang, R. C. Ewing, C. Huth, F. Wang and D. L. Shi, ACS Nano, 2010, 4, 5398–5404; (c) D. Li, J. Tang, C. Wei, J. Guo, S. L. Wang, D. Chaudhary and C. C. Wang, Small, 2012, 8, 2690–2697.
16. A. Hervault and N. T. K. Thanh, Nanoscale, 2014, 6, 11553–11573.
17. N. Lee and T. Hyeon, Chem. Soc. Rev., 2012, 41, 2575–2589.
18. S. Nikoo, M. Ebtekar, M. Jeddi-Tehrani, A. Shervin, M. Bozorgmehr, S. Kazemnejad and A. H. Zarnani, J. Obstet. Gynaecol. Res., 2012, 38, 804–809.
19. S. Kazemnejad, M.-M. Akhondi, M. Soleimani and A. H. Zarnani, Int. J. Artif. Organs, 2012, 35, 55–66.
20. C. Clarke and S. Davies, Immunomagnetic Cell Separation, 2001, 58, 17–23.
21. (a) A. Rembsum and W. Dreyer, Science, 1980, 208, 364–368; (b) D. Wang, J. He, N. Rosenzweig and Z. Rosenzweig, Nano Lett., 2004, 4, 409–413.
22. A. Dyal, K. Loos, M. Noto, S. Chang, C. Spagnoli, K. V. P. M. Shafi, A. Ulman, M. Cowman and R. A. Gross, J. Am. Chem. Soc., 2003, 125, 1684–1685.
23. (a) W. F. Ma, Y. Zhang, L. L. Li, L. J. You, P. Zhang, Y. T. Zhang, J. M. Li, M. Yu, J. Guo, H. J. Lu and C. C. Wang, ACS Nano, 2012, 6, 3179–3188; (b) Z. Liu, M. Li, X. Yang, M. Yin, J. Ren and X. Qu, Biomaterials, 2011, 32, 4683–4690; (c) Z. Liu, M. Li, F. Pu, J. Ren, X. Yang and X. Qu, J. Mater. Chem., 2012, 22, 2935–2942; (d) Z. Liu, M. Li, Z. Li, F. Pu, J. Ren and X. Qu, Nano Res., 2012, 5, 450–459.
24. Y. Zhang, Y. Yang, W. Ma, J. Guo, Y. Lin and C. Wang, ACS Appl. Mater. Interfaces, 2013, 5, 2626–2633.
25. (a) Y. Pan, X. W. Du, F. Zhao and B. Xu, Chem. Soc. Rev., 2012, 41, 2912–2942; (b) M. R. Nejadmoghaddam, M. Chamankhah, S. Zarei and A. H. Zarnani, J. Nanobiotechnol., 2011, 9, 31–42.
26. J. H. Gao, H. W. Gu and B. Xu, Acc. Chem. Res., 2009, 42, 1097–1107.
27. X. Zou, K. Li, Y. Zhao, Y. Zhang, B. Li and C. Song, J. Mater. Chem. B, 2013, 1, 5108–5113.
28. J. S. Kim, C. A. Valencia, R. Liu and W. Lin, Bioconjugate Chem., 2007, 18, 333–341.
29. Y. Liao, Y. Chenga and Q. Li, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2007, 1143, 65–71.
30. H. Aijun, L. Juanjuan, Y. Mingquan, L. Yan and P. Xinhua, Chin. J. Chem. Eng., 2011, 19, 1047–1051.
31. B. Sahoo, S. Kumar Sahu, S. Nayak, D. Dhara and P. Pramanik, Catal.: Sci. Technol., 2012, 2, 1367–1374.
32. R. Emamzadeh, M. Nazari and S. Najafzadeh, Anal. Methods, 2014, 6, 4199–4204.
33. P. Rattanaburi, B. Khumraksa and M. Pattarawarapan, Tetrahedron Lett., 2012, 53, 2689–2693.
34. M. Masteri-Farahani and N. Tayyebi, J. Mol. Catal. A: Chem., 2011, 348, 83–87.
35. G. Leofanti, M. Padovan, G. Tozzola and B. Venturelli, Catal. Today, 1998, 41, 207–219.
36. (a) M. Khalili, A. Liwo and H. A. Scheraga, J. Mol. Biol., 2006, 355, 536–547; (b) J. Butler, Y. Zhao, M. Sinkora, N. Wertz and I. Kacskovics, Dev. Comp. Immunol., 2009, 33, 321–333.
37. J. Kinet, W. Bensinger, N. Balland, M. Saint-Remy, F. Frankenne and G. Hennen, Eur. J. Clin. Invest., 1986, 16, 50–55.
38. B. V. Ayyar, S. Arora, C. Murphy and R. O'Kennedy, Methods, 2012, 56, 116–129.
39. W. Wang, D. I. C. Wang and Z. Li, Chem. Commun., 2011, 47, 8115–8117.
40. Y. Zhang, Y. Yang, W. Ma, J. Guo, Y. Lin and C. Wang, ACS Appl. Mater. Interfaces, 2013, 5, 2626–2633.
41. G. Feng, D. Hu, L. Yang, Y. Cui, X. Cui and H. Li, Sep. Purif. Technol., 2010, 74, 253–260.
42. L. Zhang, X. Zhu, D. Jiao, Y. Sun and H. Sun, Mater. Sci. Technol., 2013, 33, 1989–1992.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra25949e

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