Research article Received: 4 March 2015,
Revised: 26 July 2015,
Accepted: 4 August 2015
Published online in Wiley Online Library: 13 November 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3584
Preparation of core–shell structure Fe3O4@SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His-tag protein purification Qian Ni, Bing Chen, Shaohua Dong, Lei Tian and Quan Bai* ABSTRACT: The core–shell structure Fe3O4/SiO2 magnetic microspheres were prepared by a sol–gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu2+, Ni2+ and Zn2+, were chelated on the Fe3O4@SiO2–IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni2+-chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe3O4@SiO2–IDA–Ni2+ magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His-tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: magnetic nanoparticles; magnetic separations; limmobilized metal affinity separation; lysozyme; His-tag protein
Introduction
566
Sample preparation is a fundamental and essential step in almost all analytical procedures, especially for the analysis of complex samples like biological and environmental samples. Biological samples are particularly complicated by matrix interference. Therefore, prior to the analysis of trace biological targets, the isolation, separation and purification of raw samples is imperative (He et al., 2014; Kishikawa and Kuroda, 2014). Moreover, enrichment and purification of proteins/polypeptides is a vital area in proteomics. Many endogenous proteins/peptides and proteins/peptides with post-translational modifications are presented in extremely low abundance, and they usually suffer strong interference with highly abundant proteins/peptides as well as other contaminants, resulting in low ionization efficiency in MS analysis (Chen et al., 2010; Jiang et al., 2008; Zhao et al., 2014). There have been many qualitative and quantitative studies of endogenous peptides in complex biological samples (Cheng et al., 2013; Chen et al., 2009; Liu et al., 2012; Wolman et al., 2010). However, the detection of low-abundance proteins/polypeptides remains a great challenge owing to the limited detection limit of instruments and obvious matrix interference. Thus, the separation and enrichment of proteins/peptides from complex mixtures is of great importance to their successful identification (Zhao et al., 2014). Therefore, a rapid, convenient, gentle and efficient sample preparation is urgently needed for biological analysis (He et al., 2014).
Biomed. Chromatogr. 2016; 30: 566–573
Over recent years, core–shell structured magnetic materials have been widely utilized in various sample preparation procedures for biological analysis with advantages of superparamagnetic property, high sensitivity, good biocompatibility, outstanding binding capacity and admirable recoverability (He et al., 2014; Zhao et al., 2014; Li et al., 2013; Borlido et al., 2013). Moreover, magnetic separations are probably one of the most versatile separation processes in biotechnology as they are able to purify cells, viruses, proteins and nucleic acids directly from crude samples. The fast and gentle process in combination with its easy scale-up and automation provide unique advantages over other separation techniques (Li et al., 2013; Borlido et al., 2013). Immobilized metal ion affinity chromatography is based on the specific coordinate covalent bond of amino acids, particularly
* Correspondence to: Q. Bai, Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Science, Key Laboratory of Modern Separation Science in Shaanxi Province, Northwest University, Xi’an 710069, China. Email:
[email protected] Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of EducationInstitute of Modern Separation Science, Key Laboratory of Modern Separation Science in Shaanxi Province, Northwest University, Xi’an710069, China Abbreviations used: FTIR, Fourier-transform infrared; IDA, iminodiacetic acid; TEM, transmission electron microscopy; TEOS, tetraethoxysilane.
Copyright © 2015 John Wiley & Sons, Ltd.
Superparamagnetic microspheres for His-tag protein separation histidine, to metals. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as cobalt, nickel or copper for the purification of histidine containing proteins or peptides, and iron, zinc or gallium for the purification of phosphorylated proteins or peptides (Gaberc-Porekar and Menart, 2001; Chaga, 2001). Histag is one of the most powerful methods for the efficient purification of recombinant protein based on the affinity interactions between metal ions (e.g. Ni2+) (Yao et al., 2014) and polyhistidine affinity tag (Hengen, 1995). Recently, various magnetic materials have been successfully developed for the specific separation of His-tagged proteins (Yao et al., 2014; Frenzel et al., 2003; Zhang et al., 2013). Moreover, various metal ions such as Cu2+, Zn2+, Fe3 + , Ga3+, Zr4+, Ti4+ and Ce4+ are grafted onto magnetic silica microspheres with appropriate chelating ligands such as iminodiacetic acid (IDA) and nitrilotriacetic acid (Sun et al., 2012; Wang et al., 2014; Qi et al., 2010; Ji et al., 2012; Ma et al., 2006a, 2006b), which have the capability of selectively enriching phosphorylated peptides and protein separation. Owing to the advantages of high magnetization, good rigidity, better stability and easy separation under an external magnetic field (Shareghi et al., 2015), various nanomagnetic particles coated with different polymers (Shamim et al., 2008; Shao et al., 2009; Chen et al., 2015) and chitosan (Li et al., 2014) shells have been successfully developed for lysozyme adsorption. However, the synthesis process of these nanomagnetic particles was more complicated, and not easy to control. In this paper, a simple method to prepare the core–shell structure Fe3O4/SiO2 magnetic microspheres was presented. Firstly, the core–shell structure Fe3O4/SiO2 magnetic microspheres were prepared by a sol–gel method, and then immobiled with IDA as metal ion affinity ligands for protein adsorption. Three divalent metal ions Cu2+,Ni2+ and Zn2 + were chelated on the Fe3O4@SiO2–IDA magnetic microspheres and the resulting affinity surfaces were then tested for their propensity to adsorb lysozyme in a batch system. The adsorption– desorption behaviour of lysozyme was investigated. Furthermore, the synthesized Fe3O4@SiO2–IDA–Ni2+ magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His-tag recombinant protein Homer 1a from the inclusion extraction expressed in Escherichia coli. The study showed that the magnetic silica microspheres could be used efficiently in protein separation or purification and showed great potential in the pretreatment of the biological sample.
Experiment
expressed in E. coli was gifted by Shaanxi DongAo Biotechnology Co. Ltd (Xi’an, China). An ultrasonicator (VCF1500, Sonics, USA) was employed for disrupting the cell pellets. The electrophoresis apparatus were obtained from Bio-Rad Company (USA). A Sorvall centrifuge (RC28S, Kendro, USA) was used for centrifugation, and a dualwavelength thin layer chromatographic scanner (Cs 9310, Shimadzu, Japan) was used for the determination of the purity of protein. Synthesis of Fe3O4 particles The Fe3O4 particles were prepared via a solvothermal method as described previously (Xu et al., 2006). Briefly, 5.4 g of FeCl3 · 6H2O was first dissolved in 200 mL of ethylene glycol under violent stirring. A clear yellow solution was obtained after filtration. Then 4.0 g of PEG 10000 and 14.4 g sodium acetate were added to this solution. The mixture was stirred vigorously for 30 min and then sealed in a Teflon-lined stainless-steel autoclave (200 mL capacity). The autoclave was heated to and maintained at 200 °C for 12 h, and then allowed to cool to room temperature. The black products were washed several times with ethanol and deionized water and dried at 60 °C for 8 h. Synthesis of Fe3O4@SiO2 The core–shell Fe3O4@SiO2 nanocomposites were prepared using a modified method from Stöber et al. (1986). The core–shell Fe3O4@SiO2 magnetic microspheres were synthesized by a sol–gel process to coat a thin layer of dense amorphous silica on Fe3O4 nanoparticles. Briefly, an ethanol dispersion of the magnetite particles (150 mg) was added to a beaker and washed three times with deionized water with help of the permanent magnet. Then, 50 mL of 0.1 mol/L HCl solution was added followed by ultrasonication for 30 min. The supernatant was decanted and the superparamagnetic Fe3O4 nanoparticles were added to a three-neck round-bottom flask and dispersed in 80 mL ethanol and 20 mL deionized water by ultrasonication for about 10 min. Under continuous mechanical stirring, 1.0 mL of ammonium hydroxide (28 wt%) and 0.8 mL of TEOS were consecutively added to the reaction mixture. The reaction was allowed to proceed at 30 °C for 6 h under continuous stirring. The resultant product was obtained by magnetic separation with help of the permanent magnet and was thoroughly washed with ethanol and deionized water six times (50 mL each time). The product was then dried under vacuum at 60 °C for 8 h.
Materials and equipment
Biomed. Chromatogr. 2016; 30: 566–573
Preparation of IDA-modified Fe3O4@SiO2 magnetic microspheres The IDA-modified Fe3O4@SiO2 magnetic microspheres were prepared using the Fe3O4@SiO2 magnetic microspheres modified with IDA as chelating ligand and are shown in Scheme 1. Briefly, 2.5 g IDA was dissolved in 50 mL of a 2 mol/L solution of Na2CO3, and the pH was adjusted to 10.0 with 10 mol/L NaOH. The IDA solution was stirred for several minutes in an ice bath and then 3-glycidoxypropyltrimethoxysilane (1.6 mL) was added into the solution slowly. After 30 min of stirring at 0°C, the reaction mixture was allowed to warm up to 65°C with stirring. After 6 h, the reaction solution was cooled to room temperature and the pH was adjusted to 6.0 with concentrated HCl. One gram of Fe3O4@SiO2 magnetic microspheres was added to the above solution and
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
567
Iron chloride hexahydrate (FeCl3 · 6H2O), anhydrous sodium acetate, ethylene glycol, concentrated ammonia solution (28 wt%) and PEG 10000 were purchased from the company of Sinopharm Chemical Reagent (Beijing, China). Tetraethoxysilane (TEOS), nickel sulfate hexahydrate (NiSO4 · 6H2O), copper sulfate pentahydrate (CuSO4 · 5H2O), zinc sulfate heptahydrate (ZnSO4 · 7H2O) and IDA were obtained from Xi’an Chemical Reagent Co. Ltd (Xi’an, China). 3-Glycidoxypropyltrimethoxysilane was purchased from Aladding Chemical Reagents (Shanghai, China). Lysozyme (from chicken egg white) was purchased from Sigma-Aldrich (St Louis, MO, USA). Other chemical reagents were of analysis grade and purified by normal standard methods. The water used in the experiments was purified using a Cascada LS system (Pall Life Sciences, Anna Arbor, MI, USA). The inclusion body of the His-tag recombinant Homer 1a
Q. Ni et al.
Scheme 1. Scheme for preparation of Fe3O4@SiO2–iminodiacetic acid (IDA) magnetic microspheres.
ultrasonicated for 10 min. The suspension was heated at 95°C for 2 h with stirring. The resulting nanoparticles denoted superparamagnetic IDA silica were separated with help of the permanent magnet and washed thoroughly with deionized water six times (50 mL each time). Charging of Fe3O4@SiO2–IDA magnetic microspheres with metal ions Charging of Fe3O4@SiO2–IDA magnetic microspheres with metal ions (eg. Cu2+, Ni2+ and Zn2+ ions) was performed as follows: 60 mg of Fe3O4@SiO2–IDA magnetic microspheres were mixed with 50 mL of aqueous solution of CuSO4 (10 mg/mL). The mixture was stirred magnetically at 100 rpm for 3 h. Then the excess unbound Cu2+ was removed with water. The same method was followed to chelate Ni2+ or Zn2+ using the aqueous solution of NiSO4 or ZnSO4 (10 mg/mL). Characterization
568
The size and morphology of magnetic silica nanospheres were determined by transmission electron microscopy (TEM, Tecnai G2
wileyonlinelibrary.com/journal/bmc
F20 S-TWIN, FEI Company, USA) and scanning electron microscopy (Quanta-400, FEI Company, USA). The magnetic properties were analyzed by superconducting quantum interference device (SQUID, MPMS-XL-7, Quantum Design, USA). Fourier-transform infrared (FTIR) spectra were recorded using a FTIR spectroscopy (Tensor 27; Bruker Optik, Ettlingen, Germany). The adsorption of lysozyme with Fe3O4@SiO2–IDA-metal ions magnetic microspheres The lysozyme adsorption experiment with Fe3O4@SiO2@IDA– metal ion magnetic microspheres was carried out batchwise. A stock solution of lysozyme (10 mg/mL) was prepared in the binding buffer of phosphate-buffered saline (PBS, 20 mmol/L, pH 7.0), from which different protein concentrations were made up to a final volume of 2 mL in plastic tubes. Approximately 10 mg Fe3O4@SiO2 magnetic microspheres charged with different metal ions, such as Cu2+, Zn2+ and Ni2+, were added to the protein solution, and the mixture was shaken at 25°C for 2 h. Then the magnetic microspheres were separated by a magnet and washed with binding buffer twice. The protein concentrations in the supernatant were measured by a UV–vis spectrophotometer (UV-1700,
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 566–573
Superparamagnetic microspheres for His-tag protein separation Shimadzu, Japan) using Bradford’s (1976) method. Coomassie brilliant blue G250 was used as development reagent to measure the absorbance at 595 nm, using bovine serum albumin for the calibration curve for the determination of protein concentration. The adsorption capacity of lysozyme was calculated by mass balance. The adsorbed protein could be recovered by incubating loaded magnetic metal ions–IDA–silica nanospheres with elution buffer (20 mmol/L KH2PO4, 0.5 mol/L NaCl,pH 7.0) with shaking for 30 min. Regeneration of Fe3O4@SiO2@IDA–metal ions magnetic microspheres The lysozyme desorption experiments were performed in elution buffer (20 mmol/L KH2PO4, 0.5 mol/L NaCl, pH 7.0). The lysozyme adsorbed magnetic silica microspheres were placed in the desorption medium and stirred at 25°C for 1 h. The desorption ratio was calculated from the lysozyme adsorbed and the amount of lysozyme desorbed. To investigate the reusability of the metal ion-charged magnetic silica microspheres, the lyzoyme adsorbed nanoparticles were treated with 50 mmo/L EDTA, and the Ni2+ charging procedure was applied again. Then the Ni2+-reloaded nanoparticles were applied for the protein adsorption. This process was carried out five times. Enrichment of lysozyme from egg white with Fe3O4@SiO2@IDA–Ni2+ magnetic microspheres Egg white was obtained from fresh egg and dissolved in 20 mmol/L PBS (pH 7.0) at 1:4 (v/v) dilution and gently stirred 30 min at 4 °C to give a precipitate that was removed by centrifugation at 10,000 rpm at 4 °C for 15 min. The Fe3O4@SiO2@IDA–Ni2+ magnetic microspheres were used to isolate lysozyme from egg white. A 200 μL egg white sample and 10 mg magnetic microspheres were mixed in 1 mL 20 mmol/L PBS (pH 7.0) in a plastic tube. The capture procedure was carried out under gentle shaking at 25 °C for 2 h. The supernatant was separated with a magnet, and the microspheres were washed several times with binding buffer and then incubated with 1 mL elution buffer (20 mmol/L KH2PO4, 0.5 mol/L NaCl, pH 7.0) at 25 °C for 2 h. The final eluate and the supernatant were collected to determine the proteins concentration and assay by SDS–PAGE. The proteins concentration was measured with the above method. Enrichment of His-tag recombinant Homer 1a expressed in E. coli
Biomed. Chromatogr. 2016; 30: 566–573
Results and discussions Synthesis and characterization of Fe3O4@SiO2–IDA core–shell magnetic microspheres The synthetic procedure for Fe3O4@SiO2-IDA core–shell nanocomposite is illustrated in Scheme 1. Firstly, the Fe3O4 nanoparticles were prepared by solvothermal method. Scanning electron microscope images of the Fe3O4 particles shows that nanoparticles are spherical in shape and very uniform in both size and shape with a mean particle size of 250 nm (Fig. 1a). Owing to the magnetic dipole attractions, they tend to seriously aggregate. Subsequently, silica was coated on the Fe3O4 using a sol–gel method to form Fe3O4@SiO2 core–shell microspheres through hydrolysis of TEOS according to the Stöber method. Because the surface of Fe3O4 has a strong affinity toward silica, no primer was required to promote the deposition and adhesion of silica. The growth of silica shells on Fe3O4 nanoparticles involved the base-catalyzed hydrolysis of TEOS and subsequent condensation of silica onto the surfaces of Fe3O4 cores. Several parameters (such as the growth time and the concentration of ammonia catalyst or water) can be employed to control the thickness of silica shell on the Fe3O4 surface. It is found that the particle size can be conveniently adjusted by changing the concentration of TEOS precursor. Figure 1(b) shows the TEM image of the Fe3O4@SiO2 nanocomposite particles. From Fig. 1(b), the TEM image shows that they are spherical in shape and the sizes become larger with an average size of 500 nm. Moreover, the core–shell structure can be clearly distinguished by the color contrast between the cores and the shells. The nanoparticles are also well dispersed without aggregation, indicating the stabilization function of silica coating on the magnetic Fe3O4 nanoparticles. Silica coating stabilizes the magnetite nanoparticles in two different ways. One is by sheltering the magnetic dipole interaction through the silica shell, and the other is by bringing negative charges on the surface of silica shells (Lu et al., 2002), which enhances the Coulomb repulsion of the magnetic nanoparticles. Thus the magnetic dipole interactions between the magnetic nanoparticles were greatly screened and they could be dispersed well. Finally, IDA as a chelating ligand was immobilized on the magnetic silica nanoparticles with silylation reagent 3-glycidoxypro
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
569
The His-tag recombinant Homer 1a was expressed in E. coli. The harvested cells were washed three times at room temperature with cleaning buffer I (20 mmol/L Tris + 1 mmol/L EDTA, pH 7.4), and disrupted by ultrasonication in an ice-water bath, followed by centrifugation at 9000 rpm for 15 min, 4 °C. The isolated inclusion bodies were washed with cleaning buffer II (20 mmol/L Tris + 1 mmol/L EDTA + 2.0 mol/L urea + 1.0 mol/L NaCl, pH 7.4), III (20 mmol/L Tris + 1 mmol/L EDTA + 1.0 mol/L NaCl,pH 7.4) and I (20 mmol/L Tris + 1 mmol/L EDTA, pH 7.4) three times. After each washing step, the suspension was centrifuged at 14,000 rpm and 4°C for 15 min, and the supernatant was discarded. The inclusion bodies were suspended in solubilization buffer (20 mmol/L Tris + 8 mol/L urea + 1.0 mmol/L EDTA, pH 8.0) and stirred continuously to solubilize proteins at 4 °C overnight. The suspension was
centrifuged to remove insoluble material. The soluble supernatant was recovered and stored at 4 °C. Protein concentration in the supernatant was measured to be 8.46 mg/mL according to the Bradford method. The Fe3O4@SiO2@IDA–Ni2+ magnetic microspheres were used to enrich the His-tag recombinant Homer 1a from the extraction of the inclusion bodies. A 200 μL aliquot of Homer 1a extraction solution and 10 mg magnetic microspheres were mixed in 1 mL 20 mmol/L PBS (pH 7.0) in a plastic tube. The capture procedure was carried out under gentle shaking at 25 °C for 2 h. The supernatant was separated with a magnet, and the microspheres were washed several times with binding buffer and then incubated with 1 mL elution buffer (20 mmol/L KH2PO4, 0.5 mol/L NaCl + 50 mmol/ L imidazole, pH 7.0) at 25 °C for 2 h. The final eluate and the supernatant were collected to determine the proteins concentration and assay by SDS–PAGE. The proteins concentration was measured with the above method.
Q. Ni et al.
Figure 1. Scanning electron microscope image of Fe3O4 (a) and transmission electron microscopy images of Fe3O4@SiO2 microspheres (b).
pyltrimethoxysilane to obtain Fe3O4@SiO2–IDA core–shell nanocomposite. In order to further confirm the composition and structure of samples, FTIR spectra were measured. The FTIR spectrum of Fe3O4 (Fig. 2a) shows a strong peak at 570 cm 1, which is assigned to the vibration of the Fe–O functional group (Chi et al., 2012). After the coating of silica, Fe3O4@SiO2 microspheres show some new bands centered around 795 and 1080–1100 cm 1 (Fig. 2b). The new absorption at 795 cm 1 can be ascribed to the symmetric vibration of Si–O–Si, whereas the band at 1080–1100 cm 1 is assigned to the asymmetric stretching vibration of Si–O–Si. These results indicate that SiO2 is immobilized on the surfaces of Fe3O4 microspheres. After IDA is immoblized on the surface of Fe3O4@SiO2 microspheres, the absorption bands at 1635 and 3400 cm 1 are attributable to stretching vibrations of the ester carbonyl groups and the -OH stretching vibration, respectively (Fig. 2c). In addition, there is an absorption band at 1400 cm 1 indicating the CH2–N units (Fig. 2c). The magnetic properties of the samples were characterized using a SQUID magnetometer measured at 300 K as shown in Fig. 3. Pure Fe3O4 microspheres, Fe3O4@SiO2 microspheres and Fe3O4@SiO2–IDA nanocomposite have magnetization saturation (Ms) values of 66.5, 61.2 and 58.1 emu/g, respectively. Because of the presence of SiO2 shell, the Ms value of Fe3O4 microspheres is higher than that of Fe3O4@SiO2 microspheres and Fe3O4@SiO2– IDA nanocomposite. Compared with Fe3O4@SiO2 microspheres,
Figure 3. Magnetization curves of Fe3O4 microspheres, Fe3O4@SiO2 microspheres and Fe3O4@SiO2–IDA nanocomposite.
Fe3O4@SiO2–IDA nanocomposite exhibits a slightly smaller Ms value, which is attributed to the slight increase in the mass and size caused by immobilized IDA on the surfaces of Fe3O4@SiO2 microspheres. It should be noted that the core–shell structured Fe3O4@SiO2–IDA nanocomposite still shows strong magnetization, suggesting its suitability for magnetic separation and recovery.
570
Figure 2. Fourier-transform infrared (FTIR) spectra of Fe3O4 microspheres (a), Fe3O4@SiO2 microspheres (b) and Fe3O4@SiO2–IDA nanocomposite (c).
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 566–573
Superparamagnetic microspheres for His-tag protein separation The adsorption and desorption of lysozyme with Fe3O4@SiO2– IDA nanocomposite charged different metal ions Protein adsorption in immobilized metal ion affinity chromatography is mainly through chelation between some metal ions and amino acid residues (i.e. histidine and cysteine). In order to investigate the separation efficiency of magnetic microspheres, lysozyme was chosen as a model protein owing to its low cost and wide application. The desorption of the adsorbed lysozyme from the magnetic Fe3O4@SiO2–IDA-chelated microspheres was studied in a batch system. The adsorption performances of the magnetic Fe3O4@SiO2–IDA microspheres chelated with three different metal ions, such as Cu2 + , Ni2+ and Zn2+, were investigated with lysozyme initial concentration of 0.5 mg/mL at pH 7.0 and 25°C. As shown in Fig. 4, the maximum adsorption capacities of lyozyme for the adsorbents chelated with Cu2+, Ni2+ and Zn2+ were 48.5, 51.0 and 36.4 mg/g, respectively. The increase in ionic strength and the change of pH can both change the electrostatic or hydrophobic interactions between the protein and the microspheres. Therefore, buffer solutions containing salt are usually utilized when desorbing proteins from ligand-immobilized magnetic microspheres. The lysozyme-loaded adsorbents were placed in the desorption medium containing 20 mmol/L KH2PO4 and 0.5 mol/L NaCl at pH 7.0, and the amount of lysozyme adsorbed on the magnetic Fe3O4@SiO2–IDA microspheres chelated with Cu2+, Ni2+ and Zn2+, released in 30 min, was determined, respectively. The results are also shown in Fig. 5. It is obvious that lysozyme adsorbed on the Ni2+ and Zn2+ chelating magnetic microspheres were desorbed almost completely. The amount of the adsorbed lysozyme was desorbed by up to 95%. However, only 97.6%. The results demonstrate that the magnetic Fe3O4@SiO2–IDA–Ni2+ microspheres are promising for the separation of lysozyme from chicken egg white.
Homer 1a (H1a) is an immediate early gene involved in multiple forms of synaptic plasticity that is up-regulated in neurons after seizures (Brakeman et al., 1997) and in the hippocampus during long-term potentiation or after exploration of a novel environment. It is necessary for homeostatic scaling and regulates calcium homeostasis. H1a also induces mGluR1 and mGluR5 activity, and regulates activity-induced post- and presynaptic structural remodeling and dendritic axonal targeting of mGluR5. Moreover, Homer1 knockout mice exhibit behavioral and glutamatergic abnormalities and a schizophrenia-like phenotype (Montes-Rodriguez et al., 2013). In this work, the His-tag recombinant Homer 1a with mass weight of 30 kDa was expressed in E. coli as inclusion bodies. The magnetic Fe3O4@SiO2–IDA–Ni2+ microspheres were used to enrich the His-tag recombinant Homer 1a from the extraction of the inclusion bodies. The extract solution of inclusion bodies and the supernatant of the eluted solution were collected and analyzed by SDS–PAGE (shown in Fig. 7). Ni–IDA ligand has a stronger binding ability to His-tag, in which affinity reaction every Ni2+ and histidine form two coordinate bonds. In Fig. 7, it can be seen that the recombinant His-tag Homer 1a in the extraction solution can be enriched and isolated by the magnetic Fe3O4@SiO2–IDA–Ni2+ microspheres (lane 2). The purity was increased from 24.6 to 87.3%. The mass recovery can reach 92.3%. The result indicated that magnetic Fe3O4@SiO2–IDA–Ni2+ microspheres are an ideal sample pretreatment tool for His-tag recombinant protein purification with their stability, simplicity, good repeatablity and convenience.
Conclusion The core–shell structure Fe3O4/SiO2 magnetic microspheres were prepared by a sol–gel method, and immobilized with IDA as metal ion affinity ligands for protein adsorption. They exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu2+, Ni2+ and Zn2+, were chelated on the Fe3O4@SiO2–IDA magnetic microspheres to adsorb lysozyme, respectively. The results showed that Ni2+-chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, the adsorption equilibrium could be reached with 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe3O4@SiO2–IDA–Ni2+ magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His-tag recombinant Homer 1a expressed in E. coli. The result indicates that the magnetic microspheres show unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption and easy handling. It demonstrates that the magnetic silica microspheres could be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample.
Acknowledgments
572
Figure 7. SDS–PAGE analysis of recombinant His-tag Homer 1a from the 2+ inclusion bodies extraction solution by Ni -chelated magnetic Fe3O4@SiO2–IDA microspheres. Lane 1, the extraction solution of Homer 1a inclusion bodies; lane 2, supernatant of elution.
wileyonlinelibrary.com/journal/bmc
This work is supported by the National 863 Program (no. 2006AA02Z227), Natural Science Foundation of Shaanxi Province (2011JZ002), the Foundation of Key Laboratory in Shaanxi Province (2010JS103, 11JS097, 14JS098) and Shaanxi Provincial Science and Technology Co-ordinating innovation projects (2013SZS18-K01).
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2016; 30: 566–573
Superparamagnetic microspheres for His-tag protein separation
References Borlido L, Azevedo AM, Roque ACA and Aires-Barros MR. Magnetic separations in biotechnology. Biotechnology Advances 2013; 31: 1374–1385. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 1976; 72: 248–254. Brakeman PR, Lanahan AA, O’Brien R and Roche K. Homer: a protein that selectively binds metabotropic glutamate receptors. Nature 1997; 386: 284–288. Chaga GS. Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. Journal of Biochemistry and Biophysics: Methods 2001; 49: 313–334. Chen H, Deng C and Zhang X. Synthesis of Fe3O4@SiO2@PMMA core–shell–shell magnetic microspheres for highly efficient enrichment of peptides and proteins for MALDI-TOF MS analysis. Angewandte Chemie International Edition 2010; 49: 607–611. Chen HM, Deng CH, Li Y, Dai Y, Yang P and Zhang XM. A facile synthesis approach to C8-functionalized magnetic carbonaceous polysaccharide micro-spheres for the highly efficient and rapid enrichment of peptides and direct MALDI-TOF-MS analysis. Advanced Materials 2009; 21: 2200–2205. Chen J, Lin YX and Li J. Preparation of anionic polyelectrolyte modified mgnetic nanoparticles for rapid and efficient separation of lysozyme from egg white. Journal of Chromatography A 2015; 1388: 43–51. Cheng G, Wang ZG, Liu YL, Zhang JL, Sun DH and Ni JZ. Magnetic affinity microspheres with meso-/macroporous shells for selective enrichment and fast separation of phosphorylated biomolecules. Applied Materials Interfaces 2013; 5: 3182–3190. Chi Y, Geng W, Zhao L, Yan X, Yuan Q, Li N and Li X. Comprehensive study of mesoporous carbon functionalized with carboxylate groups and magnetic nanoparticles as a promising adsorbent. Journal of Colloid and Interface Science 2012; 369: 366–372. Frenzel A, Bergemann C, Köhl G and Reinard T. Novel purification system for 6x His-tagged proteins by magnetic affinity separation. Journal of Chromatography B 2003; 793: 325–329. Gaberc-Porekar V and Menart V. Perspectives of immobilized-metal affinity chromatography. Journal of Biochemistry and Biophysics: Methods 2001; 49: 335–360. He JC, Huang MY, Wang DM, Zhang ZM and Li GK. Magnetic separation techniques in sample preparation for biological analysis. Journal of Pharmaceutical and Biomedical Analysis 2014; 101: 84–101. Hengen PN. Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences 1995; 20: 285–286. Ji LY, Wu JH, Luo Q, Li XC, Zheng W, Zhai GJ, Wang FY, Lü S, Feng YQ, Liu JA and Xiong SX. Quantitative mass spectrometry combined with separation and enrichment of phosphopeptides by Titania coated magnetic mesoporoussilica microspheres for screening of protein kinase inhibitors. Analytical Chemistry 2012; 84: 2284–2291. Jiang XG, Ye ML and Zou HF. Technologies and methods for sample pretreatment in efficient proteome and peptidome analysis. Proteomics 2008; 8: 686–705. Kishikawa N and Kuroda N. Analytical techniques for the determination of biologically active quinones in biological and environmental samples. Journal of Pharmaceutical and Biomedical Analysis 2014; 87: 261–270. Li XS, Zhu GT, Luo YB, Yuan BF and Feng YQ. Synthesis and applications of functionalized magnetic materials in sample preparation. Trends in Analytical Chemistry 2013; 45: 233–247. Li ZH, Cao M, Zhang WG, Liu LZ, Wang JL, Ge WP, Yuan YH, Yue TL, Li RH and Yu WW. Affinity adsorption of lysozyme with Reactive Red 120 modified magnetic chitosan microspheres from thermosensitive nanomagnetic particles. Food Chemistry 2014; 145: 749–755.
Liu Q, Shi JB, Cheng MT, Li GL, Cao D and Jiang GB. Preparation of graphene-encapsulated magnetic microspheres for protein/peptide enrichment and MALDI-TOF MS analysis. Chemical Communications 2012; 48: 1874–1876. Lu Y, Yin Y, Mayers BT and Xia Y. Modifying the surface properties of superparamagnetic iron oxide nanoparticles through a sol–gel approach. Nano Letters 2002; 2: 183–186. Ma ZY, Liu XQ, Guan YP and Liu HZ. Synthesis of magnetic silica nanospheres with metal ligands and application in affinity separation of proteins. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2006a; 275: 87–91. Ma ZY, Guan YP and Liu HZ. Superparamagnetic silica nanoparticles with immobilized metal affinity ligands for protein adsorption. Journal of Magnetism and Magnetic Materials 2006b; 301: 469–477. Montes-Rodriguez CJ, Lapointe V, Trivedi V, Lu Q, Demchuk AM and McNaughton BL. Postnatal development of Homer1a in the rat hippocampus. Hippocampus 2013; 23: 890–902. Qi DW, Mao Y, Lu J, Deng CH and Zhang XM. Phosphate–functionalized 4+ magnetic microspheres for immobilization of Zr ions for selective enrichment of the phosphopeptides. Journal of Chromatography A 2010; 1217: 2606–2617. Shamim N, Liang H, Hidajat K and Uddin MS. Adsorption, desorption, and conformational changes of lysozyme. Journal of Colloid and Interface Science 2008; 320: 15–21. Shao DD, Xu KK, Song XJ, Hu JH, Yang WL and Wang CC. Effective adsorption and separation of lysozyme with PAA-modified Fe3O4@silica core/shell microspheres. Journal of Colloid and Interface Science 2009; 336: 526–532. Shareghi B, Farhadian S and Zamani N. Investigation the activity and stability of lysozyme on presence of magnetic nanoparticles. Journal of Industrial and Engineering Chemistry 2015; 21: 862–867. Stöber W, Fink A and Bohn E. Controlled growth of monodisperse silica spheres in the micron size range. Journal of Colloid and Interface Science 1986; 26: 62–69. Sun J, Liu YT, Su YJ, Xia WS and Yang YJ. Highly efficient enrichment of phosvitin phosphopeptides by novel magnetic carboxymethyl chitosan nanoparticles decorated with Fe (III) ions. Journal of Chromatography B 2012; 915: 33–38. Wang ZG, Cheng G, Liu YL, Zhang JL, Sun DH and Ni JZ. Novel core–shell Cerium (IV)-immobilized magnetic polymeric microspheres for selective enrichment and rapid separation of phosphopeptides. Journal of Colloid and Interface Science 2014; 417: 217–226. Wolman FJ, Copello GJ, Mebert AM, Targovnik AM, Miranda MV, del Cañizo N, Díaz LE and Cascone O. Egg white lysozyme puriWcation with a chitin–silica-based affinity chromatographic matrix. European Food Research Technology 2010; 231: 181–188. Xu XQ, Deng CH, Gao MX, Yu WJ, Yang PY and Zhang XM. Synthesis of magnetic microspheres with immobilized metal ions for enrichment and direct determination of phosphopeptides by matrix-assisted laser desorption ionization mass spectrometr. Advanced Materials 2006; 18: 3289–3293. Yao S, Yan X, Zhao Y, Li B and Sun L. Selective binding and magnetic 2+ separation of histidine-tagged proteins using Ni -decorated Fe3O4/ hydroxyapatite composite nanoparticles. Materials Letters 2014; 126: 97–100. Zhang L, Zhu X, Jiao D, Sun Y and Sun H. Efficient purification of His-tagged 2+ protein by superparamagnetic Fe3O4/Au-ANTA-Co nanoparticles. Materials Science and Engineering C: Materials for Biological Applications 2013; 33: 1989–1992. Zhao M, Xie YQ, Deng CH and Zhang XM. Recent advances in the application of core–shell structured magneticmaterials for the separation and enrichment of proteins and peptides. Journal of Chromatography A 2014; 1357: 182–193.
573
Biomed. Chromatogr. 2016; 30: 566–573
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
