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Facial synthesis of nickel(II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins
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Facial synthesis of nickel(II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins Xiao-Mei He, Bi-Feng Yuan, Yu-Qi Feng ∗ Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China
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Article history: Received 6 April 2016 Received in revised form 8 June 2016 Accepted 17 June 2016 Available online xxx Keywords: Ni2+ -immobilized carboxyl cotton chelator (CCC-Ni2+ ) Immobilized metal ion affinity chromatography (IMAC) His-tagged Proteins E. coli cell lysates
a b s t r a c t Immobilized metal affinity chromatography (IMAC) technique is frequently used in the purification of histidine-tagged (His-tagged) recombinant proteins. In this study, nickel(II)-immobilized carboxyl cotton chelator (CCC-Ni2+ ) fibers was synthesized by a simple method based on the coordination effect between Ni2+ and carboxyl group. The nickel content of the CCC-Ni2+ fibers was determined to be 5 times larger than that of Ni2+ -immobilized sulfhydryl cotton fiber (SCF-Ni2+ ) fibers developed in our previous work. The prepared CCC-Ni2+ fibers were then applied for the selective and rapid separation of His-tagged protein from escherichia coli (E. coli) cell lysates on the basis of the high affinity of Ni2+ to 6 × His with a labin-syringe format. Benefiting from the good biological compatibility and high nickel content, the results showed that CCC-Ni2+ fibers were able to selectively capture His-tagged proteins from complex E. coli cell lysates and exhibited a relatively large adsorption capacity toward His-tagged protein. The recoveries of His-tagged GFP in E. coli cell lysates were in the range of 89.8%-106.7% with the relative standard deviations (RSDs) less than 9.4% (intra-day) and 10.3% (inter-day). Taken together, this efficient approach for the purification of recombinant proteins extends the application of CCC-based fibrous materials in biological analysis. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Immobilized metal affinity chromatography (IMAC), an important tool for protein purification, is based on the affinity of transition metal ions on the IMAC supports toward electron-donor groups on protein [1,2]. Thereinto, IMAC technique is frequently used in the purification of histidine-tagged (His-tagged) recombinant proteins, which is generally based on the affinity of Ni2+ ions toward a string of six histidine (6 × His) fused to the recombinant proteins [3–5]. His-tagged proteins can be selectively enriched by the IMAC adsorbent while untagged proteins are removed. Among these adsorbents, nitrilotriacetic acid (NTA)-attached resin with immobilized ions Ni2+ , is one of the most used adsorbent for separating His-tagged fusion proteins [5–8]. Although the NTA-Ni2+ resin can be used in various protein expression systems, it is still limited by a long separation time, solvent consumption and high cost [9,10].
∗ Corresponding author at: Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China. E-mail address: [email protected] (Y.-Q. Feng).
To develop more rapid and convenient methods for the purpose, a variety of magnetic materials [11–15] have been exploited for the purification of His-tagged protein. However, the synthetic processes for most of the magnetic materials are complicated and time-consuming [16–18]. In addition, inorganic supports always suffer from the limitation of irreversible non-specific adsorption [1]. In this respect, natural fibrous material with excellent biological compatibility and good operability, can be an alternative matrix for biological research. Cotton fiber is an important natural material and widely used as adsorbent due to its merits including high mechanical strength, stable chemical resistance and wide availability [19,20]. For example, a novel cotton fiber-packed pipet-tip was developed by Selmen’s group and successfully applied for microscale enrichment of glycans and glycopeptides based on hydrophilic interaction [21]. However, the monotonous functional groups on the cotton fiber will restrict its broad applications. In this respect, sulfhydryl cotton fiber (SCF) was introduced as an IMAC support to immobilize Ni2+ ions based on the coordination effect between Ni2+ and thiol group, and the Ni2+ -immobilized sulfhydryl cotton fiber (SCF-Ni2+ ) adsorbent was used for the selective enrichment of His-tagged proteins in our recent work [22]. Thanks to the high reactivity of thiol group, SCF was further functional by “thiol-ene” click chemistry to
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Fig. 1. Schematic diagram of the preparation of CCC-Ni2+ fibers and lab-in-syringe SPE.
Fig. 2. SEM images of (a) cotton, (b) CCC, and (c) CCC-Ni2+ fibers; (d) IR spectra of cotton, CCC and CCC-Ni2+ fibers.
prepare two kinds of high specific adsorbents for biological analysis [23]. Very recently, carboxyl cotton chelator (CCC) with two free carboxyl groups on each structure unit was also exploited as IMAC support for the binding of metal ions in our work [24]. A novel CCC-Ti4+ fibrous material with high titanium content was prepared and subsequently proved to be a good adsorbent for phosphoproteomics analysis. Moreover, it is worth developing more CCC-based IMAC adsorbents due to its excellent binding capacity for metal ions and good biological compatibility. In this study, a novel CCC-Ni2+ fibrous adsorbent was successfully prepared with a simple method on the basis of the coordination effect between Ni2+ and carboxyl group. Due to the
high affinity of Ni2+ to 6 × His on His-tagged proteins, CCC-Ni2+ fibers were applied for selective enrichment of His-tagged protein from E. coli cell lysates using the lab-in-syringe format. 2. Experimental section 2.1. Reagents and materials Citric acid, aqueous ammonia solution (NH3 ·H2 O, 25 wt%), tris (hydromethyl) aminomethane (Tris), hydrochloric acid (HCl), sodium chloride (NaCl) and nickel(II) chloride hexahydrate (NiCl2 ·6H2 O, 98%) were all of analytical grade and purchased
Please cite this article in press as: X.-M. He, et al., Facial synthesis of nickel(II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins, J. Chromatogr. B (2016), http://dx.doi.org/10.1016/j.jchromb.2016.06.029
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Fig. 5. Calibration curve of His-tagged GFP. Fig. 3. Fluorescent spectra of His-tagged GFP. Direct analysis (a), analysis of sampling eluate after enrichment with CCC-Ni2+ fibers (b), and analysis of the released His-tagged GFP in imidazole solution (c).
2.2. Preparation of the carboxyl cotton chelator-Ni2+ (CCC-Ni2+ ) adsorbent and the lab-in-syringe SPE Carboxyl cotton chelator (CCC) was prepared according to our previous work [24]. As show in Fig. 1, CCC contains two free carboxyl groups on each structure unit, which can chelate with metal ion to make CCC-based IMAC material. In our previous work, a CCC-Ti4+ adsorbent with high titanium content was successfully prepared and applied for phosphoproteomics analysis [24]. Thanks to the high binding capacity for metal ions and good biological compatibility, CCC was further used for binding Ni2+ ions to obtain a new CCC-Ni2+ adsorbent in this study (Fig. 1). Briefly, 400 mg of CCC was mixed with 40 mL of nickel ion solution (100 mM Tris, 100 mM NaCl, 100 mM NiCl2 ·6H2 O, pH 7.2). After incubation at 25 ◦ C for 2 h, the as-prepared CCC-Ni2+ fibers were washed with distilled water to remove residual Ni2+ ions followed by drying under vacuum at 60 ◦ C for 6 h. For comparison, SCF-Ni2+ fibers proposed in our previous work were also prepared according to the literature [22]. Lab-in-syringe SPE was prepared according to our previous work [24]. 2.3. Characterization of fibers
Fig. 4. (a) Fluorescent images from the mixture solutions of His-tagged GFP and normal mouse IgG1 labeled by Cy5. Direct analysis (A), analysis of sampling eluate after enrichment with CCC-Ni2+ fibers (B), and analysis of the released proteins in imidazole solution (C). (b) Fluorescent spectra of His-tagged GFP in the mixture solutions. Direct analysis (A), analysis of sampling eluate after enrichment with CCCNi2+ fibers (B), and analysis of the released His-tagged GFP in imidazole solution (C). (c) Fluorescent spectra of normal mouse IgG1 labeled by Cy5 in the mixture solutions. Direct analysis (A) and analysis of sampling eluate after enrichment with CCC-Ni2+ fibers (B).
from Sinopharm Chemical Reagent Co. (Shanghai, China). Imidazole (C3 H4 N2 ) and nickel standard (0.5 mg mL−1 in 1% nitric acid solution) were purchased from Aladdin Chemical Reagent Co. (Shanghai, China). Purified water was obtained with a MilliQ apparatus (Millipore, Bedford, MA, USA). Histidine-tagged green fluorescent protein (His-tagged GFP) and normal mouse IgG1 conjugated by PE-Cy5 were purchased from Cusabio Biotech Co. (Wuhan, China) and Santa Cruz Biotechnology (Texas, USA), respectively.
The nickel contents of CCC-Ni2+ and SCF-Ni2+ were examined by a graphite furnace atomic absorption spectrometry (GF AAS) (Shimadzu, Japan). The microscopic morphology of fibers was determined by a Quanta 200 scanning electron microscopy (SEM) (FEI, Holand). Fourier transform infrared spectroscopy (FTIR) was carried out with a Thermo Nicolet 670 FT-IR instrument (Boston, MA, USA). 2.4. Extraction of His-tagged GFP by CCC-Ni2+ fiber-packed syringe SPE To investigate the protein separation efficiency of CCC-Ni2+ fiber, a standard mixed-protein solution of His-tagged GFP and untagged normal mouse IgG1 conjugated by PE-Cy5 was used. Briefly, 500 L of mixture solution in Tris buffer (20 mM Tris, 100 mM NaCl, pH 8.0) was pipetted up and down by CCC-Ni2+ fiberpacked syringe SPE to ensure full adsorption of proteins. Then the adsorbed His-tagged GFP was eluted by an imidazole solution (1 M). The whole enrichment process could be finished within 3 min. Finally, the sampling eluate and desorption solution were analyzed by spectrofluorometer. CCC-Ni2+ fiber-packed syringe SPE was further used for the purification of His-tagged proteins from E. coli cell lysates. The
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CCC-Ni2+ may benefit from the good stability and abundant amount of carboxyl groups on CCC, as well as the strong interaction between Ni2+ and coordinated carboxylate groups. 3.2. Evaluation of the property of CCC-Ni2+ fiber-packed syringe SPE toward His-tagged proteins
Fig. 6. Analysis of the cell lysate containing His-tagged GFP by SDS-PAGE. Direct analysis (lane 1), analysis after enrichment with CCC fibers (lane 2), or CCC-Ni2+ fibers (lane 3), or SCF-Ni2+ fibers (lane 4). Lane M, protein markers.
GW5100 escherichia coli (E. coli) cells were cultured according to our previous method [25]. In the typical experiment, His-tagged GFP (30 KDa) was spiked into lysates to make spiked-lysate samples with a series of spiking concentrations. The sample processing was the same as that of the standard mixed-protein enrichment. Finally, the recovered His-tagged GFP was analyzed by spectrofluorometer and SDS-PAGE. For comparison, CCC and SCF-Ni2+ fibers were applied to enrich His-tagged proteins from E. coli cell lysates. The extraction procedure was the same as that of CCC-Ni2+ fibers. 2.5. Instruments All photoluminescence (PL) spectra were recorded on a PerkinElmer LS 55 spectrofluorometer with an excitation wavelength of 490 nm (for GFP) and 565 nm (for Cy5). 3. Results and discussion 3.1. Synthesis and characterization of CCC-Ni2+ fibers The SEM images showed the surface morphologies of cotton (Fig. 2a), CCC (Fig. 2b), and CCC-Ni2+ (Fig. 2c) were almost the same with diameters in the range of 10 m to 20 m. In addition, the fiber strength of cotton was well maintained, thus the subsequent enrichment experiment could be carried out in the lab-in-syringe format. The infrared (IR) spectra of cotton, CCC and CCC-Ni2+ were shown in Fig. 2d. A new absorption peak (1721 cm−1 ) observed in the IR spectrum of CCC could be attributed to the stretching vibration of C O, indicating the successful synthesis of CCC via esterification. Two peaks at 1572 and 1429 cm−1 could be attributed to the asymmetric (vasym ) and symmetric (vsym ) stretching vibrations of coordinated carboxylate groups. The separation between the asymmetric and symmetric stretching frequencies (v = vasym − vsym ) was 143 cm−1 , which indicated the CCC was bidentate coordinating through both carboxylic groups [26,27]. The nickel content of fibers was examined by a graphite furnace atomic absorption spectrometry (GF AAS). In our previous work [22], a fibrous SCF-Ni2+ adsorbent was developed and applied for the purification of His-tagged protein. Here, SCF-Ni2+ fibers were used for a series of comparison with CCC-Ni2+ fibers. The nickel contents of CCC-Ni2+ fibers and SCF-Ni2+ fibers were estimated to be 58.2 and 11.1 g mg−1 , respectively. The higher content of nickel of
His-tagged GFP was used to investigate the enrichment capacity of CCC-Ni2+ fibers toward His-tagged proteins. The selectivity and efficiency of CCC-Ni2+ fibers for the enrichment of His-tagged proteins could be firstly monitored under UV excitation. The original His-tagged GFP solution showed green emission under UV extraction (A in the inset of Fig. 3). After treating with CCC-Ni2+ fibers, the protein solution became colorless (B in the inset of Fig. 3) and the fluorescent emission intensity of the sampling eluate (Fig. 3b) decreased to 4.3% of the initial intensity (Fig. 3a), demonstrating that His-tagged GFP was almost completely removed from the protein solution. After the release of His-tagged GFP from CCCNi2+ fibers with imidazole solution, the imidazole solution emitted strong green fluorescence (C in the inset of Fig. 3) and the fluorescent emission intensity had a 92.8% recovery of the initial intensity (Fig. 3c), indicating the efficient dissociability of the attached Histagged protein from CCC-Ni2+ fibers by imidazole. To investigate the enrichment specificity of CCC-Ni2+ fibers toward His-tagged proteins, a mixed-protein solution of His-tagged GFP and untagged normal mouse IgG1 (labeled by red-emitting Cy5) was used. The normal mouse IgG1 showed a red emission under UV excitation. As shown in Fig. 4a, the mixed-protein solution initially emited a yellow fluorescence under UV excitation (A in the inset of Fig. 4a). After treating with CCC-Ni2+ fibers, the remaining protein solution showed a red emission (B in the inset of Fig. 4a). The fluorescent emission intensity of His-tagged GFP in the remaining solution decreased to be 3.1% of the initial intensity (Fig. 4b), while only a 16.9% decrease in the fluorescent emission intensity of the normal mouse IgG1 was observed (Fig. 4c), indicating that His-tagged GFP was selectively separated from the mixed-protein solution and normal mouse IgG1 was left in the sampling eluate. Then the bound CCC-Ni2+ was treated with imidazole solution to release the His-tagged GFP, generating a green fluorescence (C in the inset of Fig. 4a) and a 89.2% recovery of the fluorescent emission intensity (Fig. 4b). The results showed that CCC-Ni2+ fibers exhibited superior binding property and specificity to the His-tagged proteins. The adsorption capacities of CCC-Ni2+ fibers and SCF-Ni2+ fibers toward His-tagged proteins were examined using His-tagged GFP as a probe. The adsorption capacity of CCC-Ni2+ fibers was estimated to be 1.8 g mg−1 for His-tagged GFP by the breakthrough curve measurement (Fig. S1), which was higher than that obtained by SCF-Ni2+ fibers (1.0 g mg−1 ). A comparative study of this method to previous works [9,22,28,29] was performed (Table S1). It can be seen that the developed method with lab-in-syringe SPE format was convenient and rapid, and high extraction recovery of His-tagged protein could be achieved, which could satisfy the practical application. 3.3. The reproducibility of CCC-Ni2+ fiber-packed syringe SPE The reproducibility of CCC-Ni2+ fiber-packed syringe SPE was determined by the intra- and inter-batch precisions. The intrabatch relative standard deviation (RSD) was determined on three parallel experiments by CCC-Ni2+ fiber-packed syringes prepared from the same batch, and three syringes prepared from different batches gave the inter-batch RSD. Satisfactory precisions were obtained with RSDs at 7.4% (intra-batch) and 12.5% (inter-batch), demonstrating the good reproducibility of the proposed method.
Please cite this article in press as: X.-M. He, et al., Facial synthesis of nickel(II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins, J. Chromatogr. B (2016), http://dx.doi.org/10.1016/j.jchromb.2016.06.029
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Table 1 Accuracy and precision (intra- and inter-day RSDs) for the determination of His-tagged GFP in E. coli cell lysates.
Intra-day precision (RSD, %, n = 3) Inter-day precision (RSD, %, n = 3) Recovery (%, n = 3)
Low (0.5 g mL−1 )
Medium (1.0 g mL−1 )
High (5.0 g mL−1 )
9.4 10.3 106.7 ± 8.9
8.7 7.5 98.4 ± 6.6
6.5 8.0 89.8 ± 5.9
3.4. Enrichment of His-tagged proteins from E. coli cell lysates by CCC-Ni2+ fiber-packed syringe SPE To further explore the practical application of this method, the separation of His-tagged proteins from E. coli cell lysates was examined. A calibration curve was constructed using E. coli cell lysates spiked with His-tagged GFP at different concentrations ranging from 0.1 to 7.0 g mL−1 with triplicate measurements (Fig. 5). Linearity of the calibration curve ranged from 0.1 to 7.0 g mL−1 with a correlation coefficient of 0.9961. The detection limit (3, where is the relative standard deviation of a blank solution, n = 11) was estimated to be 0.013 g mL−1 . The accuracy and precision of this method were evaluated by the recoveries and the intra- and inter-day RSDs, which were measured with His-tagged GFP spiked E. coli cell lysates at three concentrations (0.5, 1.0 and 5.0 g mL−1 ). For each concentration, triplicate measurements were performed. Intra-day RSD was calculated by repeating the analysis for three times within one day and the interday RSD was investigated on three successive days. The relative recoveries were evaluated by comparing the calculated concentration of His-tagged GFP in the E. coli cell lysates to the actual spiked concentration. As shown in Table 1, the intra- and inter-day RSDs were less than 9.4% and 10.3%, respectively, and the relative recoveries were in the range of 89.8%-106.7% with the RSDs less than 8.9%, demonstrating that good accuracy and precision were achieved. SDS-PAGE gel electrophoresis was also employed to evaluate the performance of the developed method. For the direct analysis of His-tagged GFP spiked E. coli cell lysates (5.0 g mL−1 ), His-tagged GFP couldn’t be easily distinguished due to the plenty of other proteins (Fig. 6, lane 1). After treating with CCC-Ni2+ fibers, His-tagged GFP was well separated from the complex cell lysates and clearly observed in the desorption solution (1 M of imidazole) (Fig. 6, lane 3), which visually indicated the good selectivity of CCC-Ni2+ fibers toward His-tagged proteins. By comparison, it was hardly to identify His-tagged GFP when using CCC for the extraction (Fig. 6, lane 2), demonstrating the high selectivity of CCC-Ni2+ fibers toward His-tagged protein came from the affinity between the Ni2+ ions and 6 × His. In addition, SCF-Ni2+ fibers were also applied to enrich His-tagged protein from cell lysates. Although His-tagged GFP could be well separated by SCF-Ni2+ (Fig. 6, lane 4), the recovery of Histagged GFP was lower than that by CCC-Ni2+ fibers (compared lane 3 with lane 4 in Fig. 6). The results showed that a good performance of His-tagged protein purification from complex biological sample was achieved by CCC-Ni2+ fibers, indicating a great potential of the proposed method in actual applications. 4. Conclusion In the current study, a simple approach for the facile synthesis of CCC-Ni2+ fibers was developed. Based on the high affinity of Ni2+ to 6 × His, CCC-Ni2+ fibers were applied for the selective enrichment of His-tagged protein from E. coli cell lysates using the lab-in-syringe format, which was simple, rapid and low cost. Benefiting from good biological compatibility and high content of nickel, CCC-Ni2+ fibers exhibited high selectivity and adsorption capacity toward His-tagged protein. Taken together, the proposed method expands the application of CCC-based fibrous materials in the purification of
recombinant proteins, and we expect more cotton-based materials will be exploited as adsorbents for biological analysis.
Acknowledgment The authors thank the financial support from the National Basic Research Program of China (973 Program) (2013CB910702), the National Natural Science Foundation of China (21475098), and the Natural Science Foundation of Hubei Province (2014CFA002).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2016. 06.029.
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Facial synthesis of nickel(II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins Xiao-Mei He, Bi-Feng Yuan, Yu-Qi Feng ∗ Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China
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Article history: Received 6 April 2016 Received in revised form 8 June 2016 Accepted 17 June 2016 Available online xxx Keywords: Ni2+ -immobilized carboxyl cotton chelator (CCC-Ni2+ ) Immobilized metal ion affinity chromatography (IMAC) His-tagged Proteins E. coli cell lysates
a b s t r a c t Immobilized metal affinity chromatography (IMAC) technique is frequently used in the purification of histidine-tagged (His-tagged) recombinant proteins. In this study, nickel(II)-immobilized carboxyl cotton chelator (CCC-Ni2+ ) fibers was synthesized by a simple method based on the coordination effect between Ni2+ and carboxyl group. The nickel content of the CCC-Ni2+ fibers was determined to be 5 times larger than that of Ni2+ -immobilized sulfhydryl cotton fiber (SCF-Ni2+ ) fibers developed in our previous work. The prepared CCC-Ni2+ fibers were then applied for the selective and rapid separation of His-tagged protein from escherichia coli (E. coli) cell lysates on the basis of the high affinity of Ni2+ to 6 × His with a labin-syringe format. Benefiting from the good biological compatibility and high nickel content, the results showed that CCC-Ni2+ fibers were able to selectively capture His-tagged proteins from complex E. coli cell lysates and exhibited a relatively large adsorption capacity toward His-tagged protein. The recoveries of His-tagged GFP in E. coli cell lysates were in the range of 89.8%-106.7% with the relative standard deviations (RSDs) less than 9.4% (intra-day) and 10.3% (inter-day). Taken together, this efficient approach for the purification of recombinant proteins extends the application of CCC-based fibrous materials in biological analysis. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Immobilized metal affinity chromatography (IMAC), an important tool for protein purification, is based on the affinity of transition metal ions on the IMAC supports toward electron-donor groups on protein [1,2]. Thereinto, IMAC technique is frequently used in the purification of histidine-tagged (His-tagged) recombinant proteins, which is generally based on the affinity of Ni2+ ions toward a string of six histidine (6 × His) fused to the recombinant proteins [3–5]. His-tagged proteins can be selectively enriched by the IMAC adsorbent while untagged proteins are removed. Among these adsorbents, nitrilotriacetic acid (NTA)-attached resin with immobilized ions Ni2+ , is one of the most used adsorbent for separating His-tagged fusion proteins [5–8]. Although the NTA-Ni2+ resin can be used in various protein expression systems, it is still limited by a long separation time, solvent consumption and high cost [9,10].
∗ Corresponding author at: Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China. E-mail address: [email protected] (Y.-Q. Feng).
To develop more rapid and convenient methods for the purpose, a variety of magnetic materials [11–15] have been exploited for the purification of His-tagged protein. However, the synthetic processes for most of the magnetic materials are complicated and time-consuming [16–18]. In addition, inorganic supports always suffer from the limitation of irreversible non-specific adsorption [1]. In this respect, natural fibrous material with excellent biological compatibility and good operability, can be an alternative matrix for biological research. Cotton fiber is an important natural material and widely used as adsorbent due to its merits including high mechanical strength, stable chemical resistance and wide availability [19,20]. For example, a novel cotton fiber-packed pipet-tip was developed by Selmen’s group and successfully applied for microscale enrichment of glycans and glycopeptides based on hydrophilic interaction [21]. However, the monotonous functional groups on the cotton fiber will restrict its broad applications. In this respect, sulfhydryl cotton fiber (SCF) was introduced as an IMAC support to immobilize Ni2+ ions based on the coordination effect between Ni2+ and thiol group, and the Ni2+ -immobilized sulfhydryl cotton fiber (SCF-Ni2+ ) adsorbent was used for the selective enrichment of His-tagged proteins in our recent work [22]. Thanks to the high reactivity of thiol group, SCF was further functional by “thiol-ene” click chemistry to
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Fig. 1. Schematic diagram of the preparation of CCC-Ni2+ fibers and lab-in-syringe SPE.
Fig. 2. SEM images of (a) cotton, (b) CCC, and (c) CCC-Ni2+ fibers; (d) IR spectra of cotton, CCC and CCC-Ni2+ fibers.
prepare two kinds of high specific adsorbents for biological analysis [23]. Very recently, carboxyl cotton chelator (CCC) with two free carboxyl groups on each structure unit was also exploited as IMAC support for the binding of metal ions in our work [24]. A novel CCC-Ti4+ fibrous material with high titanium content was prepared and subsequently proved to be a good adsorbent for phosphoproteomics analysis. Moreover, it is worth developing more CCC-based IMAC adsorbents due to its excellent binding capacity for metal ions and good biological compatibility. In this study, a novel CCC-Ni2+ fibrous adsorbent was successfully prepared with a simple method on the basis of the coordination effect between Ni2+ and carboxyl group. Due to the
high affinity of Ni2+ to 6 × His on His-tagged proteins, CCC-Ni2+ fibers were applied for selective enrichment of His-tagged protein from E. coli cell lysates using the lab-in-syringe format. 2. Experimental section 2.1. Reagents and materials Citric acid, aqueous ammonia solution (NH3 ·H2 O, 25 wt%), tris (hydromethyl) aminomethane (Tris), hydrochloric acid (HCl), sodium chloride (NaCl) and nickel(II) chloride hexahydrate (NiCl2 ·6H2 O, 98%) were all of analytical grade and purchased
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Fig. 5. Calibration curve of His-tagged GFP. Fig. 3. Fluorescent spectra of His-tagged GFP. Direct analysis (a), analysis of sampling eluate after enrichment with CCC-Ni2+ fibers (b), and analysis of the released His-tagged GFP in imidazole solution (c).
2.2. Preparation of the carboxyl cotton chelator-Ni2+ (CCC-Ni2+ ) adsorbent and the lab-in-syringe SPE Carboxyl cotton chelator (CCC) was prepared according to our previous work [24]. As show in Fig. 1, CCC contains two free carboxyl groups on each structure unit, which can chelate with metal ion to make CCC-based IMAC material. In our previous work, a CCC-Ti4+ adsorbent with high titanium content was successfully prepared and applied for phosphoproteomics analysis [24]. Thanks to the high binding capacity for metal ions and good biological compatibility, CCC was further used for binding Ni2+ ions to obtain a new CCC-Ni2+ adsorbent in this study (Fig. 1). Briefly, 400 mg of CCC was mixed with 40 mL of nickel ion solution (100 mM Tris, 100 mM NaCl, 100 mM NiCl2 ·6H2 O, pH 7.2). After incubation at 25 ◦ C for 2 h, the as-prepared CCC-Ni2+ fibers were washed with distilled water to remove residual Ni2+ ions followed by drying under vacuum at 60 ◦ C for 6 h. For comparison, SCF-Ni2+ fibers proposed in our previous work were also prepared according to the literature [22]. Lab-in-syringe SPE was prepared according to our previous work [24]. 2.3. Characterization of fibers
Fig. 4. (a) Fluorescent images from the mixture solutions of His-tagged GFP and normal mouse IgG1 labeled by Cy5. Direct analysis (A), analysis of sampling eluate after enrichment with CCC-Ni2+ fibers (B), and analysis of the released proteins in imidazole solution (C). (b) Fluorescent spectra of His-tagged GFP in the mixture solutions. Direct analysis (A), analysis of sampling eluate after enrichment with CCCNi2+ fibers (B), and analysis of the released His-tagged GFP in imidazole solution (C). (c) Fluorescent spectra of normal mouse IgG1 labeled by Cy5 in the mixture solutions. Direct analysis (A) and analysis of sampling eluate after enrichment with CCC-Ni2+ fibers (B).
from Sinopharm Chemical Reagent Co. (Shanghai, China). Imidazole (C3 H4 N2 ) and nickel standard (0.5 mg mL−1 in 1% nitric acid solution) were purchased from Aladdin Chemical Reagent Co. (Shanghai, China). Purified water was obtained with a MilliQ apparatus (Millipore, Bedford, MA, USA). Histidine-tagged green fluorescent protein (His-tagged GFP) and normal mouse IgG1 conjugated by PE-Cy5 were purchased from Cusabio Biotech Co. (Wuhan, China) and Santa Cruz Biotechnology (Texas, USA), respectively.
The nickel contents of CCC-Ni2+ and SCF-Ni2+ were examined by a graphite furnace atomic absorption spectrometry (GF AAS) (Shimadzu, Japan). The microscopic morphology of fibers was determined by a Quanta 200 scanning electron microscopy (SEM) (FEI, Holand). Fourier transform infrared spectroscopy (FTIR) was carried out with a Thermo Nicolet 670 FT-IR instrument (Boston, MA, USA). 2.4. Extraction of His-tagged GFP by CCC-Ni2+ fiber-packed syringe SPE To investigate the protein separation efficiency of CCC-Ni2+ fiber, a standard mixed-protein solution of His-tagged GFP and untagged normal mouse IgG1 conjugated by PE-Cy5 was used. Briefly, 500 L of mixture solution in Tris buffer (20 mM Tris, 100 mM NaCl, pH 8.0) was pipetted up and down by CCC-Ni2+ fiberpacked syringe SPE to ensure full adsorption of proteins. Then the adsorbed His-tagged GFP was eluted by an imidazole solution (1 M). The whole enrichment process could be finished within 3 min. Finally, the sampling eluate and desorption solution were analyzed by spectrofluorometer. CCC-Ni2+ fiber-packed syringe SPE was further used for the purification of His-tagged proteins from E. coli cell lysates. The
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CCC-Ni2+ may benefit from the good stability and abundant amount of carboxyl groups on CCC, as well as the strong interaction between Ni2+ and coordinated carboxylate groups. 3.2. Evaluation of the property of CCC-Ni2+ fiber-packed syringe SPE toward His-tagged proteins
Fig. 6. Analysis of the cell lysate containing His-tagged GFP by SDS-PAGE. Direct analysis (lane 1), analysis after enrichment with CCC fibers (lane 2), or CCC-Ni2+ fibers (lane 3), or SCF-Ni2+ fibers (lane 4). Lane M, protein markers.
GW5100 escherichia coli (E. coli) cells were cultured according to our previous method [25]. In the typical experiment, His-tagged GFP (30 KDa) was spiked into lysates to make spiked-lysate samples with a series of spiking concentrations. The sample processing was the same as that of the standard mixed-protein enrichment. Finally, the recovered His-tagged GFP was analyzed by spectrofluorometer and SDS-PAGE. For comparison, CCC and SCF-Ni2+ fibers were applied to enrich His-tagged proteins from E. coli cell lysates. The extraction procedure was the same as that of CCC-Ni2+ fibers. 2.5. Instruments All photoluminescence (PL) spectra were recorded on a PerkinElmer LS 55 spectrofluorometer with an excitation wavelength of 490 nm (for GFP) and 565 nm (for Cy5). 3. Results and discussion 3.1. Synthesis and characterization of CCC-Ni2+ fibers The SEM images showed the surface morphologies of cotton (Fig. 2a), CCC (Fig. 2b), and CCC-Ni2+ (Fig. 2c) were almost the same with diameters in the range of 10 m to 20 m. In addition, the fiber strength of cotton was well maintained, thus the subsequent enrichment experiment could be carried out in the lab-in-syringe format. The infrared (IR) spectra of cotton, CCC and CCC-Ni2+ were shown in Fig. 2d. A new absorption peak (1721 cm−1 ) observed in the IR spectrum of CCC could be attributed to the stretching vibration of C O, indicating the successful synthesis of CCC via esterification. Two peaks at 1572 and 1429 cm−1 could be attributed to the asymmetric (vasym ) and symmetric (vsym ) stretching vibrations of coordinated carboxylate groups. The separation between the asymmetric and symmetric stretching frequencies (v = vasym − vsym ) was 143 cm−1 , which indicated the CCC was bidentate coordinating through both carboxylic groups [26,27]. The nickel content of fibers was examined by a graphite furnace atomic absorption spectrometry (GF AAS). In our previous work [22], a fibrous SCF-Ni2+ adsorbent was developed and applied for the purification of His-tagged protein. Here, SCF-Ni2+ fibers were used for a series of comparison with CCC-Ni2+ fibers. The nickel contents of CCC-Ni2+ fibers and SCF-Ni2+ fibers were estimated to be 58.2 and 11.1 g mg−1 , respectively. The higher content of nickel of
His-tagged GFP was used to investigate the enrichment capacity of CCC-Ni2+ fibers toward His-tagged proteins. The selectivity and efficiency of CCC-Ni2+ fibers for the enrichment of His-tagged proteins could be firstly monitored under UV excitation. The original His-tagged GFP solution showed green emission under UV extraction (A in the inset of Fig. 3). After treating with CCC-Ni2+ fibers, the protein solution became colorless (B in the inset of Fig. 3) and the fluorescent emission intensity of the sampling eluate (Fig. 3b) decreased to 4.3% of the initial intensity (Fig. 3a), demonstrating that His-tagged GFP was almost completely removed from the protein solution. After the release of His-tagged GFP from CCCNi2+ fibers with imidazole solution, the imidazole solution emitted strong green fluorescence (C in the inset of Fig. 3) and the fluorescent emission intensity had a 92.8% recovery of the initial intensity (Fig. 3c), indicating the efficient dissociability of the attached Histagged protein from CCC-Ni2+ fibers by imidazole. To investigate the enrichment specificity of CCC-Ni2+ fibers toward His-tagged proteins, a mixed-protein solution of His-tagged GFP and untagged normal mouse IgG1 (labeled by red-emitting Cy5) was used. The normal mouse IgG1 showed a red emission under UV excitation. As shown in Fig. 4a, the mixed-protein solution initially emited a yellow fluorescence under UV excitation (A in the inset of Fig. 4a). After treating with CCC-Ni2+ fibers, the remaining protein solution showed a red emission (B in the inset of Fig. 4a). The fluorescent emission intensity of His-tagged GFP in the remaining solution decreased to be 3.1% of the initial intensity (Fig. 4b), while only a 16.9% decrease in the fluorescent emission intensity of the normal mouse IgG1 was observed (Fig. 4c), indicating that His-tagged GFP was selectively separated from the mixed-protein solution and normal mouse IgG1 was left in the sampling eluate. Then the bound CCC-Ni2+ was treated with imidazole solution to release the His-tagged GFP, generating a green fluorescence (C in the inset of Fig. 4a) and a 89.2% recovery of the fluorescent emission intensity (Fig. 4b). The results showed that CCC-Ni2+ fibers exhibited superior binding property and specificity to the His-tagged proteins. The adsorption capacities of CCC-Ni2+ fibers and SCF-Ni2+ fibers toward His-tagged proteins were examined using His-tagged GFP as a probe. The adsorption capacity of CCC-Ni2+ fibers was estimated to be 1.8 g mg−1 for His-tagged GFP by the breakthrough curve measurement (Fig. S1), which was higher than that obtained by SCF-Ni2+ fibers (1.0 g mg−1 ). A comparative study of this method to previous works [9,22,28,29] was performed (Table S1). It can be seen that the developed method with lab-in-syringe SPE format was convenient and rapid, and high extraction recovery of His-tagged protein could be achieved, which could satisfy the practical application. 3.3. The reproducibility of CCC-Ni2+ fiber-packed syringe SPE The reproducibility of CCC-Ni2+ fiber-packed syringe SPE was determined by the intra- and inter-batch precisions. The intrabatch relative standard deviation (RSD) was determined on three parallel experiments by CCC-Ni2+ fiber-packed syringes prepared from the same batch, and three syringes prepared from different batches gave the inter-batch RSD. Satisfactory precisions were obtained with RSDs at 7.4% (intra-batch) and 12.5% (inter-batch), demonstrating the good reproducibility of the proposed method.
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Table 1 Accuracy and precision (intra- and inter-day RSDs) for the determination of His-tagged GFP in E. coli cell lysates.
Intra-day precision (RSD, %, n = 3) Inter-day precision (RSD, %, n = 3) Recovery (%, n = 3)
Low (0.5 g mL−1 )
Medium (1.0 g mL−1 )
High (5.0 g mL−1 )
9.4 10.3 106.7 ± 8.9
8.7 7.5 98.4 ± 6.6
6.5 8.0 89.8 ± 5.9
3.4. Enrichment of His-tagged proteins from E. coli cell lysates by CCC-Ni2+ fiber-packed syringe SPE To further explore the practical application of this method, the separation of His-tagged proteins from E. coli cell lysates was examined. A calibration curve was constructed using E. coli cell lysates spiked with His-tagged GFP at different concentrations ranging from 0.1 to 7.0 g mL−1 with triplicate measurements (Fig. 5). Linearity of the calibration curve ranged from 0.1 to 7.0 g mL−1 with a correlation coefficient of 0.9961. The detection limit (3, where is the relative standard deviation of a blank solution, n = 11) was estimated to be 0.013 g mL−1 . The accuracy and precision of this method were evaluated by the recoveries and the intra- and inter-day RSDs, which were measured with His-tagged GFP spiked E. coli cell lysates at three concentrations (0.5, 1.0 and 5.0 g mL−1 ). For each concentration, triplicate measurements were performed. Intra-day RSD was calculated by repeating the analysis for three times within one day and the interday RSD was investigated on three successive days. The relative recoveries were evaluated by comparing the calculated concentration of His-tagged GFP in the E. coli cell lysates to the actual spiked concentration. As shown in Table 1, the intra- and inter-day RSDs were less than 9.4% and 10.3%, respectively, and the relative recoveries were in the range of 89.8%-106.7% with the RSDs less than 8.9%, demonstrating that good accuracy and precision were achieved. SDS-PAGE gel electrophoresis was also employed to evaluate the performance of the developed method. For the direct analysis of His-tagged GFP spiked E. coli cell lysates (5.0 g mL−1 ), His-tagged GFP couldn’t be easily distinguished due to the plenty of other proteins (Fig. 6, lane 1). After treating with CCC-Ni2+ fibers, His-tagged GFP was well separated from the complex cell lysates and clearly observed in the desorption solution (1 M of imidazole) (Fig. 6, lane 3), which visually indicated the good selectivity of CCC-Ni2+ fibers toward His-tagged proteins. By comparison, it was hardly to identify His-tagged GFP when using CCC for the extraction (Fig. 6, lane 2), demonstrating the high selectivity of CCC-Ni2+ fibers toward His-tagged protein came from the affinity between the Ni2+ ions and 6 × His. In addition, SCF-Ni2+ fibers were also applied to enrich His-tagged protein from cell lysates. Although His-tagged GFP could be well separated by SCF-Ni2+ (Fig. 6, lane 4), the recovery of Histagged GFP was lower than that by CCC-Ni2+ fibers (compared lane 3 with lane 4 in Fig. 6). The results showed that a good performance of His-tagged protein purification from complex biological sample was achieved by CCC-Ni2+ fibers, indicating a great potential of the proposed method in actual applications. 4. Conclusion In the current study, a simple approach for the facile synthesis of CCC-Ni2+ fibers was developed. Based on the high affinity of Ni2+ to 6 × His, CCC-Ni2+ fibers were applied for the selective enrichment of His-tagged protein from E. coli cell lysates using the lab-in-syringe format, which was simple, rapid and low cost. Benefiting from good biological compatibility and high content of nickel, CCC-Ni2+ fibers exhibited high selectivity and adsorption capacity toward His-tagged protein. Taken together, the proposed method expands the application of CCC-based fibrous materials in the purification of
recombinant proteins, and we expect more cotton-based materials will be exploited as adsorbents for biological analysis.
Acknowledgment The authors thank the financial support from the National Basic Research Program of China (973 Program) (2013CB910702), the National Natural Science Foundation of China (21475098), and the Natural Science Foundation of Hubei Province (2014CFA002).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2016. 06.029.
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