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Preparation of silica coated cobalt ferrite magnetic nanoparticles for the purification of histidine-tagged proteins
Author’s Accepted Manuscript Preparation of silica coated cobalt ferrite magnetic Nanoparticles for the purification of histidinetagged proteins Gülfem Aygar, Murat Kaya, Necati Özkan, Semra Kocabıyık, Mürvet Volkan www.elsevier.com/locate/jpcs
PII: DOI: Reference:
S0022-3697(15)30036-6 http://dx.doi.org/10.1016/j.jpcs.2015.08.005 PCS7608
To appear in: Journal of Physical and Chemistry of Solids Received date: 26 February 2015 Revised date: 4 August 2015 Accepted date: 9 August 2015 Cite this article as: Gülfem Aygar, Murat Kaya, Necati Özkan, Semra Kocabıyık and Mürvet Volkan, Preparation of silica coated cobalt ferrite magnetic Nanoparticles for the purification of histidine-tagged proteins, Journal of Physical and Chemistry of Solids, http://dx.doi.org/10.1016/j.jpcs.2015.08.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation of Silica Coated Cobalt Ferrite Magnetic Nanoparticles for the Purification of Histidine-Tagged Proteins
Gülfem Aygar1, Murat Kaya2*, Necati Özkan3,4,6 , Semra Kocabıyık5 and Mürvet Volkan1,6,* 1
2
Department of Chemistry, Middle East Technical University, Ankara 06800, Turkey; Department of Chemical Engineering and Applied Chemistry, Atilim University, Ankara
06836, Turkey; 3
Central Laboratory, Middle East Technical University, Ankara 06800, Turkey;
4
Polymer Science and Technology Program, Middle East Technical University, Ankara
06800, Turkey; 5
Department of Biological Sciences, Middle East Technical University, Ankara 06800,
Turkey. 6
Micro and Nanotechnology Program, Middle East Technical University, Ankara 06800,
Turkey;
*
Corresponding Authors:
Prof. Dr. Mürvet Volkan, [email protected], ODTU - Universiteler Mah. Dumlupinar Blv. No:1 Kimya Bölümü 06800, Ankara, TURKEY Tel: +90 312 210 32 28 Assist. Prof. Dr. Murat Kaya, [email protected] Atılım University Department of Chemical Engineering and Applied Chemistry, 06836 İncek, Ankara, TURKEY Tel: +90 312 586 85 61
1
ABSTRACT Surface modified cobalt ferrite (CoFe2O4) nanoparticles containing Ni-NTA affinity group were synthesized and used for the separation of histidine tag proteins from the complex matrices through the use of imidazole side chains of histidine molecules. Firstly, CoFe2O4 nanoparticles with a narrow size distribution were prepared in an aqueous solution using the controlled co-precipitation method. In order to obtain small CoFe2O4 agglomerates, oleic acid and sodium chloride were used as dispersants. The CoFe2O4 particles were coated with silica and subsequently the surface of these silica coated particles (SiO2-CoFe2O4) was modified by amine (NH2) groups in order to add further functional groups on the silica shell. Then, carboxyl (–COOH) functional groups were added to the SiO2-CoFe2O4 magnetic nanoparticles through the NH2 groups. After that Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA) was attached to carboxyl ends of the structure. Finally, the surface modified nanoparticles were labeled with nickel (Ni) (II) ions. Furthermore, the modified SiO2CoFe2O4 magnetic nanoparticles were utilized as a new system that allows purification of the N-terminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3.
Keywords: Magnetic nanoparticles, magnetic separation, surface modification, small heatshock protein, His -tagged protein
2
1.
Introduction
Magnetic nanoparticles, which exhibit magnetic properties that are different from those of their bulk counterparts, have been used successfully for wide range of applications [1] such as targeted drug delivery [2], enzyme and protein separations [3-5], magnetic resonance imaging contrast agent [6], environmental remediation [7] and biomedical and biological applications (e.g., pull down assays) [8-10]. Small particles tend to form agglomerates due to attractive van der Waals forces, therefore, it is necessary to coat magnetic nanoparticles with a protective layer such as polymer [11,], and carbon [7] in order to obtain stable particles. Protective coatings not only stabilize nanoparticles, but can also be used for further functionalization [1]. When the surfaces of magnetic nanoparticles functionalized to create substrate specific surfaces, the targeted molecules can be easily captured. Thus, magnetic separation, which does not need pretreatment operations (such as centrifugation and/or filtration), can be used as a convenient and rapid technique for the separation and purification of biomolecules, such as proteins, nucleic acids, and enzymes [13].
Non-toxic silica is often used as the coating shell to provide magnetic nanoparticles with water dispersion and biocompatibility in most applications. Silica shell can also prevent the direct contact of the magnetic core with the environment thus preventing undesired interactions. In addition, silica-coated magnetic nanoparticles have hydroxyl (–OH) groups on their surfaces, and these groups can be bonded to various functional groups; such as –NH2 and -COOH, which can’t be bound directly to magnetic nanoparticles. With the addition of functional groups onto surfaces of the particles, their use can also be extended to bio labeling, drug targeting and drug delivery. Furthermore, the silica coating of magnetic nanoparticles imparts biocompatibility which is important for biomedical applications because silica is a nontoxic material [9]. Magnetic CoFe2O4 has been synthesized using various techniques and used widely in many applications due to their unique physicochemical properties [14-17]. It is desirable that CoFe2O4 nanoparticles should have a narrow size distribution, high magnetization values, a uniform spherical shape, and superparamagnetic behavior at room temperature for biomedical applications. Many types of synthetic routes have already been employed for the preparation of CoFe2O4 nanoparticles, such as hydrothermal, co-precipitation, microemulsion, forced 3
hydrolysis, and reduction–oxidation route. However, the most important issue to be addressed for the as-prepared nanoparticles is that they are severely agglomerated with poor control of size and shape, which greatly restrict their applications [18].
The aim of this study is the preparation of surface modified CoFe2O4 nanoparticles as a magnetic separation platform for specific isolation of His-tagged proteins. To this end, first CoFe2O4 magnetic nanoparticles were synthesized by using co-precipitation method. Then, the surface of magnetic nanoparticles were coated with silica and modified with Ni-NTA affinity groups. These surface modified CoFe2O4 magnetic nanoparticles were used for purification of the N-terminal His-tagged recombinant small heat shock protein Tpv-sHSP 14.3 from cell extract of the E. coli pQE-31/775.
2. Experimental 2.1 Materials
Iron(III)chloride (FeCl3.9H2O, Riedel-de Haën), Cobalt(II) chloride 6-hydrate (CoCl2.6H2O, Surechem), Sodium hydroxide pellets (NaOH, Sigma-Aldrich), Sodium Chloride (NaCl, Fisher Scientific Company), and Oleic acid ((9Z)-Octadec-9-enoic acid, Fluka) were used for the preparation of CoFe2O4 magnetic nanoparticles. In order to prepare silica coating on CoFe2O4 nanoparticles, Tetraethyl orthosilicate (TEOS, C8H20O4Si, 98%, Aldrich),
(3-
Aminopropyl)trimethoxysilane (APTMS, H2N(CH2)3Si(OCH3)3, ≥ 98.0%, Aldrich), ands Ethanol (EtOH, C2H5OH, ≥ 99.9%, Merck) were used. For the functionalization of CoFe2O4 nanoparticles with –NH2 groups, (3-Aminopropyl) triethoxysilane (APTES, C9H23NO3Si, ≥ 98.0%, Fluka), toluene (C6H5CH3, ≥ 99.0%, Merck), and N,N- Dimethylformamide (DMF, C3H7NO, ≥ 99.8%, Sigma-Aldrich) were used. The attachment of –COOH group on the nanoparticles were done by using toluene (C6H5CH3, ≥ 99.0%, Merck), N,NDimethylformamide ( DMF, C3H7NO, ≥ 99.8%, Sigma-Aldrich), and Succinic (glutaric) anhydride (C4H4O3, ≥ 99%, Sigma- Aldrich). Surface modification of CoFe2O4 nanoparticles with NTA was performed with Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA, C10H18N2O6, ≥ 97.0%, Fluka) and 2-(4-Morpholino) ethanesulfonic acid Potassium salt (MES K salt, Sigma). For adding Ni (II) ions on the surface modified CoFe2O4 nanoparticles, Nickel
4
solution (Nickel (II) ion, 1000ppm ± 0.5%, Fisher Scientific International Company) was used.
2.2 Instruments Field Emission Scanning Electron Microscopy (FE-SEM) with energy-dispersive X-ray analyzer (EDX) (FE-SEM, Quanta 400F, FEI) and Transmission Electron Microscopy (TEM, JEOL JEM-2010F (FEG, 80-200 kV) were used for morphological and chemical characterization of CoFe2O4 and SiO2-CoFe2O4 nanoparticles. For the FE-SEM and TEM measurements, the magnetic nanoparticle suspensions were dropped on carbon-coated copper grids and then they were dried at room temperature overnight. Number-length (arithmetic) mean size (D [1,0]) and volume weighted mean size ( D[4,3]) of CoFe2O4 and SiO2-CoFe2O4 nanoparticles were determined from FE-SEM images using the following equations:
D1,0
di Ni Ni
(1)
d i4 N i D4,3 d i3 N i
(2)
Where di is the diameter of primary particles and Ni is the number of primary particles. Saturation magnetization of the CoFe2O4 magnetic nanoparticles was measured using a Vibrating Sample Magnetometer (ADE Magnetics Model EV9) at room temperature. Fourier Transform Infrared Spectroscopy (FTIR) (Alpha, Bruker) was used for characterization of nanoparticles in the range of 300 to 4500 cm-1. Determination of Ni, Cu and Ag ions attached on the surface modified CoFe2O4 nanoparticles were performed using an inductively coupled plasma spectrometer (ICP-OES, Direct Reading Echelle, Leeman Labs INC.). Particle size and distribution of the CoFe2O4 and SiO2-CoFe2O4 agglomerates were determined using the dynamic light scattering method (Malvern Nano ZS90). 2.3 Methods 2.3.1 Preparation of Cobalt Ferrite Nanoparticles CoFe2O4 nanoparticles were synthesized by the co-precipitation method [14]. According to this procedure, after mixing 0.54 g FeCl3.6H2O, 0.238 g CoCl2.6H2O and 10 mL deionized 5
water in a test tube, an orange colored solution was obtained and it was put in a 50mL beaker. The solution was then transferred into a beaker and 10mL 3M NaOH was added drop wise with Pasteur pipette by continuous stirring by using a magnetic stirrer to obtain a black colloidal suspension. 200µl oleic acid was added to the black colloidal suspension and the stirring was continued for 1 hour at 80 oC. Then the suspension was cooled to room temperature, the black precipitates (CoFe2O4 nanoparticles) were collected with a magnet and the supernatant was discarded. The CoFe2O4 nanoparticles were washed three times with deionized water –ethanol solution. The black precipitate was dried overnight at 100 oC, and then heated to 600 oC for 10 hours to remove residual water. The CoFe2O4 nanoparticles were redispersed in deionized water and stored in 15mL plastic tubes. Alternatively, sodium chloride (NaCl) was used as a dispersant. In this case, 5mL 1.5M NaCl solution was added to the orange solution together with 10mL 3M NaOH. Following the same protocol, the stirring was carried out using an ultrasonic bath instead of the magnetic stirrer to see the effect of ultrasound application on the size of CoFe2O4 nanoparticles and their agglomerates. 2.3.2 Silica Coating of Cobalt Ferrite Magnetic Nanoparticles The CoFe2O4 magnetic nanoparticles were coated with a silica shell using a sol-gel method [19]. First, 80 mL ethanol, 169 µL TEOS and 14.4 µL APTMS were mixed in a beaker and subsequently 20 mL CoFe2O4 colloidal suspension was added to the mixture and stirred for 3 hours at room temperature. Subsequently, the silica coated cobalt ferrite (SiO2-CoFe2O4) particles were collected with a permanent magnet and washed three times with deionized water. 2.3.3 Functionalization of Silica Coated Cobalt Ferrite Nanoparticles with Amine Groups After coating the CoFe2O4 nanoparticles with a silica shell, the surfaces of SiO2-CoFe2O4 nanoparticles were modified with amine groups using APTES [9]. For the coating process, 12 mL DMF and 8 mL toluene were vortexed in a 50 mL tube and this solution was put in a 50 mL beaker, and subsequently 200 µL APTES was added drop by drop into this mixture under magnetic stirring for 24 hour. Then, the functionalized particles were collected with the magnet and washed with toluene. Finally these particles were re-dispersed in 10 mL DMF. 2.3.4 Adding –COOH Functional Groups to Amine Modified Silica Coated Cobalt Ferrite Nanoparticles 6
After binding of amine group onto the SiO2-CoFe2O4 nanoparticles, carboxyl groups were added by using ring opening linker elongation reaction [20].
In this process glutaric
anhydride ring structure was opened and reacted with the amine groups. The SiO2-CoFe2O4 nanoparticles which were modified with –NH2 groups were dispersed in 10 mL DMF to obtain a homogeneous colloidal suspension. This suspension was added drop by drop to the 10 mL DMF solution containing 0.1 g glutaric anhydride and the mixture was stirred for 24 hour at room temperature. The surface modified particles were washed with DMF and kept for further use. 2.3.5 Surface Modification of Silica Coated Cobalt Ferrite Nanoparticles with NTA 5.8325g MES K salt and 7.305g NaCl were dissolved in 250 mL volumetric flask. The pH of the solution was adjusted to 6 with concentrated HCl. 7.0 mL of this solution was taken and mixed with 10 mL of -COOH functionalized SiO2-CoFe2O4 particle suspension. 0.0787g NTA was added to the suspension and the mixture was vortexed about 1 minute. The suspension was put in a 50 mL beaker and stirred for 2 hours. The surface modified SiO2CoFe2O4 particles were collected with the magnet and supernatant was removed. The collected particles were washed with deionized water. 2.3.6 Adding Ni (II) Ions to the NTA modified Silica Coated Cobalt Ferrite Magnetic Nanoparticles 20 mg/L Ni (II) ion solution was added to the NTA modified SiO2-CoFe2O4 nanoparticles by gentle mixing on a shaker platform for 90 min. The nanoparticles were collected using the external magnet and the supernatant solution was taken. The particles were washed with 15 mL deionized water and all washing solutions were kept together with supernatant solution for ICP-OES measurements to determine the amount of Ni (II) ions added on to the NTA modified SiO2-CoFe2O4 nanoparticles. 2.3.7 Preparation of E.coli pQE-31/775 cell-free extract The cell extract was prepared from the recombinant E.coli pQE-31/775 cells which expressed the small heat shock protein, tpv-HSP 14.3 from thermoacidophilic archaeon Thermoplasma volcanium, as an N-terminal 6X-His tag fusion as described earlier [21]. The cleared cell lysate was obtained by sonication of the cells containing 20 mM imidazole and 1 mg/mL 7
lysozyme, followed
by centrifugation at 10,000xg at 4 ºC for 30 min (Sigma 3K30
Centrifuge, Sigma Chemical Co., St. Louis, USA). The supernatant called “cell-free extract” was stored at -20°C until use.
2.3.8 Purification of the 6xHis tagged tpv-HSP 14.3 protein using Surface Modified Silica Coated Cobalt Ferrite Nanoparticles After thawing cell-free extract on ice a certain volume (3 mL, 0.6 mg/mL protein) was added to surface modified SiO2-CoFe2O4 nanoparticles (0.6 gm) in a glass test tube which was mixed gently in the slanted position by shaking at room temperature for 60 min. Then, the supernatant was separated from the magnetic beads by the aid of a magnet. The supernatant was completely removed and the nanoparticles were washed twice in a phospahate buffer (50 mM NaH2PO4, 300 mM NaCl , pH 8.0) to remove unbound proteins (3mL buffer was used for each washing). The bound His-tagged sHSP was eluted from the nanoparticles with 1.5 mL phosphate buffer containing 250 mM imidazole. The eluate and all other fractions were stored at -20 ºC for SDS-PAGE analysis.
2.3.9 SDS Gel Electrophoresis SDS-polyacrylamide gel electrophoresis (PAGE, 12.5 % acrylamide slab gel, 1 mm thick) was performed by the procedure of Laemmli [22]. Flowthrough, wash samples and eluted protein were mixed with 2x sample buffer and after boiling subjected to SDS-PAGE. PageRuler™ Prestained Protein Ladder, (Fermentas AB, Vilnius, Lithuania) was used as molecular weight standard. The protein bands were visualized by staining with Page Blue Protein Staining Solution (Fermentas AB, Vilnius, Lithuania).
3. Results and discussion 3.1 Morphology and Size Distibution of Cobalt Ferrite Magnetic Nanoparticles Cobalt ferrite (CoFe2O4) nanoparticles were preferred as magnetic core materials due to the easy preparation procedure.
Unlike magnetite (Fe3O4), there is no need to use inert
atmosphere or organic additives to produce CoFe2O4 nanoparticles with a narrow particle size distribution [23-25]. 8
The morphology and size distribution of the primary CoFe2O4 particles were studied using FE-SEM and TEM. Various production routes used for the preparation of CoFe2O4 particles were summarized in Table 1. Representative FE-SEM and TEM images and Energy Dispersive X-ray (EDX) pattern of the CoFe2O4 nanoparticles (for Sample 2) are shown in Figure 1. Similar FE-SEM images were also obtained for other samples (Sample 1 and Sample 3).
Figure 1. A) FE-SEM and B) TEM images and C) corresponding EDX result of CoFe2O4 nanoparticles prepared using sodium chloride as the dispersant through magnetic stirring (Sample 2).
From the SEM images, the diameters of approximately 100 primary particles were measured and the number-length (arithmetic) mean size (D[1,0]) and volume weight mean size (D[4,3]) of the primary CoFe2O4 nanoparticles were calculated. Polydispersivity index (PDI), which 9
gives information about particle size homogeneity, was calculated using the following equation.
PDI PP
D4,3 D1,0
(3)
It should be noted that the PDI calculated using Equation (3) was different from the PDI obtained from the Dynamic Light scattering technique. The mean particle sizes and PDIpp values for the primary CoFe2O4 particles prepared using various preparation routes are given in Table (1). Table 1. Mean particle sizes of the primary CoFe2O4 particles prepared using various preparation routes. Sample
Mixing
Dispersant
D[4,3] (nm) D[1,0] (nm) PDIPP*
Sample 1
Magnetic Stirring
Oleic Acid
25.1
27.4
1.1
Sample 2
Magnetic Stirring
NaCl
12.9
14.1
1.1
Sample 3
Ultrasonic
NaCl
nmnm14.06 13.9
15.1
1.1
*PDIPP refers to the polydispersivity index for the primary CoFe2O4 nanoparticles determined using the SEM images. As can be seen from Table 1, when NaCl was used as a dispersant, the primary particle size of the CoFe2O4 particles was reduced. Additionally, in the presence of NaCl, the application of the ultrasonic agitation during the preparation of CoFe2O4 particles did not influence the size of the primary CoFe2O4 particles. The special properties of magnetic nanoparticles required for biomedical applications demand precise control of particle size, dispersion and conditions that affect these properties. In principle, it is necessary to stabilize the magnetic nanoparticle dispersion in the aqueous environment. The Dynamic Light Scattering (DLS) technique was used to measure z-average mean size and Polydispersity Index (PDIA) of CoFe2O4 agglomerates. Z-average mean size is defined as the harmonic intensity averaged particle diameter. The PDI values smaller than 0.05 indicate highly monodisperse particles. The values of PDI greater than 0.7 indicate that the sample has a very broad size distribution and is probably not suitable for the DLS technique. By applying deconvolution algoritm, it is possible to obtain
particle size
distribution data from the DLS measurement. From the particle size distribution data, the 10
volume weighted mean size (D[4,3]) and surface area weighted mean size (D[3,2]) of the CoFe2O4 agglomerates was calculated from the following equation.
d im 3Vi Dm, n m 3 d i Vi
1
( mn)
(4)
Where di is the agglomerate size and Vi is the volume of agglomerates. Specific surface area of the CoFe2O4 agglomerates can be calculated using the following equation. The unit of the specific surface area can be either m2/g or m2/cm3.
SV
6 D3,2
(5)
The typical agglomerate size distribution of the CoFe2O4 agglomerates (Sample 1) measured
Volume, %
using the dynamic light scattering technique was given in Figure 2.
Size, nm Figure 2. The agglomerate size distribution of the CoFe2O4 nanoparticles
The mean particle sizes and PDIA values of the CoFe2O4 agglomerates prepared using various preparation routes are given in Table 2.
11
Table 2. Mean particle sizes of the CoFe2O4 agglomerates prepared using various preparation routes Sample
Mixing
Dispersant
Z-average
PDIA
mean size
D[4,3]
SV
(nm)
(m2/cm3)
(nm) Sample
Magnetic
Oleic Acid
147.4
0.042
139.7
47.4
1Sample
Stirring Magnetic
NaCl
127.4
0.250
102.7
65.5
2 Sample
Stirring Ultrasonic
NaCl
128.7
0.151
97.3
59.9
3
Bath
As can be seen from Tables 1 and 2, well dispersed smaller CoFe2O4 primary particles and their agglomerates were obtained using the salt-assisted solid state method in the ultrasonic bath (Sample 3) and this procedure was used throughout the study. 3.2 Silica Coating of Magnetic Cobalt Ferrite Nanoparticles CoFe2O4 nanoparticles were coated with silica in aqueous/ethanolic solution via Stöber method [19]. TEOS and APTMS were used for silica coating of the CoFe2O4 nanoparticles. Using small amount of APTMS in addition to the TEOS was very important since better silica coating was obtained compared to the procedure in which only TEOS was used. TEM image and Energy dispersive X-ray (EDX) analysis for the SiO2-CoFe2O4 nanoparticles are shown in Figure 3. The carbon signal in the EDX pattern was coming from the carbon tape used for sampling. The presence of silica peak on the spectrum was considered as the indication of silica layer formation on the surface of CoFe2O4 nanoparticles.
12
Figure 3. TEM image and EDX result of the SiO2-CoFe2O4 magnetic nanoparticles. As discussed previously the cobalt ferrite particles form agglomerates with Z-average mean size of about 128 nm, volume weighted mean size of about 97 nm, and the PDIA of 0.15. The introduction of silane into the aqueous suspension of these cobalt ferrite nanoparticles did not cause any positive influence in their dispersion. Probably agglomerates were coated with silica and subsequently an increase in the diameters of the silica coated cobalt ferrite aggregates was observed. The Z-average mean size, volume weighted mean size and the PDI of SiO2-CoFe2O4 agglomerates at pH ~7.0 were determined as 267 nm, 325 nm and 0.11 respectively. The specific surface area of SiO2-CoFe2O4 agglomerates was calculated as 21.1 m2/cm3. Further modifications such as anchoring carboxylic acid and NTA following the silica coating did not influence the agglomerate size and size distribution of the particles. 3.3 Magnetic Behavior of the Cobalt Ferrite Magnetic Particles Magnetic behavior of the CoFe2O4 particles prepared using the ultrasonic bath was tested by their collection under the influence of external magnetic field and the results are shown in Figure 4.
13
Figure 4. Magnetic behavior of the CoFe2O4 nanoparticles after the application of external magnetic field (1.6T).
As can be seen from Figure 4, the collection of the magnetic CoFe2O4 particles after the application of external magnetic field (1.6T) was completed after 60 seconds. Similar observation was noted for the SiO2-CoFe2O4 particles. The collection of the SiO2-CoFe2O4 particles was as rapid as that of the CoFe2O4 nanoparticles. The magnetic properties of the uncoated and silica coated CoFe2O4 particles were also characterized using the Vibrating Sample Magnetometer (VSM). The hysteresis curves for these particles recorded at 300K were given in Figure 5 (a) and (b). The magnetization values of the uncoated and silica coated CoFe2O4 particles at 10 kOe were measured as 46.2 emu/g and 44.7 emu/g, respectively. The coercivity values of the uncoated and silica coated CoFe2O4 particles were measured as 147.0 Oe and 202.5 Oe, respectively. There was only a slight decrease in the magnetization values at 10 kOe for the SiO2-CoFe2O4 particles compared to the uncoated ones due to nonmagnetic silica coating.
14
50
[a]
40
Magnetization (Am2/kg, emu/g)
Magnetization (Am2/kg, emu/g)
50 30 20
10
20
0
10
-10
0
-20
-10
-30
-20 -0,04
-40
0,00
0,04
-50 -3
-2
-1
0
1
2
3
[b]
40 30 20 10 0
20
-10
10
-20
0
-30
-10
-40
-20 -0,04
-50 -3
-2
-1
0
0,00
1
0,04
2
Magnetic Field (T, 10 kOe)
Magnetic Field (T, 10 kOe)
Figure 5. The magnetic hysteresis curves for [a]the uncoated and [b] SiO2-CoFe2O4 particles recorded at 300K.
3.4 Surface Modification of Silica Coated Cobalt Ferrite Nanoparticles
Besides improving the stability of the particles, silica coating of the CoFe2O4 nanoparticles also provides a platform for surface modification with various specific functional groups. By attaching functional amine groups onto the surface of the SiO2-CoFe2O4 particles, additional functional groups and biological molecules could easily be attached to the surface of these particles. After addition of amine group on the silica coating of CoFe2O4 nanoparticle, carboxyl groups were added by using ring opening linker elongation reaction [20]. In this process glutaric anhydride ring structure was opened and reacted with the amine groups. FTIR spectrometer was used for the characterization of the carboxyl groups introduced on the surface of SiO2-CoFe2O4 nanoparticles (Figure 6).
15
3
3395
1700
2870 2925
1560 1635
1400
890
1035 1135 1205
Absorbance (A) 500
1000
1500
2000
2500
3000
3500
4000
Wavenumber (cm-1)
Figure 6. FTIR results of–COOH functionalized SiO2-CoFe2O4 magnetic nanoparticles. The absorption bands at about 3395 and 1635 cm−1 in all the spectra mainly originate from the OH vibrations in H2O [9]. The spectrum of carboxyl functional groups on the modified SiO2CoFe2O4 nanoparticles contains stretching peaks around 1035 cm-1 due to the siloxane,–Si– O–Si– and silanol, Si–O groups and two small peaks (2870 and 2925 cm-1) between 2990– 2830 cm-1 for aliphatic C–H stretching vibrations of alkyl chains in APTES precursor ligand [9, 26-28]. The existance of siloxane and silanol groups that are typical groups found on the surface of CoFe2O4 nanoparticles was the proof of silica coating. Whereas the stretching vibrations corresponding to the alkyl chains are the indications of the incorporation of APTES onto silica surface through silanization. Carboxylic acid, –COOH , group was bonded to the surface through the reaction of –NH2 and glutaric anhydride, where one of the carboxylic acid group of the anhydride was converted into amide and the other one was left as a terminal –COOH group. Amides formed from primary amines are distinguished by their characteristic Amide I and Amide II bands. Amide I band is mainly attributed to the carbonyl stretching and Amide II involves contribution of several atoms including the N-H bond. The observed Amide I and Amide II bands (peaks around 1700 and 1560 cm-1) in the FTIR spectra suggest that –COOH groups were bonded to the surface through–NH2 groups. The band between 1460-1365 cm-1(around 1400 cm-1) together with the band around 890 cm-1 can be attributed to the O-H bending vibration of carboxylic acid. Although the 1460-1365 cm-1 band cannot be distinguished from C-N 16
stretching band of amide (1400 cm-1) in the same region, it may also support the presence of carboxylic acid on the surface of the SiO2-CoFe2O4 nanoparticles. The peak at 1635 cm-1 is the result of deformational vibrations of adsorbed water molecules as suggested by others [9, 26-29]
The surface modification of the SiO2-CoFe2O4 nanoparticles by amine and carboxyl groups was followed by the addition of NTA (Nα,Nα-Bis(carboxymetr56hyl)-L-lysine hydrate) groups onto the nanoparticles. NTA was covalently bound to the carboxylic acid end group of −COOH modified SiO2-CoFe2O4 particles. The surface modification steps and resulting modified SiO2-CoFe2O4 particles are shown in Figure 7.
O O Si O
APTES in DMF and toluene CoFe2O4 agglomerate coated with silica
O O Si O
NH
(CH2)3NH2
Glutaric anhydride in DMF and toluene
O O Si O
(CH2)3NHCO(CH2)3COOH
NTA attachment
Attachment of Ni (II) Ions
O N
-COOH functionalized CoFe2O4 particles
-NH2 functionalized CoFe2O4 particles
CoFe2O4 nanoparticles
O NH
NH
R1
R1
Ni+2
Figure 7. The surface modification steps and resulting modified SiO2-CoFe2O4 nanoparticles. NTA modified nanoparticles were characterized by FTIR. The FTIR spectra of NTA modified SiO2-CoFe2O4 were shown in Figure 8.
17
2915 1000
1410 1540 1635 1730
1105
Absorbance (A) 500
1500
2000
2500
3000
3500
4000
Wavenumber (cm-1)
Figure 8. FTIR results of NTA modified SiO2-CoFe2O4 nanoparticles. After modification of the surface with NTA, carboxylic acid group vibrations become predominant in the FTIR spectrum (Figure 8). The asymmetric vibration band of the deprotonated carboxylic group is located around 1410 cm-1, whereas asymmetric vibrational band of the same group is located at 1635 cm-1. The band at 1730 cm-1 was assigned to the protonated –COOH group of NTA cm-1[30-32].
As the final modification, the SiO2-CoFe2O4 nanoparticles functionalized with NTA were loaded with Ni (II) ions. The quadridentate NTA moiety covalently coupled to the surface of SiO2-CoFe2O4 nanoparticles via spacer butyl amine arm. The remaining four chelating sites of the modified NTA interact with nickel (II), which result in a tight binding of ions. Quantification of Ni (II) binding was done using an inductively coupled plasma spectrometer (ICP-OES). The concentrations of the Ni (II) ions in the loading solution prior to the addition of the magnetic nanoparticles and in the supernatant solution after removing the magnetic nanoparticles were measured by the ICP-OES. From the ICP-OES measurements it was found that 6.15mg/L Ni (II) ions were bonded to 0.0148g surface modified SiO2-CoFe2O4 nanoparticles. This result also revealed the existence of NTA on the surface of SiO2-CoFe2O4 nanoparticles. After immobilization of the Ni-NTA affinity group, the remaining two free sites of Ni (II) ions are ready to bind histidine tag proteins through the use of imidazole side chains of histidine molecules. Since the SiO2-CoFe2O4 nanoparticles retained their magnetic properties, they can be effectively used in the magnetic separation of his-tagged proteins, as explained below. 18
3.5 Purification of 6XHis-tagged Tpv/sHSP-14.3 using SiO2-CoFe2O4 nanoparticles To evaluate the surface modified SiO2-CoFe2O4 magnetic nanopaticles as an efficient platform for protein purification, the isolation of N-terminal His-tagged recombinant small heat shock protein (Tpv-sHSP 14.3) from E. coli cell extract was studied. Small heat-shock proteins are molecular chaperones which are responsible for binding of improperly folded protein-substrates [33-34] and prevent improper polypeptide associations and accumulation of aggregated proteins in the cell [35]. In this way, they further transfer entrapped unfolded proteins to the ATP dependent chaperones or to the protein degradation machines like proteasomes or autophagosomes [33, 6]. The sHSP’s chaperone activity is therefore crucial to cells’s tolerance to stress. In the main, they are dramatically up-regulated under conditions of cellular stress to being among the most abundant of all proteins [37,38]. Recent investigations indicate that sHSPs participate in regulation of many vital cellular processes, including apoptosis, cytoskeleton and carcinogenesis and have pronounced cardioprotective activity [39,40]. Furthermore, mutations of certain members of sHSP family lead to cell dysfunctions that correlate with development of different congenital diseases including certain neurodegenerative disorders [41-43]. Together these observations suggest that sHSps are on the front line of attractive therapeutic targets. Thus, development of such a convenient purification system would be useful for preparation protein drugs such as pharmachaperones. The purification protocol applied in this study yielded highly purified his-tagged recombinant small heat shock protein as revealed by SDS-PAGE (Figure 9). The purified sample form the E.coli cell extract produced a protein band of about 14.5 kDa on the gel, which corresponds to the protein molecular weight deduced from the predicted amino acid sequence. The Ni (II) ion loaded SiO2-CoFe2O4 nanoparticles system developed in this study can be convenient and useful tool for biological and clinical research and applications.
19
M
1
2
3
4
Figure 9. SDS-PAGE analysis of His-tagged sHSP form E.coli extract. Lanes: 1: Crude protein, 2: Wash sample, 3: 2nd elution, 4: 1 st elution, M. Prestained Protein Ladder, (170-10 kDa, Fermentas); Arrow indicates bands of sHSP proteins
4. Conclusion CoFe2O4 magnetic nanoparticles with a narrow particle size distribution were synthesized by using the co-precipitation technique without using any surfactant. The sizes of CoFe2O4 agglomerates, which were synthesized under the control of magnetic stirring and ultrasonic agitation by using different dispersing agents, were characterized in detail. By combining the SiO2-CoFe2O4 magnetic nanoparticles with the specific affinity offered by Ni-NTA metalchelate functional group, it has been demonstrated for the first time that the Ni-NTA modified SiO2-CoFe2O4 can isolate histidine-tagged proteins directly from the crude samples without any purification steps. NTA-attachment was done on the surfaces of SiO2-CoFe2O4 particles using carboxylic acid as a robust and cost-effective anchor instead of expensive reagents (like EDC/NHS couple) with long procedure. The new purification system developed has several merits that may not be found in other protein purification systems. There is no need for specialized binding buffers and substrates for protein elution. All of the purification steps can be easily and quickly performed by using a magnet instead of a centrifuge. The prepared magnetic materials show excellent performance in the separation of a N-terminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3. The results obtained in this study 20
suggest that the magnetic materials with different surface functionalities may also find applications in a wide variety of biological applications.
Acknowledgements The authors thank Central Laboratory at METU for FE-SEM, TEM and particle size measurements.
References
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Research highlights Novel magnetic nanoplatform was applied for the first time in the purification of Nterminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3. successfully. NTA-attachment with robust and cost-effective anchor with easy procedure. Detailed study of the size of the agglomerates under the control of magnetic stirring and ultrasonic agitation by using different dispersing agents.
24
PII: DOI: Reference:
S0022-3697(15)30036-6 http://dx.doi.org/10.1016/j.jpcs.2015.08.005 PCS7608
To appear in: Journal of Physical and Chemistry of Solids Received date: 26 February 2015 Revised date: 4 August 2015 Accepted date: 9 August 2015 Cite this article as: Gülfem Aygar, Murat Kaya, Necati Özkan, Semra Kocabıyık and Mürvet Volkan, Preparation of silica coated cobalt ferrite magnetic Nanoparticles for the purification of histidine-tagged proteins, Journal of Physical and Chemistry of Solids, http://dx.doi.org/10.1016/j.jpcs.2015.08.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation of Silica Coated Cobalt Ferrite Magnetic Nanoparticles for the Purification of Histidine-Tagged Proteins
Gülfem Aygar1, Murat Kaya2*, Necati Özkan3,4,6 , Semra Kocabıyık5 and Mürvet Volkan1,6,* 1
2
Department of Chemistry, Middle East Technical University, Ankara 06800, Turkey; Department of Chemical Engineering and Applied Chemistry, Atilim University, Ankara
06836, Turkey; 3
Central Laboratory, Middle East Technical University, Ankara 06800, Turkey;
4
Polymer Science and Technology Program, Middle East Technical University, Ankara
06800, Turkey; 5
Department of Biological Sciences, Middle East Technical University, Ankara 06800,
Turkey. 6
Micro and Nanotechnology Program, Middle East Technical University, Ankara 06800,
Turkey;
*
Corresponding Authors:
Prof. Dr. Mürvet Volkan, [email protected], ODTU - Universiteler Mah. Dumlupinar Blv. No:1 Kimya Bölümü 06800, Ankara, TURKEY Tel: +90 312 210 32 28 Assist. Prof. Dr. Murat Kaya, [email protected] Atılım University Department of Chemical Engineering and Applied Chemistry, 06836 İncek, Ankara, TURKEY Tel: +90 312 586 85 61
1
ABSTRACT Surface modified cobalt ferrite (CoFe2O4) nanoparticles containing Ni-NTA affinity group were synthesized and used for the separation of histidine tag proteins from the complex matrices through the use of imidazole side chains of histidine molecules. Firstly, CoFe2O4 nanoparticles with a narrow size distribution were prepared in an aqueous solution using the controlled co-precipitation method. In order to obtain small CoFe2O4 agglomerates, oleic acid and sodium chloride were used as dispersants. The CoFe2O4 particles were coated with silica and subsequently the surface of these silica coated particles (SiO2-CoFe2O4) was modified by amine (NH2) groups in order to add further functional groups on the silica shell. Then, carboxyl (–COOH) functional groups were added to the SiO2-CoFe2O4 magnetic nanoparticles through the NH2 groups. After that Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA) was attached to carboxyl ends of the structure. Finally, the surface modified nanoparticles were labeled with nickel (Ni) (II) ions. Furthermore, the modified SiO2CoFe2O4 magnetic nanoparticles were utilized as a new system that allows purification of the N-terminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3.
Keywords: Magnetic nanoparticles, magnetic separation, surface modification, small heatshock protein, His -tagged protein
2
1.
Introduction
Magnetic nanoparticles, which exhibit magnetic properties that are different from those of their bulk counterparts, have been used successfully for wide range of applications [1] such as targeted drug delivery [2], enzyme and protein separations [3-5], magnetic resonance imaging contrast agent [6], environmental remediation [7] and biomedical and biological applications (e.g., pull down assays) [8-10]. Small particles tend to form agglomerates due to attractive van der Waals forces, therefore, it is necessary to coat magnetic nanoparticles with a protective layer such as polymer [11,], and carbon [7] in order to obtain stable particles. Protective coatings not only stabilize nanoparticles, but can also be used for further functionalization [1]. When the surfaces of magnetic nanoparticles functionalized to create substrate specific surfaces, the targeted molecules can be easily captured. Thus, magnetic separation, which does not need pretreatment operations (such as centrifugation and/or filtration), can be used as a convenient and rapid technique for the separation and purification of biomolecules, such as proteins, nucleic acids, and enzymes [13].
Non-toxic silica is often used as the coating shell to provide magnetic nanoparticles with water dispersion and biocompatibility in most applications. Silica shell can also prevent the direct contact of the magnetic core with the environment thus preventing undesired interactions. In addition, silica-coated magnetic nanoparticles have hydroxyl (–OH) groups on their surfaces, and these groups can be bonded to various functional groups; such as –NH2 and -COOH, which can’t be bound directly to magnetic nanoparticles. With the addition of functional groups onto surfaces of the particles, their use can also be extended to bio labeling, drug targeting and drug delivery. Furthermore, the silica coating of magnetic nanoparticles imparts biocompatibility which is important for biomedical applications because silica is a nontoxic material [9]. Magnetic CoFe2O4 has been synthesized using various techniques and used widely in many applications due to their unique physicochemical properties [14-17]. It is desirable that CoFe2O4 nanoparticles should have a narrow size distribution, high magnetization values, a uniform spherical shape, and superparamagnetic behavior at room temperature for biomedical applications. Many types of synthetic routes have already been employed for the preparation of CoFe2O4 nanoparticles, such as hydrothermal, co-precipitation, microemulsion, forced 3
hydrolysis, and reduction–oxidation route. However, the most important issue to be addressed for the as-prepared nanoparticles is that they are severely agglomerated with poor control of size and shape, which greatly restrict their applications [18].
The aim of this study is the preparation of surface modified CoFe2O4 nanoparticles as a magnetic separation platform for specific isolation of His-tagged proteins. To this end, first CoFe2O4 magnetic nanoparticles were synthesized by using co-precipitation method. Then, the surface of magnetic nanoparticles were coated with silica and modified with Ni-NTA affinity groups. These surface modified CoFe2O4 magnetic nanoparticles were used for purification of the N-terminal His-tagged recombinant small heat shock protein Tpv-sHSP 14.3 from cell extract of the E. coli pQE-31/775.
2. Experimental 2.1 Materials
Iron(III)chloride (FeCl3.9H2O, Riedel-de Haën), Cobalt(II) chloride 6-hydrate (CoCl2.6H2O, Surechem), Sodium hydroxide pellets (NaOH, Sigma-Aldrich), Sodium Chloride (NaCl, Fisher Scientific Company), and Oleic acid ((9Z)-Octadec-9-enoic acid, Fluka) were used for the preparation of CoFe2O4 magnetic nanoparticles. In order to prepare silica coating on CoFe2O4 nanoparticles, Tetraethyl orthosilicate (TEOS, C8H20O4Si, 98%, Aldrich),
(3-
Aminopropyl)trimethoxysilane (APTMS, H2N(CH2)3Si(OCH3)3, ≥ 98.0%, Aldrich), ands Ethanol (EtOH, C2H5OH, ≥ 99.9%, Merck) were used. For the functionalization of CoFe2O4 nanoparticles with –NH2 groups, (3-Aminopropyl) triethoxysilane (APTES, C9H23NO3Si, ≥ 98.0%, Fluka), toluene (C6H5CH3, ≥ 99.0%, Merck), and N,N- Dimethylformamide (DMF, C3H7NO, ≥ 99.8%, Sigma-Aldrich) were used. The attachment of –COOH group on the nanoparticles were done by using toluene (C6H5CH3, ≥ 99.0%, Merck), N,NDimethylformamide ( DMF, C3H7NO, ≥ 99.8%, Sigma-Aldrich), and Succinic (glutaric) anhydride (C4H4O3, ≥ 99%, Sigma- Aldrich). Surface modification of CoFe2O4 nanoparticles with NTA was performed with Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA, C10H18N2O6, ≥ 97.0%, Fluka) and 2-(4-Morpholino) ethanesulfonic acid Potassium salt (MES K salt, Sigma). For adding Ni (II) ions on the surface modified CoFe2O4 nanoparticles, Nickel
4
solution (Nickel (II) ion, 1000ppm ± 0.5%, Fisher Scientific International Company) was used.
2.2 Instruments Field Emission Scanning Electron Microscopy (FE-SEM) with energy-dispersive X-ray analyzer (EDX) (FE-SEM, Quanta 400F, FEI) and Transmission Electron Microscopy (TEM, JEOL JEM-2010F (FEG, 80-200 kV) were used for morphological and chemical characterization of CoFe2O4 and SiO2-CoFe2O4 nanoparticles. For the FE-SEM and TEM measurements, the magnetic nanoparticle suspensions were dropped on carbon-coated copper grids and then they were dried at room temperature overnight. Number-length (arithmetic) mean size (D [1,0]) and volume weighted mean size ( D[4,3]) of CoFe2O4 and SiO2-CoFe2O4 nanoparticles were determined from FE-SEM images using the following equations:
D1,0
di Ni Ni
(1)
d i4 N i D4,3 d i3 N i
(2)
Where di is the diameter of primary particles and Ni is the number of primary particles. Saturation magnetization of the CoFe2O4 magnetic nanoparticles was measured using a Vibrating Sample Magnetometer (ADE Magnetics Model EV9) at room temperature. Fourier Transform Infrared Spectroscopy (FTIR) (Alpha, Bruker) was used for characterization of nanoparticles in the range of 300 to 4500 cm-1. Determination of Ni, Cu and Ag ions attached on the surface modified CoFe2O4 nanoparticles were performed using an inductively coupled plasma spectrometer (ICP-OES, Direct Reading Echelle, Leeman Labs INC.). Particle size and distribution of the CoFe2O4 and SiO2-CoFe2O4 agglomerates were determined using the dynamic light scattering method (Malvern Nano ZS90). 2.3 Methods 2.3.1 Preparation of Cobalt Ferrite Nanoparticles CoFe2O4 nanoparticles were synthesized by the co-precipitation method [14]. According to this procedure, after mixing 0.54 g FeCl3.6H2O, 0.238 g CoCl2.6H2O and 10 mL deionized 5
water in a test tube, an orange colored solution was obtained and it was put in a 50mL beaker. The solution was then transferred into a beaker and 10mL 3M NaOH was added drop wise with Pasteur pipette by continuous stirring by using a magnetic stirrer to obtain a black colloidal suspension. 200µl oleic acid was added to the black colloidal suspension and the stirring was continued for 1 hour at 80 oC. Then the suspension was cooled to room temperature, the black precipitates (CoFe2O4 nanoparticles) were collected with a magnet and the supernatant was discarded. The CoFe2O4 nanoparticles were washed three times with deionized water –ethanol solution. The black precipitate was dried overnight at 100 oC, and then heated to 600 oC for 10 hours to remove residual water. The CoFe2O4 nanoparticles were redispersed in deionized water and stored in 15mL plastic tubes. Alternatively, sodium chloride (NaCl) was used as a dispersant. In this case, 5mL 1.5M NaCl solution was added to the orange solution together with 10mL 3M NaOH. Following the same protocol, the stirring was carried out using an ultrasonic bath instead of the magnetic stirrer to see the effect of ultrasound application on the size of CoFe2O4 nanoparticles and their agglomerates. 2.3.2 Silica Coating of Cobalt Ferrite Magnetic Nanoparticles The CoFe2O4 magnetic nanoparticles were coated with a silica shell using a sol-gel method [19]. First, 80 mL ethanol, 169 µL TEOS and 14.4 µL APTMS were mixed in a beaker and subsequently 20 mL CoFe2O4 colloidal suspension was added to the mixture and stirred for 3 hours at room temperature. Subsequently, the silica coated cobalt ferrite (SiO2-CoFe2O4) particles were collected with a permanent magnet and washed three times with deionized water. 2.3.3 Functionalization of Silica Coated Cobalt Ferrite Nanoparticles with Amine Groups After coating the CoFe2O4 nanoparticles with a silica shell, the surfaces of SiO2-CoFe2O4 nanoparticles were modified with amine groups using APTES [9]. For the coating process, 12 mL DMF and 8 mL toluene were vortexed in a 50 mL tube and this solution was put in a 50 mL beaker, and subsequently 200 µL APTES was added drop by drop into this mixture under magnetic stirring for 24 hour. Then, the functionalized particles were collected with the magnet and washed with toluene. Finally these particles were re-dispersed in 10 mL DMF. 2.3.4 Adding –COOH Functional Groups to Amine Modified Silica Coated Cobalt Ferrite Nanoparticles 6
After binding of amine group onto the SiO2-CoFe2O4 nanoparticles, carboxyl groups were added by using ring opening linker elongation reaction [20].
In this process glutaric
anhydride ring structure was opened and reacted with the amine groups. The SiO2-CoFe2O4 nanoparticles which were modified with –NH2 groups were dispersed in 10 mL DMF to obtain a homogeneous colloidal suspension. This suspension was added drop by drop to the 10 mL DMF solution containing 0.1 g glutaric anhydride and the mixture was stirred for 24 hour at room temperature. The surface modified particles were washed with DMF and kept for further use. 2.3.5 Surface Modification of Silica Coated Cobalt Ferrite Nanoparticles with NTA 5.8325g MES K salt and 7.305g NaCl were dissolved in 250 mL volumetric flask. The pH of the solution was adjusted to 6 with concentrated HCl. 7.0 mL of this solution was taken and mixed with 10 mL of -COOH functionalized SiO2-CoFe2O4 particle suspension. 0.0787g NTA was added to the suspension and the mixture was vortexed about 1 minute. The suspension was put in a 50 mL beaker and stirred for 2 hours. The surface modified SiO2CoFe2O4 particles were collected with the magnet and supernatant was removed. The collected particles were washed with deionized water. 2.3.6 Adding Ni (II) Ions to the NTA modified Silica Coated Cobalt Ferrite Magnetic Nanoparticles 20 mg/L Ni (II) ion solution was added to the NTA modified SiO2-CoFe2O4 nanoparticles by gentle mixing on a shaker platform for 90 min. The nanoparticles were collected using the external magnet and the supernatant solution was taken. The particles were washed with 15 mL deionized water and all washing solutions were kept together with supernatant solution for ICP-OES measurements to determine the amount of Ni (II) ions added on to the NTA modified SiO2-CoFe2O4 nanoparticles. 2.3.7 Preparation of E.coli pQE-31/775 cell-free extract The cell extract was prepared from the recombinant E.coli pQE-31/775 cells which expressed the small heat shock protein, tpv-HSP 14.3 from thermoacidophilic archaeon Thermoplasma volcanium, as an N-terminal 6X-His tag fusion as described earlier [21]. The cleared cell lysate was obtained by sonication of the cells containing 20 mM imidazole and 1 mg/mL 7
lysozyme, followed
by centrifugation at 10,000xg at 4 ºC for 30 min (Sigma 3K30
Centrifuge, Sigma Chemical Co., St. Louis, USA). The supernatant called “cell-free extract” was stored at -20°C until use.
2.3.8 Purification of the 6xHis tagged tpv-HSP 14.3 protein using Surface Modified Silica Coated Cobalt Ferrite Nanoparticles After thawing cell-free extract on ice a certain volume (3 mL, 0.6 mg/mL protein) was added to surface modified SiO2-CoFe2O4 nanoparticles (0.6 gm) in a glass test tube which was mixed gently in the slanted position by shaking at room temperature for 60 min. Then, the supernatant was separated from the magnetic beads by the aid of a magnet. The supernatant was completely removed and the nanoparticles were washed twice in a phospahate buffer (50 mM NaH2PO4, 300 mM NaCl , pH 8.0) to remove unbound proteins (3mL buffer was used for each washing). The bound His-tagged sHSP was eluted from the nanoparticles with 1.5 mL phosphate buffer containing 250 mM imidazole. The eluate and all other fractions were stored at -20 ºC for SDS-PAGE analysis.
2.3.9 SDS Gel Electrophoresis SDS-polyacrylamide gel electrophoresis (PAGE, 12.5 % acrylamide slab gel, 1 mm thick) was performed by the procedure of Laemmli [22]. Flowthrough, wash samples and eluted protein were mixed with 2x sample buffer and after boiling subjected to SDS-PAGE. PageRuler™ Prestained Protein Ladder, (Fermentas AB, Vilnius, Lithuania) was used as molecular weight standard. The protein bands were visualized by staining with Page Blue Protein Staining Solution (Fermentas AB, Vilnius, Lithuania).
3. Results and discussion 3.1 Morphology and Size Distibution of Cobalt Ferrite Magnetic Nanoparticles Cobalt ferrite (CoFe2O4) nanoparticles were preferred as magnetic core materials due to the easy preparation procedure.
Unlike magnetite (Fe3O4), there is no need to use inert
atmosphere or organic additives to produce CoFe2O4 nanoparticles with a narrow particle size distribution [23-25]. 8
The morphology and size distribution of the primary CoFe2O4 particles were studied using FE-SEM and TEM. Various production routes used for the preparation of CoFe2O4 particles were summarized in Table 1. Representative FE-SEM and TEM images and Energy Dispersive X-ray (EDX) pattern of the CoFe2O4 nanoparticles (for Sample 2) are shown in Figure 1. Similar FE-SEM images were also obtained for other samples (Sample 1 and Sample 3).
Figure 1. A) FE-SEM and B) TEM images and C) corresponding EDX result of CoFe2O4 nanoparticles prepared using sodium chloride as the dispersant through magnetic stirring (Sample 2).
From the SEM images, the diameters of approximately 100 primary particles were measured and the number-length (arithmetic) mean size (D[1,0]) and volume weight mean size (D[4,3]) of the primary CoFe2O4 nanoparticles were calculated. Polydispersivity index (PDI), which 9
gives information about particle size homogeneity, was calculated using the following equation.
PDI PP
D4,3 D1,0
(3)
It should be noted that the PDI calculated using Equation (3) was different from the PDI obtained from the Dynamic Light scattering technique. The mean particle sizes and PDIpp values for the primary CoFe2O4 particles prepared using various preparation routes are given in Table (1). Table 1. Mean particle sizes of the primary CoFe2O4 particles prepared using various preparation routes. Sample
Mixing
Dispersant
D[4,3] (nm) D[1,0] (nm) PDIPP*
Sample 1
Magnetic Stirring
Oleic Acid
25.1
27.4
1.1
Sample 2
Magnetic Stirring
NaCl
12.9
14.1
1.1
Sample 3
Ultrasonic
NaCl
nmnm14.06 13.9
15.1
1.1
*PDIPP refers to the polydispersivity index for the primary CoFe2O4 nanoparticles determined using the SEM images. As can be seen from Table 1, when NaCl was used as a dispersant, the primary particle size of the CoFe2O4 particles was reduced. Additionally, in the presence of NaCl, the application of the ultrasonic agitation during the preparation of CoFe2O4 particles did not influence the size of the primary CoFe2O4 particles. The special properties of magnetic nanoparticles required for biomedical applications demand precise control of particle size, dispersion and conditions that affect these properties. In principle, it is necessary to stabilize the magnetic nanoparticle dispersion in the aqueous environment. The Dynamic Light Scattering (DLS) technique was used to measure z-average mean size and Polydispersity Index (PDIA) of CoFe2O4 agglomerates. Z-average mean size is defined as the harmonic intensity averaged particle diameter. The PDI values smaller than 0.05 indicate highly monodisperse particles. The values of PDI greater than 0.7 indicate that the sample has a very broad size distribution and is probably not suitable for the DLS technique. By applying deconvolution algoritm, it is possible to obtain
particle size
distribution data from the DLS measurement. From the particle size distribution data, the 10
volume weighted mean size (D[4,3]) and surface area weighted mean size (D[3,2]) of the CoFe2O4 agglomerates was calculated from the following equation.
d im 3Vi Dm, n m 3 d i Vi
1
( mn)
(4)
Where di is the agglomerate size and Vi is the volume of agglomerates. Specific surface area of the CoFe2O4 agglomerates can be calculated using the following equation. The unit of the specific surface area can be either m2/g or m2/cm3.
SV
6 D3,2
(5)
The typical agglomerate size distribution of the CoFe2O4 agglomerates (Sample 1) measured
Volume, %
using the dynamic light scattering technique was given in Figure 2.
Size, nm Figure 2. The agglomerate size distribution of the CoFe2O4 nanoparticles
The mean particle sizes and PDIA values of the CoFe2O4 agglomerates prepared using various preparation routes are given in Table 2.
11
Table 2. Mean particle sizes of the CoFe2O4 agglomerates prepared using various preparation routes Sample
Mixing
Dispersant
Z-average
PDIA
mean size
D[4,3]
SV
(nm)
(m2/cm3)
(nm) Sample
Magnetic
Oleic Acid
147.4
0.042
139.7
47.4
1Sample
Stirring Magnetic
NaCl
127.4
0.250
102.7
65.5
2 Sample
Stirring Ultrasonic
NaCl
128.7
0.151
97.3
59.9
3
Bath
As can be seen from Tables 1 and 2, well dispersed smaller CoFe2O4 primary particles and their agglomerates were obtained using the salt-assisted solid state method in the ultrasonic bath (Sample 3) and this procedure was used throughout the study. 3.2 Silica Coating of Magnetic Cobalt Ferrite Nanoparticles CoFe2O4 nanoparticles were coated with silica in aqueous/ethanolic solution via Stöber method [19]. TEOS and APTMS were used for silica coating of the CoFe2O4 nanoparticles. Using small amount of APTMS in addition to the TEOS was very important since better silica coating was obtained compared to the procedure in which only TEOS was used. TEM image and Energy dispersive X-ray (EDX) analysis for the SiO2-CoFe2O4 nanoparticles are shown in Figure 3. The carbon signal in the EDX pattern was coming from the carbon tape used for sampling. The presence of silica peak on the spectrum was considered as the indication of silica layer formation on the surface of CoFe2O4 nanoparticles.
12
Figure 3. TEM image and EDX result of the SiO2-CoFe2O4 magnetic nanoparticles. As discussed previously the cobalt ferrite particles form agglomerates with Z-average mean size of about 128 nm, volume weighted mean size of about 97 nm, and the PDIA of 0.15. The introduction of silane into the aqueous suspension of these cobalt ferrite nanoparticles did not cause any positive influence in their dispersion. Probably agglomerates were coated with silica and subsequently an increase in the diameters of the silica coated cobalt ferrite aggregates was observed. The Z-average mean size, volume weighted mean size and the PDI of SiO2-CoFe2O4 agglomerates at pH ~7.0 were determined as 267 nm, 325 nm and 0.11 respectively. The specific surface area of SiO2-CoFe2O4 agglomerates was calculated as 21.1 m2/cm3. Further modifications such as anchoring carboxylic acid and NTA following the silica coating did not influence the agglomerate size and size distribution of the particles. 3.3 Magnetic Behavior of the Cobalt Ferrite Magnetic Particles Magnetic behavior of the CoFe2O4 particles prepared using the ultrasonic bath was tested by their collection under the influence of external magnetic field and the results are shown in Figure 4.
13
Figure 4. Magnetic behavior of the CoFe2O4 nanoparticles after the application of external magnetic field (1.6T).
As can be seen from Figure 4, the collection of the magnetic CoFe2O4 particles after the application of external magnetic field (1.6T) was completed after 60 seconds. Similar observation was noted for the SiO2-CoFe2O4 particles. The collection of the SiO2-CoFe2O4 particles was as rapid as that of the CoFe2O4 nanoparticles. The magnetic properties of the uncoated and silica coated CoFe2O4 particles were also characterized using the Vibrating Sample Magnetometer (VSM). The hysteresis curves for these particles recorded at 300K were given in Figure 5 (a) and (b). The magnetization values of the uncoated and silica coated CoFe2O4 particles at 10 kOe were measured as 46.2 emu/g and 44.7 emu/g, respectively. The coercivity values of the uncoated and silica coated CoFe2O4 particles were measured as 147.0 Oe and 202.5 Oe, respectively. There was only a slight decrease in the magnetization values at 10 kOe for the SiO2-CoFe2O4 particles compared to the uncoated ones due to nonmagnetic silica coating.
14
50
[a]
40
Magnetization (Am2/kg, emu/g)
Magnetization (Am2/kg, emu/g)
50 30 20
10
20
0
10
-10
0
-20
-10
-30
-20 -0,04
-40
0,00
0,04
-50 -3
-2
-1
0
1
2
3
[b]
40 30 20 10 0
20
-10
10
-20
0
-30
-10
-40
-20 -0,04
-50 -3
-2
-1
0
0,00
1
0,04
2
Magnetic Field (T, 10 kOe)
Magnetic Field (T, 10 kOe)
Figure 5. The magnetic hysteresis curves for [a]the uncoated and [b] SiO2-CoFe2O4 particles recorded at 300K.
3.4 Surface Modification of Silica Coated Cobalt Ferrite Nanoparticles
Besides improving the stability of the particles, silica coating of the CoFe2O4 nanoparticles also provides a platform for surface modification with various specific functional groups. By attaching functional amine groups onto the surface of the SiO2-CoFe2O4 particles, additional functional groups and biological molecules could easily be attached to the surface of these particles. After addition of amine group on the silica coating of CoFe2O4 nanoparticle, carboxyl groups were added by using ring opening linker elongation reaction [20]. In this process glutaric anhydride ring structure was opened and reacted with the amine groups. FTIR spectrometer was used for the characterization of the carboxyl groups introduced on the surface of SiO2-CoFe2O4 nanoparticles (Figure 6).
15
3
3395
1700
2870 2925
1560 1635
1400
890
1035 1135 1205
Absorbance (A) 500
1000
1500
2000
2500
3000
3500
4000
Wavenumber (cm-1)
Figure 6. FTIR results of–COOH functionalized SiO2-CoFe2O4 magnetic nanoparticles. The absorption bands at about 3395 and 1635 cm−1 in all the spectra mainly originate from the OH vibrations in H2O [9]. The spectrum of carboxyl functional groups on the modified SiO2CoFe2O4 nanoparticles contains stretching peaks around 1035 cm-1 due to the siloxane,–Si– O–Si– and silanol, Si–O groups and two small peaks (2870 and 2925 cm-1) between 2990– 2830 cm-1 for aliphatic C–H stretching vibrations of alkyl chains in APTES precursor ligand [9, 26-28]. The existance of siloxane and silanol groups that are typical groups found on the surface of CoFe2O4 nanoparticles was the proof of silica coating. Whereas the stretching vibrations corresponding to the alkyl chains are the indications of the incorporation of APTES onto silica surface through silanization. Carboxylic acid, –COOH , group was bonded to the surface through the reaction of –NH2 and glutaric anhydride, where one of the carboxylic acid group of the anhydride was converted into amide and the other one was left as a terminal –COOH group. Amides formed from primary amines are distinguished by their characteristic Amide I and Amide II bands. Amide I band is mainly attributed to the carbonyl stretching and Amide II involves contribution of several atoms including the N-H bond. The observed Amide I and Amide II bands (peaks around 1700 and 1560 cm-1) in the FTIR spectra suggest that –COOH groups were bonded to the surface through–NH2 groups. The band between 1460-1365 cm-1(around 1400 cm-1) together with the band around 890 cm-1 can be attributed to the O-H bending vibration of carboxylic acid. Although the 1460-1365 cm-1 band cannot be distinguished from C-N 16
stretching band of amide (1400 cm-1) in the same region, it may also support the presence of carboxylic acid on the surface of the SiO2-CoFe2O4 nanoparticles. The peak at 1635 cm-1 is the result of deformational vibrations of adsorbed water molecules as suggested by others [9, 26-29]
The surface modification of the SiO2-CoFe2O4 nanoparticles by amine and carboxyl groups was followed by the addition of NTA (Nα,Nα-Bis(carboxymetr56hyl)-L-lysine hydrate) groups onto the nanoparticles. NTA was covalently bound to the carboxylic acid end group of −COOH modified SiO2-CoFe2O4 particles. The surface modification steps and resulting modified SiO2-CoFe2O4 particles are shown in Figure 7.
O O Si O
APTES in DMF and toluene CoFe2O4 agglomerate coated with silica
O O Si O
NH
(CH2)3NH2
Glutaric anhydride in DMF and toluene
O O Si O
(CH2)3NHCO(CH2)3COOH
NTA attachment
Attachment of Ni (II) Ions
O N
-COOH functionalized CoFe2O4 particles
-NH2 functionalized CoFe2O4 particles
CoFe2O4 nanoparticles
O NH
NH
R1
R1
Ni+2
Figure 7. The surface modification steps and resulting modified SiO2-CoFe2O4 nanoparticles. NTA modified nanoparticles were characterized by FTIR. The FTIR spectra of NTA modified SiO2-CoFe2O4 were shown in Figure 8.
17
2915 1000
1410 1540 1635 1730
1105
Absorbance (A) 500
1500
2000
2500
3000
3500
4000
Wavenumber (cm-1)
Figure 8. FTIR results of NTA modified SiO2-CoFe2O4 nanoparticles. After modification of the surface with NTA, carboxylic acid group vibrations become predominant in the FTIR spectrum (Figure 8). The asymmetric vibration band of the deprotonated carboxylic group is located around 1410 cm-1, whereas asymmetric vibrational band of the same group is located at 1635 cm-1. The band at 1730 cm-1 was assigned to the protonated –COOH group of NTA cm-1[30-32].
As the final modification, the SiO2-CoFe2O4 nanoparticles functionalized with NTA were loaded with Ni (II) ions. The quadridentate NTA moiety covalently coupled to the surface of SiO2-CoFe2O4 nanoparticles via spacer butyl amine arm. The remaining four chelating sites of the modified NTA interact with nickel (II), which result in a tight binding of ions. Quantification of Ni (II) binding was done using an inductively coupled plasma spectrometer (ICP-OES). The concentrations of the Ni (II) ions in the loading solution prior to the addition of the magnetic nanoparticles and in the supernatant solution after removing the magnetic nanoparticles were measured by the ICP-OES. From the ICP-OES measurements it was found that 6.15mg/L Ni (II) ions were bonded to 0.0148g surface modified SiO2-CoFe2O4 nanoparticles. This result also revealed the existence of NTA on the surface of SiO2-CoFe2O4 nanoparticles. After immobilization of the Ni-NTA affinity group, the remaining two free sites of Ni (II) ions are ready to bind histidine tag proteins through the use of imidazole side chains of histidine molecules. Since the SiO2-CoFe2O4 nanoparticles retained their magnetic properties, they can be effectively used in the magnetic separation of his-tagged proteins, as explained below. 18
3.5 Purification of 6XHis-tagged Tpv/sHSP-14.3 using SiO2-CoFe2O4 nanoparticles To evaluate the surface modified SiO2-CoFe2O4 magnetic nanopaticles as an efficient platform for protein purification, the isolation of N-terminal His-tagged recombinant small heat shock protein (Tpv-sHSP 14.3) from E. coli cell extract was studied. Small heat-shock proteins are molecular chaperones which are responsible for binding of improperly folded protein-substrates [33-34] and prevent improper polypeptide associations and accumulation of aggregated proteins in the cell [35]. In this way, they further transfer entrapped unfolded proteins to the ATP dependent chaperones or to the protein degradation machines like proteasomes or autophagosomes [33, 6]. The sHSP’s chaperone activity is therefore crucial to cells’s tolerance to stress. In the main, they are dramatically up-regulated under conditions of cellular stress to being among the most abundant of all proteins [37,38]. Recent investigations indicate that sHSPs participate in regulation of many vital cellular processes, including apoptosis, cytoskeleton and carcinogenesis and have pronounced cardioprotective activity [39,40]. Furthermore, mutations of certain members of sHSP family lead to cell dysfunctions that correlate with development of different congenital diseases including certain neurodegenerative disorders [41-43]. Together these observations suggest that sHSps are on the front line of attractive therapeutic targets. Thus, development of such a convenient purification system would be useful for preparation protein drugs such as pharmachaperones. The purification protocol applied in this study yielded highly purified his-tagged recombinant small heat shock protein as revealed by SDS-PAGE (Figure 9). The purified sample form the E.coli cell extract produced a protein band of about 14.5 kDa on the gel, which corresponds to the protein molecular weight deduced from the predicted amino acid sequence. The Ni (II) ion loaded SiO2-CoFe2O4 nanoparticles system developed in this study can be convenient and useful tool for biological and clinical research and applications.
19
M
1
2
3
4
Figure 9. SDS-PAGE analysis of His-tagged sHSP form E.coli extract. Lanes: 1: Crude protein, 2: Wash sample, 3: 2nd elution, 4: 1 st elution, M. Prestained Protein Ladder, (170-10 kDa, Fermentas); Arrow indicates bands of sHSP proteins
4. Conclusion CoFe2O4 magnetic nanoparticles with a narrow particle size distribution were synthesized by using the co-precipitation technique without using any surfactant. The sizes of CoFe2O4 agglomerates, which were synthesized under the control of magnetic stirring and ultrasonic agitation by using different dispersing agents, were characterized in detail. By combining the SiO2-CoFe2O4 magnetic nanoparticles with the specific affinity offered by Ni-NTA metalchelate functional group, it has been demonstrated for the first time that the Ni-NTA modified SiO2-CoFe2O4 can isolate histidine-tagged proteins directly from the crude samples without any purification steps. NTA-attachment was done on the surfaces of SiO2-CoFe2O4 particles using carboxylic acid as a robust and cost-effective anchor instead of expensive reagents (like EDC/NHS couple) with long procedure. The new purification system developed has several merits that may not be found in other protein purification systems. There is no need for specialized binding buffers and substrates for protein elution. All of the purification steps can be easily and quickly performed by using a magnet instead of a centrifuge. The prepared magnetic materials show excellent performance in the separation of a N-terminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3. The results obtained in this study 20
suggest that the magnetic materials with different surface functionalities may also find applications in a wide variety of biological applications.
Acknowledgements The authors thank Central Laboratory at METU for FE-SEM, TEM and particle size measurements.
References
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Research highlights Novel magnetic nanoplatform was applied for the first time in the purification of Nterminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3. successfully. NTA-attachment with robust and cost-effective anchor with easy procedure. Detailed study of the size of the agglomerates under the control of magnetic stirring and ultrasonic agitation by using different dispersing agents.
24