hydroxyapatite composite nanoparticles

Materials Letters 126 (2014) 97–100

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Materials Letters journal homepage: www.elsevier.com/locate/matlet

Selective binding and magnetic separation of histidine-tagged proteins using Ni2 þ -decorated Fe3O4/hydroxyapatite composite nanoparticles Shasha Yao a, Xiqing Yan b, Yanbao Zhao a,n, Binjie Li a, Lei Sun a a b

Key Laboratory for Special Functional Materials, Henan University, Kaifeng 475004, PR China College of Pharmacy, Xinxiang Medical University, Xinxiang 453003, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 24 December 2013 Accepted 5 April 2014 Available online 13 April 2014

Fe3O4/hydroxyapatite (Fe3O4/HAP) nanoparticles (NPs) were synthesized by hydrothermal method. The obtained NPs have excellent magnetic responsibility and can immobilize metal ions. After chelating Ni2 þ ions, Fe3O4/HAP-Ni2 þ NPs can be used to enrich histidine-tagged (His-tagged) proteins directly from the mixture of lysed cells. Results show that Fe3O4/HAP-Ni2 þ NPs present negligible nonspecific protein adsorption and high protein binding activity with the saturation capacity being 12.98 mmol/g. Their separation capcity towards His-tagged proteins was remained after 4 times recycling. They are especially suitable for rapid purification of His-tagged proteins. & 2014 Elsevier B.V. All rights reserved.

Keywords: Magnetic materials Fe3O4/HAP Nanoparticles Nanocomposites

1. Introduction Proteins play a variety of crucial roles in organisms and the development of efficient methods to separate target proteins from a biological source is currently a hot topic [1,2]. The biotechnologies today enable proteins to express with a tag very easily, and many protein–purification methods employ a separation step based on specific interactions between immobilized ligands and affinity tags on the protein [3]. One of the most popular tags is polyhistidine which binds strongly to divalent metal ions such as Ni2 þ and Co2 þ ions [4]. Nickel ion affinity chromatography has therefore become the most commonly method to separate Histagged proteins [5]. Although this method is easily adaptable to any protein expression system, it has some limitations, including the need for pretreatment to remove the cell debris, colloid contaminants and time-consuming. Magnetic NPs have been extensively studied for various biological applications, including magnetic resonance imaging [6], drug delivery [7], and biomolecule separation [8]. To magnetically separate His-tagged proteins, the key is to couple the appropriate affinity agents on the surface of magnetic NPs. For example, nickel nitrilotriacetic acid (Ni-NTA) modified Fe3O4 NPs were used to separate biological targets at low concentrations, but such particles with small size are difficult to be collected from solution [9,10]. In addition, NiO coated NPs were also used to purify Histagged proteins [11], but it is necessary to improve their stability

n

Corresponding author. Tel.: þ 86 378 22820579; fax: þ 86 378 23881358. E-mail address: [email protected] (Y. Zhao).

http://dx.doi.org/10.1016/j.matlet.2014.04.022 0167-577X/& 2014 Elsevier B.V. All rights reserved.

against oxidation in an aqueous environment. To improve the magnetic separation efficiency, several novel separation system based on magnetic NPs have been developed, such as Fe3O4@SiO2@P(St-MA)/Ni-NTA [12], Fe3O4@SiO2@Polymer brush/Ni-NTA [13] and Fe3O4@SiO2@NiAl-LDH [14], which display excellent magnetic separation performance of His-tagged proteins. These approaches, although promising, are often limited by complicated synthesis routes and time-consuming experiment techniques. Due to its favorable biocompatibility and adsorption capacity, HAP NPs have been studied for drug delivery, tissue engineering and protein adsorption [15,16]. Especially, HAP has rich surface active sites, and can immobilize metal ions through chelating bond. After binding Ni2 þ ions, HAP-Ni2 þ NPs are used to separate His-tagged proteins. Here, we report a facile route to prepare novel absorbent Fe3O4/HAP NPs with magnetic responsibility. Compared with Ni-NTA based absorbent, the preparation of HAP based absorbent is facile, cost effective and highly efficient.

2. Experimental Synthesis: All reagents are used as-received (SI 1). Typically, FeCl2  4H2O (0.37 g) and FeCl3  6H2O (1.0 g) were dissolved in distilled water (30 mL) and then 10 mL NH3  H2O was added to it with mechanical stirring. After reaction at 50 1C for 30 min, the Fe3O4 sample was collected by magnet. The magnetic sample was dispersed into citric acid solution (90 mL, 0.05 M). After stirring at 90 1C for 1.5 h, 100 mL mixed solution (17 mmol Ca(NO3)2, 10 mmol (NH4)2HPO4) was added drop-wise into it over 30 min with continuous stirring. Subsequently, the solution was transferred into a

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S. Yao et al. / Materials Letters 126 (2014) 97–100

250 mL Teflon-lined stainless-steel autoclave, sealed and heated at 180 1C for 12 h. Finally, the separated Fe3O4/HAP sample was dispersed in NiCl2 solution (50 mL, 2 M) to chelate Ni2 þ ions. Expression and purification: His-tagged thioredoxin 9 (Histagged TRX9) [17] was prepared. We cloned TRX from Arabidopsis thaliana and constructed them into the PET-32a plasmid. Histagged recombinant plasmids were transformed into Escherichia coli strain Rosetta (DE3) (Novagen) for protein expression using standard protocols [18]. After being washed with Tris buffer, Fe3O4/HAP-Ni NPs (10 mg) were added directly into 2 mL mixture of cell lysate and incubated with shaking for 2 h. Then, Fe3O4/HAP NPs were isolated from the solution by magnet and washed with Tris buffer to remove residual proteins. Subsequently, Fe3O4/HAP NPs were washed with 300 μL imidazole (1.0 M) to disassociate His-tagged proteins from their surface. Fe3O4/HAP NPs are reused by washing sequentially with EDTA (0.1 M) and NiCl2 solution (2 M). Characterizations: The morphology and composition were characterized by field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), fourier transform infrared

(FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) spectra, atomic adsorption spectrophotometer (AAS) and thermogravimetric analysis (TG), respectively. Particle size distribution was measured by Malvern Nano ZS90. The separated Histagged proteins were detected with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of His-tagged proteins was analyzed by UV–vis Spectrophotometer.

3. Results and discussion Fig. 1a and b gives the FESEM and TEM images of the Fe3O4/HAP NPs. It is clear that the sample is spherical in shape and has an average diameter of 80 nm. The size distribution curve presents single peak at 80 nm (the PDI is 0.35), which is agreement with FESEM observation (Fig. 1c). The hysteresis loop of Fe3O4/HAP NPs measured at 300 K show paramagnetic behavior, and their saturation magnetization (Ms) is 8.5 emu/g, which is smaller than that of Fe3O4 NPs (Fig. 1d). This reduction might be caused by the formation of Fe3O4/HAP composite. In addition, fast aggregation of Fe3O4/HAP NPs from homogeneous dispersion was observed in the presence of external magnetic field (Fig. 1d, inset), which is important in terms of their practical manipulation. Fig. 2 displays the FT-IR spectra (a) and XRD patterns of Fe3O4/ HAP NPs. For comparison, the FT-IR spectrum of Fe3O4 NPs is also given. For Fe3O4 NPs, the broad bands at 3444 cm  1 and 1631 cm  1 are assigned to the absorbed water. There is an obvious adsorption peak at 580 cm  1, which is characteristic of Fe–O vibrations of Fe3O4. In the FT-IR spectrum of Fe3O4/HAP NPs, there

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H(Oe) Fig. 1. FESEM image (a), TEM image (b) and size distribution histogram (c) of Fe3O4/HAP NPs. Room-temperature (300 K) magnetic hysteresis loops (d) and Magnetic response photos (inset) of Fe3O4/HAP NPs.

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2 theta (degree) Fig. 2. FT-IR spectra (a) XRD pattern (b) of Fe3O4/HAP NPs.

S. Yao et al. / Materials Letters 126 (2014) 97–100

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Captured His-tagged Protein (%)

Fig. 3. SDS-PAGE analysis of purified His-tagged proteins by Fe3O4/HAP-Ni2 þ NPs. Lane L, E. coli lysate. Lane M Marker. (a) Lane 1–4, the fractions washed off from the Fe3O4/ HAP-Ni2 þ NPs with different amount (lane 1, 1.0 mg; lane 2, 5.0 mg; lane 3, 10.0 mg; lane 4, 15.0 mg). (b) Lane 1–6, the fractions washed off from the Fe3O4/HAP-Ni2 þ NPs treated with different concentration of Ni2 þ (lane 1, 2 mol/L; lane 2, 1 mol/L; lane 3, 0.5 mol/L lane 4, 0.2 mol/L; lane 5, 0.1 mol/L; lane 6, 0.05 mol/L).

100 80 60 40 20 0 1

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Recycling Times Fig. 4. SDS-PAGE analysis (left) and recycling efficiency (right) of proteins by reused materials.

also appear adsorption peaks of water. The bands at 1029, 601, and 563 cm  1 are assigned to vibrations of the phosphate group [19]. The weak peaks at 1460 and 1384 cm  1 are attributed to vibration modes of CO23  absorption bands [20]. In the XRD pattern, Fe3O4/ HAP sample presents multiple peaks, and these peaks are attributed to face-centered cubic (fcc) Fe3O4 phase (JCPDS 19-0629) and crystalline HAP (JCPDS 09-0432) NCs, respectively, which further confirmed the formation of Fe3O4/HAP NPs. In addition, XPS analyses (SI2) reveal that the Ca/P and Ni/P ratio in Fe3O4/HAPNi NPs is 1.27 and 0.27, respectively, and the Ni content determined by AAS is 9.6 mmol/g. Due to the high affinity of Ni2 þ toward histidine, Fe3O4/HAP-Ni NPs can be used for purification of His-tagged proteins in crude E. coli cell lysate. Fig. 3 gives SDS-PAGE analysis of the purified His-tagged TRX9 proteins. Lane M is molecular weight markers and lane L is E. coli cell lysate. The MW of His-tagged TRX was 14.2 KD. There is an intense strip at 14.2 kDa on the lane 1–4 in Fig. 3a, which indicates that the Fe3O4/HAP-Ni NPs have excellent Histagged protein binding properties with negligible nonspecific adsorption. It is clear that the amount of the captured proteins was increased with increasing the weight of Fe3O4/HAP NPs, but their binding capacity was reduced. For instance, when Fe3O4/HAP NPs was 1.0, 5.0, 10 and 15 mg, their capacity was 12.98 mmol/g, 9.19 mmol/g, 7.73 mmol/g and 5.97 mmol/g, respectively. Such a high efficiency comes from the high content of Ni2 þ ions on the surface of NPs, which depends the concentration of Ni solution. It is found that the amount of the captured His-tagged TRX9 was increased with increasing the concentration of Ni2 þ in the range of

0.05 M to 2 M (Fig. 3b), and the max value was 10.24 mmol/g. The amount of proteins captured by Fe3O4/HAP NPs is highly dependent on the concentration of proteins in cell lysate and concentration of imidazole (SI 3). To further test the practical utility of Fe3O4/HAP NPs, the recyclability of these NPs in His-tagged protein separation system was also investigated (Fig. 4). After each separation, Fe3O4/HAP NPs were rinsed with PBS to release excess adsorbed imidazole onto the surface upon sonication. The separation capacity of Histagged TRX protein from the lysate was  82% after recycling four times, indicating that Fe3O4/HAP NPs have excellent separation efficiency.

4. Conclusion In this paper, magnetic responsive Fe3O4/HAP NPs were prepared through a hydrothermal route. After chelating Ni2 þ ions, Fe3O4/HAP NPs can capture His-tagged proteins directly from the mixture of lysed cells. Their selectivity and recyclability for the His-tagged proteins are maintained after 4 times recycling.

Acknowledgement Financial support of this work from National Natural Science Foundation of China (21271062) and Program for Changjiang

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Scholars and Innovative Research Team (PCS IRT1126) are gratefully acknowledged.

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