Materials Science and Engineering C 34 (2014) 468–473
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis of petal-like ferric oxide/cysteine architectures and their application in affinity separation of proteins Xueyan Zou a, Kun Li c,1, Yanbin Yin b, Yanbao Zhao a,⁎, Yu Zhang c, Binjie Li d, Shasha Yao a, Chunpeng Song c a
Key Laboratory for Special Functional Materials, Henan University, Kaifeng 475004, PR China School of Mathematics and Information Sciences, Henan University, Kaifeng 475004, PR China Key Laboratory of Plant Stress Biology, Henan University, Kaifeng 475004, PR China d Key Laboratory of Cellular and Molecular Immunology, Henan University, Kaifeng 475004, PR China b c
a r t i c l e
i n f o
Article history: Received 11 April 2013 Received in revised form 28 August 2013 Accepted 29 September 2013 Available online 8 October 2013 Keywords: Ferric oxide/cysteine Preparation His-tagged protein Separation
a b s t r a c t Petal-like ferric oxide/cysteine (FeOOH/Cys) architectures were prepared through a solvothermal route, which possessed high thiol group density. These thiol groups as binding sites can chelate Ni2+ ions, which can be further used to enrich and separate his-tagged proteins directly from the mixture of lysed cells without sample pretreatment. These results show that the FeOOH/Cys architectures with immobilized Ni2+ ions present negligible nonspecific protein adsorption and high protein adsorption capacity, with the saturation capacity being 88 mg/g, which are especially suitable for purification of his-tagged proteins. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The easy separation and purification of proteins, especially lowabundance proteins, are very important in proteomics [1–4]. The traditional protein separation and purification methods are usually highly selective for target proteins [5,6]. However, they usually contain multistage processes such as precipitation, dialysis, ultrafiltration, and chromatography, which are generally complicated, time-consuming, and expensive for large-scale production. Therefore, there is a need to develop fast separation method of target proteins which are easy to deteriorate. The biotechnologies nowadays enable proteins to easily express with a tag, and many protein-purification methods are based on the specific interactions between immobilized ligands and affinity tags [7,8]. Among affinity tags, histidine tags are particularly popular, which can be readily incorporated into desired proteins by the expression of the target gene in microorganism cells using commercial expression vectors [9,10]. In the case of his-tagged proteins, histidine chains have affinity for certain metal ions such as nickel or copper, and they can specifically interact with the immobilized metal ions to create strong, yet reversible binding [11,12]. More recently, nanoparticles or nanorods have emerged as a promising separation nano-tool in the proteome research [13–16]. Isolation of his-tagged proteins typically involves
⁎ Corresponding author. Tel./fax: +86 378 3881358. E-mail address: [email protected] (Y. Zhao). 1 Identical first author. 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.046
immobilizing metal ions, selectively binding proteins and effectively releasing recombinant proteins from the support surface. Generally, iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED) and nitrolotriacetic acid (NTA) are used as metal chelators [17–24]. However, it is difficult to obtain high density of functional groups on the support surface by post-grafting method and these materials show low surface metal ion density, leading to low purification efficiency. To increase the surface metal ion density, herein we employed Lcysteine as metal chelator to prepare petal-like ferric oxide (FeOOH/ Cys) architectures via a solvothermal route. The prepared FeOOH/Cys architectures possess high density of surface thiol groups which as binding sites can chelate Ni2+ ions. The FeOOH/Cys-Ni2+ architectures as affinity adsorbent are used to directly enrich and separate three kinds of his-tagged proteins from the mixture of cell lysate, which displayed much higher enrichment efficiency than commercial Ni-NTA agarose.
2. Experimental section 2.1. Materials L-Cysteine (≥99.0%) was from Aladdin, China. Nickelous chloride (NiCl2·6H2O, ≥98.0%), ferric chloride crystal (FeCl3·6H2O, ≥99.0%), sodium acetate anhydrous (NaAc, ≥99.0%), ethylene glycol, and absolute alcohol were purchased from Tianjin Kermel Chemical Reagent Co., China. Ni-NTA agarose and BCA protein assay kit were purchased from QIAGEN, Beijing CoWin Biotech. Co. Protein molecular weight marker (Low) was purchased from TakaRa Biotech. Co.
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2.2. Preparation of FeOOH/Cys architectures and adsorption of Ni2+ ions In a typical synthesis, 1.0 g FeCl3·6H2O, 2.7 g NaAc and 1.0 g L-Cys were dissolved in 30 mL ethylene glycol and magnetically stirred for 0.5 h at room temperature. Then, the solution was transferred into a Teflon-lined stainless-steel autoclave, sealed and heated at 200 °C for 8 h. Subsequently, the solution was cooled down to room temperature, centrifuged and washed to get FeOOH/Cys product. Finally, 3 mg FeOOH/Cys was dispersed in 50 mL of 2 M NiCl2 solution to chelate Ni2+ ions for 1 h at room temperature. After separation from the NiCl2 solution by centrifugation, the precipitate was washed six times with water. 2.3. Preparation and separation of his-tagged proteins In this study, three different 6 × his-tagged proteins were prepared (SI1): his-tagged ABA INSENSITIVE 2 (his-tagged ABI2ΔN99) [25], histagged OPEN STOMATA 1 (his-tagged OST1) [26] and his-tagged Thioredoxin (his-tagged TRX) [27]. We cloned ABI2ΔN99 and OST1 from Arabidopsis thaliana and constructed them into the pET-28a plasmid. ABI2ΔN99 is the N terminal cut off 99 amino acids from ABI2 and his-TRX is from PET-32a plasmid [28]. The 6 × his-tagged recombinant plasmids were transformed into Escherichia coli strain Rosetta (DE3) (Novagen) for protein expression using standard protocols [29]. Theoretically, all the proteins with 6×his-tagged should be captured by metal-chelate affinity separation method. In the present work, first the FeOOH/Cys-Ni2+ architectures were separated from the solution. After being washed three times with Tris buffer (20 mM), they were added directly into 1.0 mL mixture of cell lysate and shaken for 2 h at a rotation speed of 90 rpm at room temperature. Second, the FeOOH/ Cys-Ni2+ architectures having captured his-tagged proteins were isolated from the solution by centrifugation and washed three times with Tris buffer in order to remove any residual uncaptured proteins. Then, the targeting architectures were washed with 300 μL imidazole solution (0.5 M) to disassociate his-tagged proteins from their surface. Separately collected protein solutions were detected by SDS-PAGE. The architectures as adsorbents can be recovered and reused by washing sequentially with EDTA (0.1 M) and NiCl2 solution (2 M). 2.4. Characterization The morphology and composition were characterized by scanning electron microscopy (SEM, JSM 5600LV, Japan), Fourier transform infrared (FT-IR, AVATAR360, America), X-ray diffraction (XRD, X-Pertpro, Holland) and thermogravimetric analysis (TGA, EXSTAR
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6000), respectively. The separated his-tagged proteins were detected with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE, Power PAC 300, China), with the preconcentration voltage of 70 V and the separation voltage of 120 V. The binding protein concentration was analyzed by BCA Protein Assay Kit, and BSA was a standard sample to make standard curve. 3. Results and discussion The preparation of FeOOH/Cys-Ni2+ architectures and their purification of proteins include three steps. First, FeOOH/Cys architectures were synthesized by solvothermal route. Second, FeOOH/Cys architectures were surface immobilized with a number of Ni2+ through chelate bond, denoted as FeOOH/Cys-Ni2+. Finally, these architectures were directly used to enrich and separate his-tagged proteins from the mixture of cell lysate (Scheme 1). Fig. 1 gives the SEM image (a) and XRD pattern (b) of the product prepared in 1.0g L-Cys. As shown in Fig. 1a, the main products are petals or sheets with mean diameter of several micrometers, which have larger specific surface. It is clear that the XRD pattern exhibits prominent multiple peaks (Fig. 1b). The intense peaks at scattering angles (2θ) of 14.16, 27.08, 36.24 and 46.76 are respectively indexed to scattering from the 020, 021, 130 and 150 crystal planes of the FeOOH crystals (JCPDS No. 76-2301), and the other peaks can be distinguished for the L-cysteine sample (JCPDS No. 13-0722). The result suggests that the as-synthesized products are FeOOH/Cys composite. FT-IR and Raman spectra of the prepared FeOOH/Cys architectures are given in Fig. 2. In Fig. 2a, the band at 890 cm− 1 and 795 cm− 1 corresponds to the \OH of FeOOH vibration [30]. The peaks at 630–670 cm− 1 and 700–745 cm− 1 are attributed to the C\S stretching vibration [31]. There appears a sharp and intense peak at 3000–3500 cm− 1, which corresponds to \NH2 and \OH stretching vibration [32]. The strong peak at 2921 cm− 1 and 2854 cm−1 belongs to the stretching vibration of \CH bond of L-Cys. The peak around 1631 cm−1 is due to \C_O group stretching vibration. In the Raman spectrum (Fig. 2b), L-Cys presents the characteristic peak of thiol group at 2560 cm−1 [33], but FeOOH/Cys architectures show no absorption peak of thiol group, revealing that thiol groups in FeOOH/Cys architectures might exist in the form of S\S bond or others. The FT-IR and Raman analysis indicate the coexistence of L-Cys and FeOOH in the obtained product. After forming free thiol groups by reduction, the content of thiol group of FeOOH/Cys architectures can be quantitatively determined by DTNB method [34]. We studied whether the amount of L-Cys could influence the density of thiol group on the surface of the FeOOH/Cys
Scheme 1. Schematic representation of the preparation procedure of FeOOH/Cys-Ni2+ architectures and their separation of his-tagged proteins.
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Content / µmol/g
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Fig. 1. SEM image (a) and XRD pattern (b) of the prepared sample.
architectures. Fig. 3 plots the content of thiol group of FeOOH/Cys architectures versus the amount of L-Cys in the range of 0.1–8.0 g while the rest of parameters remain unchanged. It can be seen that the content of surface thiol group of FeOOH/Cys architectures declines initially with the increase of L-Cys dosage, and it remains unchanged when the
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amount of L-Cys reaches 6.0 g. Especially, the content of surface thiol group reaches the high value of 637.7 μmol/g when the amount of L-Cys is 1.0 g. TG analysis (Fig. 4) was employed to estimate the weight ratio of inorganic and organic components in FeOOH/Cys sample. For pure L-Cys (curve 1), there is an obvious mass loss (about 80%) from 200 °C to 300 °C, which is attributed to the thermal decomposition of L-Cys. The prepared FeOOH/Cys sample also presents major mass loss in the range of 200 °C to 300 °C (curve 2), which is mainly attributed to the thermal decomposition of L-Cys component. The mass loss is about 35%, which suggests that the sample contains about 45% L-Cys molecules (mole ratio of FeOOH:Cys is 1:1.8). Fig. 5 gives the SEM images of the products prepared with different amounts of L-Cys. At the amount of L-Cys between 1.0 g–2.0 g, the main product was sheet or petal with mean diameter of several micrometers (Fig. 5a, b). When the amount of L-Cys was increased to 4.0g, the sample presented flower-like architecture, consisting of several sheets (Fig. 5c). Further increasing the amount of L-Cys to 6.0 g, regular hexagonal sample was obtained (Fig. 5d). When the amount of L-Cys was 8.0g, the product became flower, which was composed of thicker petals (Fig. 5e). It is clear that the morphology of product is strongly depended on the amount of L-Cys in the reaction system. To estimate the adsorption capacity of FeOOH/Cys architectures, AAS was employed as a general method for determination of metal ions. Fig. 6 is the relationship between the capacity and amount of L-Cys. It can be seen that the Ni2+ ion adsorptive capacity of FeOOH/Cys grows weaker with the increasing of the amount of L-Cys. The adsorption capacity reaches a maximum (444.6 mg/g) when the amount of L-Cys is 1.0 g. In order to verify whether the bonding between FeOOH/Cys and Ni2+ ions is weak, the FeOOH/Cys-Ni2+ samples after chelating Ni2+ ions were collected, which were washed for six times to remove the physical adsorption. The results show that there is little change on the 100
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Fig. 5. SEM images of FeOOH/Cys architectures prepared at different amount of L-Cys: a. 1.0 g; b. 2.0 g; c. 4.0 g; d. 6.0 g; e. 8.0 g.
capacity of FeOOH/Cys after being washed for six times (431.2 mg/g). It suggests that the force between FeOOH/Cys and Ni2+ is strong and FeOOH/Cys-Ni2+ is stable. TG analysis was employed to estimate the weight ratio of inorganic and organic components of as-synthesized FeOOH/Cys and FeOOH/ Cys-Ni2+ samples. Fig. 7 presents the TG curves of FeOOH/Cys (curve
1) and FeOOH/Cys-Ni2+ (curve 2) samples. Both samples undergo a successive weight loss process from room temperature to 700 °C, and their total mass loss is 55% and 37%, respectively. The difference in mass loss is from binding Ni2+ ions and the amount of binding Ni2+ ions is about 18%. FeOOH/Cys-Ni2+ architectures can be used for direct separation of his-tagged proteins from crude cell lysate. To ensure the saturated
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Fig. 8. SDS-PAGE analysis of purified recombinant OST1 proteins. Lane 1, marker; lane 2, E. coli lysate; lanes 3–5, the fractions washed off from the FeOOH/Cys-Ni2+ architectures when different amounts of E. coli lysate were used (lane 3, 250 μL; lane 4, 500 μL; lane 5, 1000 μL); lanes 6–8, the remainder of different amounts of E. coli lysate separated by FeOOH/Cys-Ni (lane 6, 250 μL; lane 7, 500 μL; lane 8, 1000 μL).
adsorption of target proteins, recombinant OST1 solution of different concentrations was used. Fig. 8 gives the SDS-PAGE analysis of recombinant OST1 purified by FeOOH/Cys-Ni2+ architectures. It can be seen that the quantity of captured OST1 proteins was depended directly on the concentration of proteins in cell lysate (Fig. 8, lanes 3–5). The concentration of cell lysate was 655.83 μg/mL. When OST1 proteins were eluted from 250 μL, 500 μL and 1000 μL of E. coli lysate, the capacities of FeOOH/Cys-Ni2+ architectures (3 mg) were 33.61 μg/mg, 39.86 μg/mg and 41.95 μg/mg, respectively (SI2). Lanes 6–8 are the corresponding remainder of cell lysate separated by FeOOH/Cys-Ni2+ architectures (3mg). When the amount of E. coli lysate was very low, the supernatant after separation with FeOOH/Cys-Ni2+ architectures scarcely contained remnant target protein capture (Fig. 8, lane 6). When the amount was increased to 500 μL, there is a weak his-tagged OST1 band (Fig. 8, lane 7), showing that it reaches the max. Fig. 9 gives the SDS-PAGE analysis of recombinant protein purified by FeOOH/Cys-Ni2+ architectures. The molecular weight of his-tagged OST1, his-tagged ABI2 and his-tagged TRX was 34 kDa, 40 kDa and 22 kDa, respectively. It has been found that the quantities of OTS1 bound to FeOOH/Cys-Ni2+ architectures by non-specific adsorption were much less than those specifically captured by these architectures (Fig. 9, lanes 3–6), suggesting that the FeOOH/Cys-Ni2+ architectures have a very high specificity. Obviously, the amount of FeOOH/Cys-Ni2+ architectures was also an important factor on the adsorption quantity of OST1 proteins (Fig. 9, lanes 3–6). The capacity of FeOOH/Cys-Ni2+ architectures (1mg) capturing OTS1 at about 88μgproteinmg−1 from lane 3 in Fig. 9a (SI3) is much higher than that of commercial microbeads at
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Fig. 10. The fractions washed off from FeOOH/Cys-Ni2+ architectures: lane 1, marker; lanes 2–4, E. coli lysate; lanes 5–7, the fractions washed off from the FeOOH/Cys-Ni2+ architectures when different kinds of his-tagged proteins were used (lane 5, his-tagged OST1; lane 6, his-tagged ABI2; lane 7, his-tagged TRX).
only 10–12 μg of protein mg−1 [14]. Such a high efficiency comes from the highly specific surface of the architectures and the corresponding high content of Ni2+ on the surface of the architectures. In addition, the FeOOH/Cys-Ni2+ architectures can be repeatedly used in the experiment, if it was treated with EDTA and NiCl2 on demand. As shown in Fig. 9b, the specificity and affinity of the FeOOH/Cys-Ni2+ architectures remained unaffected after 4 times of recycling use. In order to verify the universality of FeOOH/Cys-Ni2+ architectures, we use FeOOH/Cys-Ni2+ architectures to separate three kinds of histagged proteins (his-tagged OST1, his-tagged ABI2, and his-tagged TRX) from the E. coli lysate solution. Fig. 10 gives the SDS-PAGE analysis of three kinds of recombinant proteins purified by FeOOH/Cys-Ni2+ architectures. It is clear that the three kinds of recombinant proteins can be separated specifically from the E. coli lysate and there was no nonspecific. The results indicate that these architectures can be used effectively in his-tagged protein affinity separation or purification (Fig. 10, lanes 5–7).
4. Conclusion L-Cysteine-functionalized FeOOH architectures (FeOOH/Cys) were prepared through a solvothermal route. The prepared FeOOH/Cys architectures possessed high thiol group density, which can chelate Ni2+ ions to form FeOOH/Cys-Ni2+. The FeOOH/Cys-Ni2+ architectures can capture his-tagged proteins directly from the mixture of lysed cells. The results indicate that the architectures have a high specificity and adsorption capacity, and are especially suitable for purification of histagged proteins.
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Fig. 9. SDS-PAGE analysis of purified recombinant protein by synthesized architectures. (a) The fractions washed off from FeOOH/Cys-Ni2+ architectures: lane 1, marker; lane 2, E. coli lysate; lanes 3–6, the fractions washed off from the FeOOH/Cys-Ni2+ architectures when different amount of FeOOH/Cys-Ni2+ architectures were used (lane 3, 1.0 mg; lane 4, 3.0 mg; lane 5, 5.0 mg; lane 6, 10.0 mg). (b) The fractions released from reused FeOOH/Cys-Ni2+ architectures up to four times: lane 1, marker; lane 2, E. coli lysate; lane 3, 1st; lane 4, 2nd; lane 5, 3rd; lane 6, 4th.
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Acknowledgment Financial support of this work from the National Natural Science Foundation of China (20671029, 21271062), Construction Fund of Henan Province and Department of Education (SBGJ090515) and National Key Basic Special Funds (2012CB114301 to C.-P.S.) are gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.09.046. References [1] H. Seto, Y. Ogata, T. Murakami, Y. Hoshino, Y. Miura, ACS Appl. Mater. Interfaces 4 (2012) 411–417. [2] U.L. Lao, A. Mulchandani, W. Chen, J. Am. Chem. Soc. 128 (2006) 14756–14757. [3] C. Liu, C.F. Monson, T. Yang, H. Pace, P.S. Cremer, Anal. Chem. 83 (2011) 7876–7880. [4] B. Li, X. Zou, Y. Zhao, L. Sun, S. Li, Mater. Sci. Eng. C (2013), http://dx.doi.org/10.1016/ j.msec.2013.02.030. [5] H.Y. Xie, R. Zhen, B. Wang, Y.J. Feng, P. Chen, J. Hao, J. Phys. Chem. C 114 (2010) 4825–4830. [6] L. Yang, C. Guo, S. Chen, F. Wang, J. Wang, Z. An, C. Liu, H. Liu, Ind. Eng. Chem. Res. 48 (2009) 944–950. [7] I.S. Lee, N. Lee, J. Park, B.H. Kim, Y.W. Yi, T. Kim, T.K. Kim, I.H. Lee, S.R. Paik, T. Hyeon, J. Am. Chem. Soc. 128 (2006) 10658–10659. [8] K.S. Lee, I.S. Lee, Chem. Commun. (2008) 709–711. [9] J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Nature 258 (1975) 598–599. [10] F. Xu, J.H. Geiger, G.L. Baker, M.L. Bruening, Langmuir 27 (2011) 3106–3112. [11] R. Ahrends, S. Pieper, B. Neumann, C. Scheler, M.W. Linscheid, Anal. Chem. 81 (2009) 2176–2184.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis of petal-like ferric oxide/cysteine architectures and their application in affinity separation of proteins Xueyan Zou a, Kun Li c,1, Yanbin Yin b, Yanbao Zhao a,⁎, Yu Zhang c, Binjie Li d, Shasha Yao a, Chunpeng Song c a
Key Laboratory for Special Functional Materials, Henan University, Kaifeng 475004, PR China School of Mathematics and Information Sciences, Henan University, Kaifeng 475004, PR China Key Laboratory of Plant Stress Biology, Henan University, Kaifeng 475004, PR China d Key Laboratory of Cellular and Molecular Immunology, Henan University, Kaifeng 475004, PR China b c
a r t i c l e
i n f o
Article history: Received 11 April 2013 Received in revised form 28 August 2013 Accepted 29 September 2013 Available online 8 October 2013 Keywords: Ferric oxide/cysteine Preparation His-tagged protein Separation
a b s t r a c t Petal-like ferric oxide/cysteine (FeOOH/Cys) architectures were prepared through a solvothermal route, which possessed high thiol group density. These thiol groups as binding sites can chelate Ni2+ ions, which can be further used to enrich and separate his-tagged proteins directly from the mixture of lysed cells without sample pretreatment. These results show that the FeOOH/Cys architectures with immobilized Ni2+ ions present negligible nonspecific protein adsorption and high protein adsorption capacity, with the saturation capacity being 88 mg/g, which are especially suitable for purification of his-tagged proteins. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The easy separation and purification of proteins, especially lowabundance proteins, are very important in proteomics [1–4]. The traditional protein separation and purification methods are usually highly selective for target proteins [5,6]. However, they usually contain multistage processes such as precipitation, dialysis, ultrafiltration, and chromatography, which are generally complicated, time-consuming, and expensive for large-scale production. Therefore, there is a need to develop fast separation method of target proteins which are easy to deteriorate. The biotechnologies nowadays enable proteins to easily express with a tag, and many protein-purification methods are based on the specific interactions between immobilized ligands and affinity tags [7,8]. Among affinity tags, histidine tags are particularly popular, which can be readily incorporated into desired proteins by the expression of the target gene in microorganism cells using commercial expression vectors [9,10]. In the case of his-tagged proteins, histidine chains have affinity for certain metal ions such as nickel or copper, and they can specifically interact with the immobilized metal ions to create strong, yet reversible binding [11,12]. More recently, nanoparticles or nanorods have emerged as a promising separation nano-tool in the proteome research [13–16]. Isolation of his-tagged proteins typically involves
⁎ Corresponding author. Tel./fax: +86 378 3881358. E-mail address: [email protected] (Y. Zhao). 1 Identical first author. 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.046
immobilizing metal ions, selectively binding proteins and effectively releasing recombinant proteins from the support surface. Generally, iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED) and nitrolotriacetic acid (NTA) are used as metal chelators [17–24]. However, it is difficult to obtain high density of functional groups on the support surface by post-grafting method and these materials show low surface metal ion density, leading to low purification efficiency. To increase the surface metal ion density, herein we employed Lcysteine as metal chelator to prepare petal-like ferric oxide (FeOOH/ Cys) architectures via a solvothermal route. The prepared FeOOH/Cys architectures possess high density of surface thiol groups which as binding sites can chelate Ni2+ ions. The FeOOH/Cys-Ni2+ architectures as affinity adsorbent are used to directly enrich and separate three kinds of his-tagged proteins from the mixture of cell lysate, which displayed much higher enrichment efficiency than commercial Ni-NTA agarose.
2. Experimental section 2.1. Materials L-Cysteine (≥99.0%) was from Aladdin, China. Nickelous chloride (NiCl2·6H2O, ≥98.0%), ferric chloride crystal (FeCl3·6H2O, ≥99.0%), sodium acetate anhydrous (NaAc, ≥99.0%), ethylene glycol, and absolute alcohol were purchased from Tianjin Kermel Chemical Reagent Co., China. Ni-NTA agarose and BCA protein assay kit were purchased from QIAGEN, Beijing CoWin Biotech. Co. Protein molecular weight marker (Low) was purchased from TakaRa Biotech. Co.
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2.2. Preparation of FeOOH/Cys architectures and adsorption of Ni2+ ions In a typical synthesis, 1.0 g FeCl3·6H2O, 2.7 g NaAc and 1.0 g L-Cys were dissolved in 30 mL ethylene glycol and magnetically stirred for 0.5 h at room temperature. Then, the solution was transferred into a Teflon-lined stainless-steel autoclave, sealed and heated at 200 °C for 8 h. Subsequently, the solution was cooled down to room temperature, centrifuged and washed to get FeOOH/Cys product. Finally, 3 mg FeOOH/Cys was dispersed in 50 mL of 2 M NiCl2 solution to chelate Ni2+ ions for 1 h at room temperature. After separation from the NiCl2 solution by centrifugation, the precipitate was washed six times with water. 2.3. Preparation and separation of his-tagged proteins In this study, three different 6 × his-tagged proteins were prepared (SI1): his-tagged ABA INSENSITIVE 2 (his-tagged ABI2ΔN99) [25], histagged OPEN STOMATA 1 (his-tagged OST1) [26] and his-tagged Thioredoxin (his-tagged TRX) [27]. We cloned ABI2ΔN99 and OST1 from Arabidopsis thaliana and constructed them into the pET-28a plasmid. ABI2ΔN99 is the N terminal cut off 99 amino acids from ABI2 and his-TRX is from PET-32a plasmid [28]. The 6 × his-tagged recombinant plasmids were transformed into Escherichia coli strain Rosetta (DE3) (Novagen) for protein expression using standard protocols [29]. Theoretically, all the proteins with 6×his-tagged should be captured by metal-chelate affinity separation method. In the present work, first the FeOOH/Cys-Ni2+ architectures were separated from the solution. After being washed three times with Tris buffer (20 mM), they were added directly into 1.0 mL mixture of cell lysate and shaken for 2 h at a rotation speed of 90 rpm at room temperature. Second, the FeOOH/ Cys-Ni2+ architectures having captured his-tagged proteins were isolated from the solution by centrifugation and washed three times with Tris buffer in order to remove any residual uncaptured proteins. Then, the targeting architectures were washed with 300 μL imidazole solution (0.5 M) to disassociate his-tagged proteins from their surface. Separately collected protein solutions were detected by SDS-PAGE. The architectures as adsorbents can be recovered and reused by washing sequentially with EDTA (0.1 M) and NiCl2 solution (2 M). 2.4. Characterization The morphology and composition were characterized by scanning electron microscopy (SEM, JSM 5600LV, Japan), Fourier transform infrared (FT-IR, AVATAR360, America), X-ray diffraction (XRD, X-Pertpro, Holland) and thermogravimetric analysis (TGA, EXSTAR
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6000), respectively. The separated his-tagged proteins were detected with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE, Power PAC 300, China), with the preconcentration voltage of 70 V and the separation voltage of 120 V. The binding protein concentration was analyzed by BCA Protein Assay Kit, and BSA was a standard sample to make standard curve. 3. Results and discussion The preparation of FeOOH/Cys-Ni2+ architectures and their purification of proteins include three steps. First, FeOOH/Cys architectures were synthesized by solvothermal route. Second, FeOOH/Cys architectures were surface immobilized with a number of Ni2+ through chelate bond, denoted as FeOOH/Cys-Ni2+. Finally, these architectures were directly used to enrich and separate his-tagged proteins from the mixture of cell lysate (Scheme 1). Fig. 1 gives the SEM image (a) and XRD pattern (b) of the product prepared in 1.0g L-Cys. As shown in Fig. 1a, the main products are petals or sheets with mean diameter of several micrometers, which have larger specific surface. It is clear that the XRD pattern exhibits prominent multiple peaks (Fig. 1b). The intense peaks at scattering angles (2θ) of 14.16, 27.08, 36.24 and 46.76 are respectively indexed to scattering from the 020, 021, 130 and 150 crystal planes of the FeOOH crystals (JCPDS No. 76-2301), and the other peaks can be distinguished for the L-cysteine sample (JCPDS No. 13-0722). The result suggests that the as-synthesized products are FeOOH/Cys composite. FT-IR and Raman spectra of the prepared FeOOH/Cys architectures are given in Fig. 2. In Fig. 2a, the band at 890 cm− 1 and 795 cm− 1 corresponds to the \OH of FeOOH vibration [30]. The peaks at 630–670 cm− 1 and 700–745 cm− 1 are attributed to the C\S stretching vibration [31]. There appears a sharp and intense peak at 3000–3500 cm− 1, which corresponds to \NH2 and \OH stretching vibration [32]. The strong peak at 2921 cm− 1 and 2854 cm−1 belongs to the stretching vibration of \CH bond of L-Cys. The peak around 1631 cm−1 is due to \C_O group stretching vibration. In the Raman spectrum (Fig. 2b), L-Cys presents the characteristic peak of thiol group at 2560 cm−1 [33], but FeOOH/Cys architectures show no absorption peak of thiol group, revealing that thiol groups in FeOOH/Cys architectures might exist in the form of S\S bond or others. The FT-IR and Raman analysis indicate the coexistence of L-Cys and FeOOH in the obtained product. After forming free thiol groups by reduction, the content of thiol group of FeOOH/Cys architectures can be quantitatively determined by DTNB method [34]. We studied whether the amount of L-Cys could influence the density of thiol group on the surface of the FeOOH/Cys
Scheme 1. Schematic representation of the preparation procedure of FeOOH/Cys-Ni2+ architectures and their separation of his-tagged proteins.
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Content / µmol/g
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Fig. 1. SEM image (a) and XRD pattern (b) of the prepared sample.
architectures. Fig. 3 plots the content of thiol group of FeOOH/Cys architectures versus the amount of L-Cys in the range of 0.1–8.0 g while the rest of parameters remain unchanged. It can be seen that the content of surface thiol group of FeOOH/Cys architectures declines initially with the increase of L-Cys dosage, and it remains unchanged when the
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amount of L-Cys reaches 6.0 g. Especially, the content of surface thiol group reaches the high value of 637.7 μmol/g when the amount of L-Cys is 1.0 g. TG analysis (Fig. 4) was employed to estimate the weight ratio of inorganic and organic components in FeOOH/Cys sample. For pure L-Cys (curve 1), there is an obvious mass loss (about 80%) from 200 °C to 300 °C, which is attributed to the thermal decomposition of L-Cys. The prepared FeOOH/Cys sample also presents major mass loss in the range of 200 °C to 300 °C (curve 2), which is mainly attributed to the thermal decomposition of L-Cys component. The mass loss is about 35%, which suggests that the sample contains about 45% L-Cys molecules (mole ratio of FeOOH:Cys is 1:1.8). Fig. 5 gives the SEM images of the products prepared with different amounts of L-Cys. At the amount of L-Cys between 1.0 g–2.0 g, the main product was sheet or petal with mean diameter of several micrometers (Fig. 5a, b). When the amount of L-Cys was increased to 4.0g, the sample presented flower-like architecture, consisting of several sheets (Fig. 5c). Further increasing the amount of L-Cys to 6.0 g, regular hexagonal sample was obtained (Fig. 5d). When the amount of L-Cys was 8.0g, the product became flower, which was composed of thicker petals (Fig. 5e). It is clear that the morphology of product is strongly depended on the amount of L-Cys in the reaction system. To estimate the adsorption capacity of FeOOH/Cys architectures, AAS was employed as a general method for determination of metal ions. Fig. 6 is the relationship between the capacity and amount of L-Cys. It can be seen that the Ni2+ ion adsorptive capacity of FeOOH/Cys grows weaker with the increasing of the amount of L-Cys. The adsorption capacity reaches a maximum (444.6 mg/g) when the amount of L-Cys is 1.0 g. In order to verify whether the bonding between FeOOH/Cys and Ni2+ ions is weak, the FeOOH/Cys-Ni2+ samples after chelating Ni2+ ions were collected, which were washed for six times to remove the physical adsorption. The results show that there is little change on the 100
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Fig. 5. SEM images of FeOOH/Cys architectures prepared at different amount of L-Cys: a. 1.0 g; b. 2.0 g; c. 4.0 g; d. 6.0 g; e. 8.0 g.
capacity of FeOOH/Cys after being washed for six times (431.2 mg/g). It suggests that the force between FeOOH/Cys and Ni2+ is strong and FeOOH/Cys-Ni2+ is stable. TG analysis was employed to estimate the weight ratio of inorganic and organic components of as-synthesized FeOOH/Cys and FeOOH/ Cys-Ni2+ samples. Fig. 7 presents the TG curves of FeOOH/Cys (curve
1) and FeOOH/Cys-Ni2+ (curve 2) samples. Both samples undergo a successive weight loss process from room temperature to 700 °C, and their total mass loss is 55% and 37%, respectively. The difference in mass loss is from binding Ni2+ ions and the amount of binding Ni2+ ions is about 18%. FeOOH/Cys-Ni2+ architectures can be used for direct separation of his-tagged proteins from crude cell lysate. To ensure the saturated
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Fig. 8. SDS-PAGE analysis of purified recombinant OST1 proteins. Lane 1, marker; lane 2, E. coli lysate; lanes 3–5, the fractions washed off from the FeOOH/Cys-Ni2+ architectures when different amounts of E. coli lysate were used (lane 3, 250 μL; lane 4, 500 μL; lane 5, 1000 μL); lanes 6–8, the remainder of different amounts of E. coli lysate separated by FeOOH/Cys-Ni (lane 6, 250 μL; lane 7, 500 μL; lane 8, 1000 μL).
adsorption of target proteins, recombinant OST1 solution of different concentrations was used. Fig. 8 gives the SDS-PAGE analysis of recombinant OST1 purified by FeOOH/Cys-Ni2+ architectures. It can be seen that the quantity of captured OST1 proteins was depended directly on the concentration of proteins in cell lysate (Fig. 8, lanes 3–5). The concentration of cell lysate was 655.83 μg/mL. When OST1 proteins were eluted from 250 μL, 500 μL and 1000 μL of E. coli lysate, the capacities of FeOOH/Cys-Ni2+ architectures (3 mg) were 33.61 μg/mg, 39.86 μg/mg and 41.95 μg/mg, respectively (SI2). Lanes 6–8 are the corresponding remainder of cell lysate separated by FeOOH/Cys-Ni2+ architectures (3mg). When the amount of E. coli lysate was very low, the supernatant after separation with FeOOH/Cys-Ni2+ architectures scarcely contained remnant target protein capture (Fig. 8, lane 6). When the amount was increased to 500 μL, there is a weak his-tagged OST1 band (Fig. 8, lane 7), showing that it reaches the max. Fig. 9 gives the SDS-PAGE analysis of recombinant protein purified by FeOOH/Cys-Ni2+ architectures. The molecular weight of his-tagged OST1, his-tagged ABI2 and his-tagged TRX was 34 kDa, 40 kDa and 22 kDa, respectively. It has been found that the quantities of OTS1 bound to FeOOH/Cys-Ni2+ architectures by non-specific adsorption were much less than those specifically captured by these architectures (Fig. 9, lanes 3–6), suggesting that the FeOOH/Cys-Ni2+ architectures have a very high specificity. Obviously, the amount of FeOOH/Cys-Ni2+ architectures was also an important factor on the adsorption quantity of OST1 proteins (Fig. 9, lanes 3–6). The capacity of FeOOH/Cys-Ni2+ architectures (1mg) capturing OTS1 at about 88μgproteinmg−1 from lane 3 in Fig. 9a (SI3) is much higher than that of commercial microbeads at
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Fig. 10. The fractions washed off from FeOOH/Cys-Ni2+ architectures: lane 1, marker; lanes 2–4, E. coli lysate; lanes 5–7, the fractions washed off from the FeOOH/Cys-Ni2+ architectures when different kinds of his-tagged proteins were used (lane 5, his-tagged OST1; lane 6, his-tagged ABI2; lane 7, his-tagged TRX).
only 10–12 μg of protein mg−1 [14]. Such a high efficiency comes from the highly specific surface of the architectures and the corresponding high content of Ni2+ on the surface of the architectures. In addition, the FeOOH/Cys-Ni2+ architectures can be repeatedly used in the experiment, if it was treated with EDTA and NiCl2 on demand. As shown in Fig. 9b, the specificity and affinity of the FeOOH/Cys-Ni2+ architectures remained unaffected after 4 times of recycling use. In order to verify the universality of FeOOH/Cys-Ni2+ architectures, we use FeOOH/Cys-Ni2+ architectures to separate three kinds of histagged proteins (his-tagged OST1, his-tagged ABI2, and his-tagged TRX) from the E. coli lysate solution. Fig. 10 gives the SDS-PAGE analysis of three kinds of recombinant proteins purified by FeOOH/Cys-Ni2+ architectures. It is clear that the three kinds of recombinant proteins can be separated specifically from the E. coli lysate and there was no nonspecific. The results indicate that these architectures can be used effectively in his-tagged protein affinity separation or purification (Fig. 10, lanes 5–7).
4. Conclusion L-Cysteine-functionalized FeOOH architectures (FeOOH/Cys) were prepared through a solvothermal route. The prepared FeOOH/Cys architectures possessed high thiol group density, which can chelate Ni2+ ions to form FeOOH/Cys-Ni2+. The FeOOH/Cys-Ni2+ architectures can capture his-tagged proteins directly from the mixture of lysed cells. The results indicate that the architectures have a high specificity and adsorption capacity, and are especially suitable for purification of histagged proteins.
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Fig. 9. SDS-PAGE analysis of purified recombinant protein by synthesized architectures. (a) The fractions washed off from FeOOH/Cys-Ni2+ architectures: lane 1, marker; lane 2, E. coli lysate; lanes 3–6, the fractions washed off from the FeOOH/Cys-Ni2+ architectures when different amount of FeOOH/Cys-Ni2+ architectures were used (lane 3, 1.0 mg; lane 4, 3.0 mg; lane 5, 5.0 mg; lane 6, 10.0 mg). (b) The fractions released from reused FeOOH/Cys-Ni2+ architectures up to four times: lane 1, marker; lane 2, E. coli lysate; lane 3, 1st; lane 4, 2nd; lane 5, 3rd; lane 6, 4th.
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Acknowledgment Financial support of this work from the National Natural Science Foundation of China (20671029, 21271062), Construction Fund of Henan Province and Department of Education (SBGJ090515) and National Key Basic Special Funds (2012CB114301 to C.-P.S.) are gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.09.046. References [1] H. Seto, Y. Ogata, T. Murakami, Y. Hoshino, Y. Miura, ACS Appl. Mater. Interfaces 4 (2012) 411–417. [2] U.L. Lao, A. Mulchandani, W. Chen, J. Am. Chem. Soc. 128 (2006) 14756–14757. [3] C. Liu, C.F. Monson, T. Yang, H. Pace, P.S. Cremer, Anal. Chem. 83 (2011) 7876–7880. [4] B. Li, X. Zou, Y. Zhao, L. Sun, S. Li, Mater. Sci. Eng. C (2013), http://dx.doi.org/10.1016/ j.msec.2013.02.030. [5] H.Y. Xie, R. Zhen, B. Wang, Y.J. Feng, P. Chen, J. Hao, J. Phys. Chem. C 114 (2010) 4825–4830. [6] L. Yang, C. Guo, S. Chen, F. Wang, J. Wang, Z. An, C. Liu, H. Liu, Ind. Eng. Chem. Res. 48 (2009) 944–950. [7] I.S. Lee, N. Lee, J. Park, B.H. Kim, Y.W. Yi, T. Kim, T.K. Kim, I.H. Lee, S.R. Paik, T. Hyeon, J. Am. Chem. Soc. 128 (2006) 10658–10659. [8] K.S. Lee, I.S. Lee, Chem. Commun. (2008) 709–711. [9] J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Nature 258 (1975) 598–599. [10] F. Xu, J.H. Geiger, G.L. Baker, M.L. Bruening, Langmuir 27 (2011) 3106–3112. [11] R. Ahrends, S. Pieper, B. Neumann, C. Scheler, M.W. Linscheid, Anal. Chem. 81 (2009) 2176–2184.
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