Facile and high-efficient immobilization of histidine-tagged multimeric protein G on magnetic nanoparticles
© Lee and Chang; licensee Springer. 2014
Received: 17 November 2014
Accepted: 4 December 2014
Published: 10 December 2014
This work reports the high-efficient and one-step immobilization of multimeric protein G on magnetic nanoparticles. The histidine-tagged (His-tag) recombinant multimeric protein G was overexpressed in Escherichia coli BL21 by the repeated linking of protein G monomers with a flexible linker. High-efficient immobilization on magnetic nanoparticles was demonstrated by two different preparation methods through the amino-silane and chloro-silane functionalization on silica-coated magnetic nanoparticles. Three kinds of multimeric protein G such as His-tag monomer, dimer, and trimer were tested for immobilization efficiency. For these tests, bicinchoninic acid (BCA) assay was employed to determine the amount of immobilized His-tag multimeric protein G. The result showed that the immobilization efficiency of the His-tag multimeric protein G of the monomer, dimer, and trimer was increased with the use of chloro-silane-functionalized magnetic nanoparticles in the range of 98% to 99%, rather than the use of amino-silane-functionalized magnetic nanoparticles in the range of 55% to 77%, respectively.
KeywordsMagnetic nanoparticle Immobilization Protein G Histidine-tag Efficiency
Magnetic nanoparticles (MNPs) have been widely used in various biomaterial applications, such as the immobilization of enzymes and proteins, drug delivery systems, labeling and sorting of cells, and the purification of DNA and proteins [1–8]. One of the most recent applications being investigated extensively is the use of functionalized MNPs as magnetic carriers for specific protein purification [9–14]. Among them, immobilization methods for purification specific protein have used metal ion affinity-tagged protein, such as histidine (His)-tagged proteins. The functionalized magnetic nanoparticles and tagged protein offer several advantages for protein purification, such as easy handling and simple procedures. The protein purification can be carried out in a single tube without expensive instruments, centrifuges, or other such systems.
In this work, we report the one-step preparation of multimeric protein G with a His-tagged (His-tag) monomer, dimer, and trimer on magnetic nanoparticles for high-efficient immobilization. This immobilization was demonstrated by two different pathways of amino-silane and chloro-silane functionalization on silica-coated magnetic nanoparticles. To calculate the efficiency, bicinchoninic acid (BCA) assay was used and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Synthesis of the CTMS-MNPs@SiO2
Iron (II) chloride tetrahydrate (99%), iron (III) chloride hexahydrate (98%), tetraethyl orthosilicate (98%), and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich chemical (St. Louis, MO, USA). (Chloromethyl) phenylethyltrimethoxysilane (CTMS) was purchased from Gelest (Morrisville, PA, USA). Pierce BCA™ protein assay kit was purchased from Thermo Scientific Inc. (Pittsburgh, PA, USA). HPLC-grade solvents were used in both the reaction and washing steps. All aqueous solution was prepared with double-distilled water.
MNPs were prepared by coprecipitation with Fe2+ and Fe3+. To prepare the MNPs, a mixture of 2 M FeCl2 · 4H2O and 1 M FeCl3 · 6H2O was added to 0.7 M ammonia solution with vigorous stirring at room temperature. The obtained MNPs were separated by magnet, and the supernatant was decanted. The collected MNPs were washed several times with ethanol and then air-dried. In order to synthesize the silica coating of MNPs, the 100 mg of MNPs were dispersed in 100 mL of cyclohexane under sonication for 3 h in the presence of 3.3 mL of oleic acid. The 44 g of Igepal CO-520 (Sigma-Aldrich chemical) dissolved solution in 900 mL of cyclohexane was added to MNP solution. After stirring for 15 min, 8 mL of aqueous ammonia solution was added dropwise. The mixed solution was further stirred for 20 h at room temperature. Methanol was added to the solution to form dark precipitates, which were collected through magnetic separation after the removal of supernatants. The dark precipitate was washed repeatedly with ethanol and then vacuum dried.
The MNPs@SiO2 was then modified with silane coupling agent to introduce chloride group. Briefly, 100 mg of MNPs@SiO2 was dispersed in 9 mL of acetone containing 1 mL of CTMS, and the mixture was rotated at room temperature for 12 h in the dark. After silanization, the resultants were separation by magnetic attraction and washed repeatedly with acetone to remove unreacted silane coupling agent, and then dried at room temperature overnight in the dark.
Immobilization of histidine-tagged multimeric protein G
His-tagged recombinant dimer and trimer of protein G were overexpressed in Escherichia coli BL21 by the repeated linking of protein G monomers with flexible (GGGGS)3((glycine/glycine/serine)3) . The sizes for His-tagged monomer, dimer, and trimer protein G were 16, 32, and 46 kDa, respectively.
The immobilization of His-tagged multimeric protein G experiments were carried out in 200 μL of protein solution dissolved in 10 mM phosphate buffer (pH 7.2) and were added to disperse the chloride group functionalized MNPs@SiO2 (5 mg). The initial concentration of protein G was 100 μg/mL. The reaction mixtures were incubated within a rotator at room temperature for 1 h with continual agitation to the suspending particles. After removal of particles by magnetic separation, the multimeric protein G concentrations in the supernatants were determined by fluorescence intensity using a microplate reader (Infinite M200, Tecan Ltd; Männedorf, Switzerland) at 595 nm. The fluorescence intensity of sample was measured five times with a 20-μs integration time by using a microplate reader and BCA protein assay kit.
SDS-PAGE of the samples were performed using standard protocols for conformation of protein binding efficiency . Samples were incubated at 100°C for 5 min in a sample buffer and then separated by SDS-PAGE (12% gels).
The particle size and morphology of silica-coated magnetic nanoparticles were determined by transmission electronic microscopy (TEM) using a JEM-4010 (JEOL; Tokyo, Japan) at an accelerating voltage of 400 kV. The magnetic nanoparticles and silica-coated magnetic nanoparticles were characterized by powder X-ray diffraction (XRD), using a Rigaku D/MAX-2500 diffractometer (Rigaku; Tokyo Japan) using filtered Cu Kα radiation, and the data were collected for 2θ of 20.0° to 80.0°. The magnetization of silica-coated magnetic nanoparticles at room temperature up to 10 kOe was measured by vibrating sample magnetometer (VSM) using a VSM 4179 (Oxford Instruments; Oxfordshire, UK). Fourier transform infrared (FT-IR) spectroscopy was used to identify the functionalized MNPs@SiO2. The FT-IR spectra were recorded on a V-460 (JASCO; Easton, MD, USA) FT-IR spectrometer using KBr pellets. Spectra were obtained at a resolution of 4 cm-1, and the wavenumber range from 4,000 to 650 cm-1. The fluorescence intensity and absorbance of the samples were measured using microplate reader Infinite M200 (Tecan Ltd). The fluorescence was measured five times for each sample with a 20-μs integration time.
Results and discussion
Figure 1 shows the synthetic procedures of conventional preparation with (a) a sulfo-SMCC cross-linker from amino-silaneMNPs@SiO2 and (b) a one-step preparation with a chloro-silane functional group on MNPs@SiO2. This CTMS strongly anchors the MNPs@SiO2 surface, while the chloro functions on the outer surface induce coupling between the MNPs@SiO2 and His-tagged biomolecules. This method was simple and low cost compared to the Sulfo-SMCC-MNPs@SiO2.
In order to confirm the modification of the MNPs' surface, the FT-IR spectroscopy of the prepared MNPs@SiO2 and CTMS-MNPs@SiO2 materials was obtained (Figure 3B). The bands at 3,400 to 3,500 cm-1 were due to -OH stretching on silanol. The peaks at 2,930 cm-1 were attributed to the stretching vibration of C-H in aromatic and C-H in alkyl . The peaks at 1,400 to 1,550 cm-1 were assigned to the stretching vibration of C-C in an aromatic ring. The presence of additional absorption bands at 800, 950, and 1,110 cm-1 were most likely due to the symmetric and asymmetric stretching vibration of framework and terminal Si-O groups. These all confirm that CTMS has been successfully conjugated on the surface of the MNPs@SiO2.
In summary, a facile and high-efficient one-step immobilization of His-tagged multimeric protein G on the magnetic nanoparticle surface was achieved with two kinds of APTMS and CTMS silanes. Based on the BCA test, the immobilization efficiencies of the His-tagged multimeric protein G with CTMS-MNPs@SiO2 were higher than those of NH2-MNPs@SiO2. The immobilization efficiency with CTMS-MNPs@SiO2 nanoparticles was 99.4 ± 0.015%, 99.2 ± 0.006%, and 98.1 ± 0.005% for the His-tagged monomer, dimer, and trimer of protein G, respectively.
This work was supported by a grant from the Fundamental R&D program and funded by the Korea Institute of Ceramic Engineering and Technology (KICET) and Ministry of Trade, Industry and Energy (MOTIE), Republic of Korea.
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