Synthesis of biofunctionalized silica nanospheres to separate GST-tagged proteins

Process Biochemistry 51 (2016) 804–808

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Synthesis of biofunctionalized silica nanospheres to separate GST-tagged proteins Xueyan Zou a , Liangliang Li b , Haitao Lu a,c , Yutao Zhang a,c , Yanbao Zhao a , Yu Zhang b , Quanhui Guo d,∗ a

Engineering Research Center for Nanomaterials, Henan University, Kaifeng, 475004, PR China Key Laboratory of Plant Stress Biology, Henan University, Kaifeng, 475004, PR China c Henan University Minsheng College, Kaifeng, 475004, PR China d College of Chemistry and Chemical Engineering, Henan University, Kaifeng, 475004, PR China b

a r t i c l e

i n f o

Article history: Received 22 November 2015 Received in revised form 13 March 2016 Accepted 14 March 2016 Available online 21 March 2016 Keywords: Silica hollow nanospheres GST-tagged Surface functionalization Purification

a b s t r a c t A Thiol-functionalized silica hollow nanospheres(denoted as SiO2 -SH NSs) were prepared through a hydrothermal route. The SiO2 -SH NSs were conjugated with glutathione group (denoted as GSH) to afford SiO2 -GSH NSs. The as-prepared SiO2 -GSH sample has hollow structure and exhibits an average diameter of about 45 nm and a wall thickness of 10 nm. The SiO2 -GSH NSs were used to separate three kinds of GST-tagged proteins (GST-tagged GPX, GST-tagged LOV and GST-tagged 210-6P). These SiO2 -GSH NSs exhibit negligible non-specific adsorption, high binding capacity (89.9 ␮mol/g), the low detection limit (1.0 × 10−6 mol/L) and reuse property, showing great potentiality in purifying GST-tagged proteins. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Glutathione S-transferase (denoted as GST) represents a major group of detoxification isoenzymes which participate in a wide range of processes including xenobiotic biotransformation, drug metabolism, degradation ofaromatic amino acids and so on [1]. In the meantime, GST can be used as a marker of early phase tumors such as brain tumor, gangliogliomas, protate cacer and so on [2–4]. This could partly account for the significance of the separation and purification of GST and GST-tagged proteins in biological medicine and disease detection. Recently many materials, such as microfiber, magnetic microparticles and gels, have been employed to separate and purify GST and GST-tagged fusion proteins [5,6]. These materials, although adaptable to many protein expression systems, have some limitations, such as the need for pretreatment to remove cell debris and colloid contaminants, a relatively long operation time, and poor protein solubility. Today, nanoparticles or nanorods as promising nano-devices for the separation of target proteins could be advantageous over conventional counterparts [7–10]; however, surface group density of solid nano-adsorbents was low due to the

∗ Corresponding author. E-mail address: [email protected] (Q. Guo). http://dx.doi.org/10.1016/j.procbio.2016.03.010 1359-5113/© 2016 Elsevier Ltd. All rights reserved.

muti-step surface modification reaction. In the present research, we focus on the preparation of functionalized silica hollow NSs to enhance the surface group density. As-synthesized silica NSs can directly enrich and separate GST-tagged proteins from the E. coil cell lysate, showing great potential as novel adsorbents.

2. Experimental 2.1. Materials 3-Mercaptopropyltrimethoxysilane (MPS) was purchased from Alfa-Aesar, Amarica. Tetraethyl orthosilicate (TEOS) was from Tianjin Fuchen Chemicals; Hexadecyltrimethylammonium Bromide (CTAB) was purchased from Sinopharm Chemicals; Glutatione (GSH) was purchased from Amresco Company. Triethanolammine (TEA) was purchased from Tianjin Kermel Chemicals. Ethylene diamine tetraacetic acid (denoted as EDTA), absolute alcohol and aqueous ammonia (28 wt%) were purchased from Tianjin Kermel Chemical Reagent Company, China. Phosphate buffer salines (abridged as PBS; concentration 0.1 mol/L, pH 8.0; concentration 0.01 mol/L, pH 7.4) and 5,5 -dithiobis-(2-nitrobenzoic acid) (abridged as DTNB, purity ≥98%) were purchased from Sigma–Aldrich, Amarica. All the reagents are of analytical grade and used as-received.

X. Zou et al. / Process Biochemistry 51 (2016) 804–808

2.2. Preparation of SiO2 -GSH Briefly, 0.03 g CTAB, 2.8 mL of ethanol and 1.1 mL of TEA were dissovled in 17 mL of H2 O to form a transparent solution. Then mixture of TEOS and MPS (different volume ratios) was slowly dropped into above solution at 60 ◦ C. After the resultant solution heated at 60 ◦ C for 3 h, the solution was transferred into a Teflon-lined stainless-steel autoclave, sealed and heated at 110 ◦ C for 24 h. Upon completion of the reaction, the solution was cooled, centrifuged, washed and dried at 60 ◦ C in an oven to afford SiO2 -SH product. 0.3 g of SiO2 -SH product was dispersed in 10 mL 80 mg/mL of GSH solution. The dispersion was oscillated at 37 ◦ C for 24 h (60 rev/min), followed by centrifuging to afford desired SiO2 -GSH product. Subsequently, the obtained SiO2 -GSH product was fully washed with PBS solution to remove physically adsorbed GSH, followed by dispersion in ethanol (25%, v/v) and storing at 4 ◦ C. 2.3. Separation and detection of GST-tagged proteins The to-be-tested mixed proteins were collected from the cell lysate of Escherichia coli, which is by water lysis. (concentration 0.01 mol/L, pH 7.4). 1 mL 0.02 g/mL of SiO2 -GSH sample fully washed with PBS solution was directly introduced into 1000 ␮L of the cell lysate and shaken at a rotary speed of 90 rev/min at 4 ◦ C for 1 h to capture GST-tagged proteins. Upon completion of capturing GST-tagged proteins, the SiO2 -GSH sample with captured GST-tagged proteins was isolated by centrifugation and fully washed with PBS solution to remove residual uncaptured proteins, followed by washing with 300 ␮L of 50 mmol/L GSH solution to

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disassociate the GST-tagged proteins from the surface. Then the separated GST-tagged proteins were detected with SDS-PAGE, with the preconcentration voltage of 70 V and the separation voltage of 120 V. The binding proteins concentration was analyzed at 280 nm and 400 nm by UV–vis spectrophotometer. 2.4. Characterization The morphology and composition were characterized by transmission electron microscopy (TEM, JEM-2010, Japan), Fourier transform infrared (FT-IR, AVATAR360, America) and thermogravimetric analysis (TG, EXSTAR 6000, Japan), respectively. The surface area was measured by the Brunauer-Emmett-Teller (BET) method (QUADRASORB, American). The separated GST-tagged proteins were detected with SDS-PAGE (Power PAC 300, China). The content of SH group of the prepared nanospheres and the binding proteins concentration was analyzed by UV–vis spectrophotometer (nanodrop 2000c, America). 3. Results and discussion 3.1. TEM images of SiO2 -SH NSs< A sereies of SiO2 -SH samples were prepared under different TEOS/MPS volume ratios so as to investigate the effects of reaction condition on the morphology of the SiO2 -SH products. As shown in Table 1 and Fig. 1, SiO2 -SH sample obtained at a TEOS/MPS volume ratio of 10:1 (TEOS 1.4 mL, MPS 0.14 mL) exhibits hollow spherical shape as well as an average diameter of about 45 nm and a wall

Fig. 1. TEM images of SiO2 -SH and SiO2 -GSH product obtained under different reaction conditions: a- SiO2 -SH at TEOS/MPS volume ratio 10:1, b- SiO2 -SH at TEOS/MPS volume ratio 1:1, c- SiO2 -SH at TEOS/MPS volume ratio 1:10, d- SiO2 -SH with 1.4 mL of MPS alone, e,f-the SiO2 -GSH NSs prepared from the SiO2 -SH obtained at a TEOS/MPS volume ratio of 10:1.

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102

1.0

2

96

1

2550

Weight / %

Transmission

0.8

99

0.6 0.4 0.2 0.0

1: SiO2-SH 2: SiO2-GSH 3500

3000

2500

2000

1500

Wavenumber / cm-1

1000

500

93

2 1

90 87 84 81

1-SiO2-SH

78

2-SiO2-GSH

75

100

200

300

400

500

600

700

800

Temperature / oC Fig. 2. FT-IR spectra of as-prepared SiO2 -SH sample (curve 1) and SiO2 -GSH sample (curve 2), both samples prepared at a TEOS/MPS volume ratio 10:1.

thickness of 10 nm (Fig. 1a). When the volume ratio of TEOS/MPS is 1:1 (TEOS 1.4 mL, MPS 1.4 mL), as-obtained SiO2 -SH sample still retains hollow spherical shape, but its wall thickness rises to about 30 nm (Fig. 1b). Further increase of the ratio of MPS generates SiO2 SH solid spheres with an average diameter of about 100 nm (see Fig. 1c (TEOS 0.14 mL, MPS 1.4 mL) and Fig. 1d (TEOS 0 mL, MPS 1.4 mL)). It can be seen that the morphology of SiO2 -SH and the wall thickness of these hollow spheres can be controlled by TEOS/MPS volume ratio. Furthermore, the formation mechanism of hollow SiO2 -SH spheres can be explained by Ostwald Ripening theory [11]. The internal pressure of these SiO2 -SH spheres gradually increased under the action of CTAB template to forming the hollow structure. 3.2. Analysis of SiO2 -GSH NSs Subsequently, SiO2 -GSH NSs are prepared from the SiO2 -SH obtained at a TEOS/MPS volume ratio of 10:1 (Fig. 1a, the same hereafter except for special explanation) and exhibits similar hollow spherical shape (see Fig. 1e,f). It can be seen that the average diameter was about 45 nm, with shell thickness of 10 nm, which indicates that the functionalization of SiO2 -SH hollow NSs with GSH group under properly selected reaction condition leads to little change in their morphology. In order to detect the surface area and pore properties of prepared SiO2 -GSH NSs, BET surface area from N2 adsorption/desorption analysis was surveyed. The BET surface area, pore volume and pore size of the sample are 260.0 m2 /g, 0.73 cm3 /g and 0.4 nm, respectively. In order to evaluate the content of the GSH group of SiO2 -GSH samples, we detect the content of SH of SiO2 -SH by DTNB method. DTNB can react quantitatively with SH group of GSH to form 5-thiobis-(2-nitrobenzoic acid), a characteristic chromophoric product which can be monitored by measuring the absorbance change at 412 nm. According to the relationship between the absorbance and GSH concentration, the working plot of SH concentration versus absorbance can be established, and the surface SH content of SiO2 -SH spheres can thus be calculated from the working plot by taking into account the absorbance of the reaction solution containing SiO2 -SH and DTNB. It can be seen that the content of surface thiol group of SiO2 -SH NSs was about 202.3 ␮mol/g, which is approximate to the content of GSH group of SiO2 -GSH NSs. Fig. 2 gives the FT-IR spectra of a typical SiO2 -SH sample (Fig. 1a) and corresponding SiO2 -GSH sample (Fig. 1e). The peak at 3436 cm−1 is attributed to the OH stretching vibration of silica hollow spheres, and those around 1129 cm−1 , 801 cm−1 , and 473 cm−1 are assigned to Si O Si stretching vibration [12]. Particularly, SiO2 -SH and SiO2 -GSH exhibit very similar with FT-IR peaks,

Fig. 3. TG curves of as-prepared SiO2 -SH sample (curve 1) and SiO2 -GSH sample (curve 2), both samples prepared at a TEOS/MPS volume ratio 10:1.

which indicates that they both consist of SiO2 as the major component. However, there are some differences in the FT-IR spectra of SiO2 -SH and SiO2 -GSH. Namely, as-obtained SiO2 -SH NSs show an intense peak of S H stretching vibration at 2550 cm−1 [13], but this peak disappears after SiO2 -SH NSs is surface functionalized by GSH group (see Fig. 2), possibly because the SH group is consumed upon reaction with GSH generating SiO2 -GSH NSs [5]. Fig. 3 gives the TG curves of the SiO2 -SH and SiO2 -GSH samples. These samples successively lose weight from room temperature to 850 ◦ C. The initial mass loss below 250 ◦ C can be attributed to the desorption of adsorbed water from the surface of the sample powder and the following mass loss is related to the further release of the inner adsorbed or crystal water. It can be seen that the mass losses of both samples occur mainly in the temperature range of 250–850 ◦ C. Besides, the total mass loss of SiO2 -SH and SiO2 -GSH is about 14.6% and 19.9%, respectively; and the larger mass loss (about 5.3%) of the latter is possibly due to the enhanced thermal decomposition of GSH group than SH group, well conforming to their surface thiol group content determined by DTNB method. 3.3. Schematic representation Affinity interaction between the ligand and target proteins has been accepted as an effective means of protein separation. Scheme 1 gives the schematic representation of the preparation of SiO2 -GSH NSs and their separation of GST-tagged proteins. First, hollow SiO2 -SH NSs were prepared through a hydrothermal route. Second, GSH was conjugated on the surface SiO2 -SH NSs to SiO2 GSH NSs. Subsequently, the SiO2 -GSH NSs were directly used to enrich and separate GST-tagged proteins from the cell lysate. In the end, the SiO2 -GSH NSs can be reused to separate the target proteins for several times by the same method. All the experimental process is facile and easy to operate. 3.4. SDS-PAGE analysis To estimate the separation efficiency of the as-prepared samples for target proteins (GST-tagged LOV proteins), we conducted SDS-PAGE analysis under different separation conditions. Fig. 4 shows the SDS-PAGE analytical results of GST-tagged LOV proteins separated and purified by SiO2 -GSH NSs. It can be seen that the as-synthesized SiO2 -GSH NSs can efficiently enrich the target proteins from the cell lysate. Namely, the quantity of the dissociated proteins increases with the increase of the concentration of GSH elution solution in the range of 10–100 mmol/L (10, 20, 50, and

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TEOS MPS

+GSH

hytrolysis

SiO2-SH

centrifuge

SiO2-GSH

add

incubate

reuse

+GSH

mix proteins

+

GST-tagged LOV Note: -SH;

-GSH;

SiO2-GSH GST-tagged LOV;

mix proteins

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proteins is 1.0 × 10−4 mol/L, 1.0 × 10−5 mol/L and 1.0 × 10−6 mol/L, the as-prepared SiO2 -GSH sample (0.02 g) has an adsorption capacity of 55.3 ␮mol/g, 32.2 ␮mol/g and 32.0 ␮mol/g, respectively. This means that the as-prepared SiO2 -GSH NSs can be used to detect the target proteins at a detection limit of lower than 1.0 × 10−6 mol/L (Fig. 4a lane 7–9). In order to investigate the reusability of as-prepared SiO2 -GSH NSs, we also conducted recycle SDS-PAGE experiments to evaluate the separation efficiency of the typical SiO2 -GSH product for the target proteins (see lanes 10–12 in Fig. 4a). It is seen that asprepared SiO2 -GSH NSs exhibits good selectivity to GST-tagged LOV in the E. coli lysate, and their specificity and affinity remain barely changed after 3 cycles of reuse. Namely, it exhibits binding capacities of 39.3 ␮mol/g, 32.0 ␮mol/g and 31.8 ␮mol/g for GST-tagged LOV protein after 1, 2 and 3 cycles of reuse. Fig. 4b gives the SDS-PAGE analytical results of the three kinds of recombinant proteins (GST-tagged GPX, GST-tagged LOV and GSTtagged 210-6P1) separated and purified by SiO2 -GSH NSs, where the universality of the SiO2 -GSH NSs is evaluated based on the separation of the three kinds of GST-tagged proteins from the E. coli lysate solution. It is seen that the three kinds of recombinant proteins can be well separated from the E. coli lysate without non-specificity. This means that the SiO2 -GSH NSs are applicable to efficient affinity separation and purification of GST-tagged proteins (see lanes 5–7 in Fig. 4b), with the separation capacity (0.02 g of SiO2 -GSH NSs) for GPX, LOV and 210-6P1 being 22.8 ␮mol/g, 47.3 ␮mol/g and 27.9 ␮mol/g, respectively.

Scheme 1. Preparation of SiO2 -GSH NSs and their separation of GST-tagged LOV proteins.

4. Conclusions

100 mmol/L) while the other conditions are kept unchanged (Fig. 4a lane 3–6), and the maximum quantity of the dissociated proteins is as much as 89.9 ␮mol/g. Furthermore, when the concentration of GSH elution solution is kept constant (50 mmol/L), the detection limit of the prepared SiO2 -GSH NSs for GST-tagged LOV proteins in the cell lysate was surveyed. In the selected concentration range of GST-tagged LOV proteins, as-prepared SiO2 -GSH NSs exhibit negligible non-specific protein adsorption and can specifically bind target proteins with a high efficiency. For instance, when the concentration of the LOV

A facile hydrothermal method was established to fabricate thiolfunctionalized silica hollow NSs. Resultant thiol-functionalized silica hollow spheres were allowed to conjugate with GSH molecule thereby affording SiO2 -GSH hollow NSs as potential adsorbents for GST-tagged proteins. Structural characterizations in combination with SDS-PAGE analyses under different conditions indicate that SiO2 -GSH products obtained under properly selected reaction conditions have hollow spherical shape and exhibit good separation ability as well as good reusability towards GST-tagged proteins, showing great potential in the efficient separation and purification of the target proteins.

Fig. 4. SDS-PAGE analysis of purified GST-tagged LOV proteins by SiO2 -GSH NSs prepared under a TEOS/MPS volume ratio of 10:1. a: lane 1, Maker; lane 2, E. coli. lysate; lane 3–6, the fractions washed off from the SiO2 -GSH NSs with different concentration of GSH solution (lane 3, 10 mmol/L; lane 4, 20 mmol/L; lane 5, 50 mmol/L; lane 6, 100 mmol/L); Lane 7–9, the fraction washed off from SiO2 -GSH NSs when different concentration of GST-tagged LOV proteins were used (lane 7, 1.0 × 10−4 mol/L; lane 8, 1.0 × 10−5 mol/L; lane 9, 1.0 × 10−6 mol/L); Lane 10–12, the fraction washed off from SiO2 -GSH NSs by reused materials. lane 10, 1st; lane 11, 2nd; lane 12, 3rd. b: lane 1, Marker; lane 2, GST-tagged GPX E. coli lysate; lane 3, GST-tagged LOV E. coli lysate; lane 4, GST-tagged 210-6P1 E. coli lysate; lane 5–7, the fractions washed off from the SiO2 -GSH NSs when different kinds of GST-tagged proteins were used (lane 5, GST-tagged GPX; lane 6, GST-tagged LOV; lane 7, GST-tagged 210-6P1).

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