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Facile Oriented Immobilization of Histidine-Tagged Proteins on Nonfouling Cobalt Polyphenolic Self-Assembly Surfaces Lulu Han, Qi Liu, Liwei Yang, Tong Ye, Zhien He, and Lingyun Jia ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00691 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 18, 2017

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ACS Biomaterials Science & Engineering

Facile Oriented Immobilization of Histidine-Tagged Proteins on Nonfouling Cobalt Polyphenolic Self-Assembly Surfaces Lulu Han, Qi Liu, Liwei Yang, Tong Ye, Zhien He and Lingyun Jia*

Liaoning Key Laboratory of Molecular Recognition and Imaging, School of Life science and Biotechnology, Dalian University of Technology, Dalian 116023, P. R. China *Corresponding authors

E-mail address: [email protected]

Phone: (86) 411 84706125

Fax: (86) 411 84706125

1

ACS Paragon Plus Environment


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ABSTRACT In this study, a completely green and facile protocol to oriented immobilization of histidine-tagged (His-tagged) proteins based on plant polyphenolic tannic acid (TA) was described. This is the first time, that TA was being applied as ionic chelators to immobilize His-tagged proteins. To reduce the non-specific interactions between the TA and immobilized proteins, nonfouling zwitterionic poly(sulfobetaine methacrylate) (PSBMA) was assembled on the TA surface. The use of PSBMA could maintain the high activity of the His-tagged proteins and inhibit the adsorption of untagged protein to the TA surface. Subsequently, the obtained TA/PSBMA film was further chelated with CoII for specific binding to a His-tagged protein. As CoIII is more stable and inert than CoII, the chelated CoII was oxidized to CoIII. Using this approach, His-tagged chitinase was anchored to TA/PSBMA/CoIII film as a catalyst for the hydrolysis of chitin. The loading capacity of the film for the His-tagged chitinase can reach ~4.0 µg/cm2. Moreover, the oriented immobilized chitinase had high catalytic activity and excellent thermal and storage stability as well as being more resistant to proteolytic digestion by papain. This low-cost and green protein-oriented immobilization strategy may serve as a versatile platform for a range of applications, such as biomaterials, biocatalysis, sensors, drug delivery and so on.

KEYWORDS: Tannic acid; Cobalt; His-tagged proteins; Oriented immobilization; Nonfouling

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1. INTRODUCTION Immobilization of proteins on a solid surface is of great importance in numerous applications, including biomaterials, sensors,1 drug delivery,2-3 and biocatalysis.4-5 In general, proteins are often immobilized onto a surface via nonspeciﬁc adsorption through hydrophobic interaction and electrostatic force,6-8 as well as reaction between specific amino acids of the proteins and the complementary reactive groups on the surface of the solid support.4,

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However, non-specific protein immobilization, both via nonspeciﬁc adsorption and chemical reaction, may lead to partial or complete loss of protein activity resulting from random orientation and structural deformation of the immobilized proteins.10-11 To overcome these limitations, attention has focused on oriented protein immobilization6, which can result in greater exposure of the functional domains and higher activity for the immobilized proteins. At present, both covalent and non-covalent strategies are being developed for oriented protein immobilization.12 Covalent approaches have taken advantage of the Staudinger ligation reaction13, “click” chemistry14-15, epoxy groups16-17and so on. Representative non-covalent systems are exemplified by the biotin-streptavidin method18, DNA-directed immobilization12, 19, as well as antibodies to epitope peptides.20 However, traditional methods suffer from limitations related to the purification or preparation of functional groups prior to conjugation or the use of exogenous catalysts, unconventional amino acids, and toxic chemicals such as epichlorohydrin and ethylenediamine. One alternative approach that could avoid these limitations is to adopt the method used in immobilized metal ion affinity chromatography (IMAC).21 The principle is that metal ions first bind to a chelator-modified surface, and then bind the recombinant protein containing six 3



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consecutive histidine residues (His6-tagged) at the N- or C-terminus. This process usually occurs under very mild condition.22 However, binding of the protein to a chelator-modified surface is mediated by metal ions such as NiII , CoII and ZnII via the histidine residues is unstable, and the bound protein can be displaced by a competitive-binding agent such as imidazole.23-27 Recently, CoIII-mediated binding of His-tagged protein with excellent affinity has been used as a general approach for stable, oriented, and specific protein immobilization.28-30 In this method, metal ion chelators such as nitrilotriacetic acid (NTA) or porphyrin-phospholipid are used to bind CoII, which can be oxidized to (or directly transfer to) CoIII after binding with a His-tagged protein.28, 30 However, a monolayer of the chelators that limits the loading capacity of the supporting material, as well as the complex and exogenous chemical approaches and high cost of this method may limit its large-scale applications. Polyphenols are widely found in many plant products, such as green tea, cocoa and fruits.31 They are readily available, inexpensive and generally recognized as safe by the U.S. Food and Drug Administration. These natural polyphenolics have recently attracted widespread interests in material science due to their robust anchoring and coordinating ability on various substrates.32-37 Tannic acid (TA) is a widely used polyphenol that has abundant catechol (1,2-dihydroxyphenyl) and galloyl (1,2,3-trihydroxyphenyl) groups (Figure S1a). These groups can effectively chelate 18 different metal ions, including CoII ion.38 From these properties, we inferred that TA could be used as a new affinity ligand to chelate abundant CoII/CoIII for the purpose of immobilizing His-tagged proteins. However, TA has been reported to have strong interaction with proteins, a property that can induce nonspecific protein adhesion.39-40 To avoid this undesirable effect, antifouling polymers should be 4


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introduced to the TA surface through electrostatic/hydrogen bonding interaction to resist the nonspecific protein adhesion. To verify the above concept, we first prepared TA film through coordination with FeIII ions (Figure 1a). Subsequently, zwitterionic poly(sulfobetaine methacrylate) (PSBMA) was assembled on the TA film (Figure 1b), and CoII ions were then chelated to the TA film (Figure 1c) to selectively capture and immobilize a His-tagged protein. After CoII was oxidzed to CoIII, the binding mass, activity and stability of the His-tagged protein were investigated. This method is simple and easy to implement. On the other hand, less reagents are used in this method for protein conjugation and cannot cause a huge source of hazardous chemical waste. Moreover, the large number of binding sites for CoII ions on the TA film would increase the amount of bound CoII ions, eventually leading to improved binding capacity for the protein to be immobilized.

Figure 1. Scheme of the assembly of TA/PSBMA/CoII film. a) TA film was prepared through the coordination interaction between TA and FeIII ions. b) Zwitterionic poly(sulfobetaine methacrylate) (PSBMA) was assembled on the TA film. c) CoII ions were chelated to the TA film. 5



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2. EXPERIMENTAL SECTION 2.1 Materials Tannic

acid

(TA,

Mw=1701

Da),

FeCl3·6H2O

and

N-(3-Sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium Betaine (SBMA, 97%), 4-methylumbelliferyl N,N-diacetyl-b-D-chitobioside were purchased from Sigma-Aldrich (USA) and used without further purification. Cobalt chloride (CoCl2), bovine serum albumin (BSA), fluorescein isothiocyanate (FITC) were purchased from Solarbio (Beijing, China). Silicon wafers were obtained from Zhejiang Lijing Tech Co., Ltd (Zhejing, China). Water with a resistivity of 18.2 MΩ.cm was obtained via passage of tape water through a Millipore water purification system (Millipore, USA). PSBMA (Figure S1b) was synthesized following a published procedure.41 The gene-encoding chitinase was inserted into the bacterial inducible shuttle vector pET28a and E. coli strain BL21 (DE3) was then transformed with the recombinant plasmid. A single colony selected from the positive transformants was inoculated into 10 mL of LB plus ampicillin and grown for overnight at 37 °C. The overnight culture was inoculated into 1 L fresh TB plus ampicillin and incubated for 4 h at 37 °C followed by the addition of IPTG to 1 mM and another 16 h of incubation at 16 oC. The cells were then harvested and lysed by sonication, and the His-tagged chitinase was purified by immobilized metal affinity chromatography (IMAC; GE Healthcare) 2.2 Preparation of TA films Typically, quartz slides were cut to approximately 1 cm × 1-3 cm pieces and used as a substrate for the film fabrication. Prior to surface modification, the pieces of quartz slides 6


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were cleaned in boiling Piranha solution [98% H2SO4: 30% H2O2 = 3:1 (v/v), Caution: piranha solution reacts violently with most organic materials and must be handled with extreme care!] and then washed with DI water. The piranha-treated quartz slides were immersed in trimethylchlorosilane (4%, v/v, dichloromethane) for 4 h, followed by thorough rinsing in ethanol and DI water, and drying with nitrogen gas. TA films were prepared based on the coordination between polyphenol and metal ions.42-43 Moreover, polystyrene tissue culture plate (TCPS) was used as the substrate without further treatment. Typically, a cut quartz slide was placed in a 5-mL tube, and 2 mL of aqueous FeCl3·6H2O solution (0.8 mg mL-1) and 2 mL of TA aqueous solution (3.2 mg mL-1) were added to the tube. The tube was gently shaken for 3 min and the quartz slide was then removed and thoroughly rinsed with DI water. This whole process was defined as one coating cycle. The resulting film obtained from seven cycles of coating was designated as TA film. 2.3 Preparation of TA/PSBMA films The TA film was further immersed in 1 mg/mL PSBMA aqueous solution for 40 min at room temperature, and then washed with water. This film was referred to as TA/PSBMA film. 2.4 Preparation of TA/PSBMA/CoII films The TA/PSBMA film was treated with a solution containing 1 mL of CoCl2 solution (2.5 mg/mL) and 3 mL of Tris-HCl (100 mM, pH=7.4) inside a 5 mL-tube. The sample was then gently shaken for 15 min. The resulting film was thoroughly rinsed with water and was designated as TA/PSBMA/CoII film. 2.5 His-Tagged Protein Binding to TA/PSBMA/CoII film TA/PSBMA/CoII film was incubated with 1 mL of 400 µg/mL His-tagged protein for 2 h 7



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at 4 oC, and then thoroughly rinsed with water. 2.6 Characterization All the different films prepared and from different treatments were completely vacuum-dried prior to characterization. UV-Vis absorption measurement was conducted using a Lambda 35 UV-Vis spectrophotometer (PerkinElmer, USA). The film sample (1 cm × 3 cm) was inserted into the sample cell and then scanned at wavelengths ranging from 200 nm to 800 nm. X-ray photoelectron spectroscopy (XPS) was carried out with ESCALAB™ 250Xi (ThermoFisher, USA). Static contact angle was measured at room temperature with 3 µL of water (Milli-Q) droplets using the sessile drop method (Dataphysics OCA20, Germany). 2.7 QCM-D measurements QCM-D measurement was performed on a Q-Sense E4 system (Q-Sense, Sweden) with QCM-D crystals (Q-sense, Sweden). The QCM-D measurements can be divided into real time QCM-D measurements in buffer and offline QCM measurements in air. The real time QCM-D measurements were performed on a Q-Sense E4 system with QCM-D crystals at a flow rate of 100 µL min-1 and 25 oC. However, in experiments that measured the immobilization of protein onto the film and the stability of the immobilized protein, a flow rate of 20 µL min-1 was used instead. Gold QCM-D crystals (5 MHz) were cleaned with a solution containing NH4OH, 30% H2O2, and H2O in a 1:1:5 (v/v/v) ratio, then thoroughly rinsed with DI water and dried in a stream of nitrogen. The treated QCM-D crystals were deposited onto the TA film, and then placed into the QCM-D instrument. PSBMA, Co2+, BSA and His-tagged chitinase stepwise binding to TA films was monitored in

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real-time on QCM-D (Figure 2a and Figure 4). Voigt viscoelastic model was used to fit the 1st, 3rd and 5th overtones of QCM-D data for calculating a bound hydrated mass and thickness. 44 Naked gold crystals and vacuum-dried TA, TA/PSBMA and TA/PSBMA/Co2+-coated gold crystals were measured on offline QCM in air. Due to the rigid properties of these dry films, the dry masses of TA, PSBMA, Co2+ were calculated by the Sauerbrey equation.45

∆m = −

஼∆௙

(1)

௡

where n is the overtone number (n = 3) and C denotes a constant characteristic of the sensitivity of the resonator to changes in mass (17.7 ng Hz−1 cm−2 for the used 5 MHz quartz crystals). ∆݂ is the frequency difference obtaining from merging the two corresponding QCM curves by using the ‘Stitch files’ function of QSoft 401 software. Curves from the third overtone (n = 3) were used as representative curves for all of the graphs. 2.8 The activity of the chitinase In this experiment, 96-well polystyrene tissue culture plate (TCPS) was used as the substrate. Coating of the plate was carried out as described in Section 2.2 to Section 2.4, except that the inner wall of the well instead of quartz slide, was coated with the TA/PSBMA/CoII film. Chitinase activity was assayed by hydrolysis of 4-methylumbelliferone according

to a

previously

published method.46

Ten microliters

of 5

mM of

4-methylumbelliferyl N,N-diacetyl-b-D-chitobioside and 90 µL of 0.1 M acetate buffer (pH 6.0) were dispensed into a 96-well plate that had been pre-coated with TA/PSBMA/CoII film, and immobilized with His-tagged chitinase for 3 min. The reaction was stopped by the addition of 100 µL of 4% Na2CO3, and the fluorescence was measured at 440 nm with an emission wavelength of 360 nm. The activity of the immobilized chitinase was normalized to 9



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the activity of the soluble chitinase with an equal mass. All the assays were carried out in triplicates. 2.9 Thermal stability of the immobilized chitinase The thermal stability of the immobilized chitinase was determined after incubation at 60 o

C for 5 h, whereas the storage stability of the enzyme was determined by measuring the

activity of the enzyme after incubation at 4 oC for different lengths of time over a one-month period. The soluble enzyme was used as a control in the activity assay. All the assays were carried out in triplicates. 2.10 Susceptibility of the immobilized chitinase to protease digestion Chitinase-immobilized film was incubated with 100 µg/mL papain at 37 oC for 2 h. The activity of the chitinase was measured before and after treatment with papain. The assay was carried out in triplicates.

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3. RESULTS AND DISCUSSION 3.1 Assembly of TA/PSBMA/CoII film

Figure 2. Characterization of TA/PSBMA/CoII film. a) Real-time QCM-D data of frequency and dissipation shifts of TA/PSBMA/CoII film. b) Dry mass of TA, PSBMA and cobalt ions calculated by the Sauerbrey equation. c) Static water contact-angle. d) UV-Vis absorption spectra. Data are the means ± SDs of three samples.

Tannic acid (TA, Figure S1a) can easily adhere to a wide variety of substrates, such as metal, polymer, rubber and inorganic substances because of the general surface-binding affinity of the catechol (1,2-dihydroxyphenyl) and galloyl (1,2,3-trihydroxyphenyl) groups.47,32 These groups can strongly coordinate with metal ions. Based on the metal (FeIII) ion-TA coordination, a cross-linked metal-organic TA film was prepared by incubating a 11



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quartz slide in a FeIII ion-containing TA solution, and the thickness of the TA film was increased by simply repeating the rapid coating cycle (Figure S2). Since the feed molar ratio of TA to FeIII ion was almost 1:8, excess TA was present on the surface of the quartz slide to further chelate with the FeIII ions. The TA film was subsequently dipped in CoII solution to allow the CoII ions to chelate with the TA, serving as an affinity ligand for binding with a His-tagged protein. Moreover, the excess free catechol and galloyl groups of the TA film could also interact with the zwitterionic poly(sulfobetaine methacrylate) (PSBMA) via electrostatic interaction to prevent any subsequent nonspecific protein adsorption.41 The scheme for the generation of TA/PSBMA/CoII film is shown in Figure 1. The processes of PSBMA deposition and CoII ion loading were monitored in situ on QCM-D chips pre-coated with the TA film (Figure 2a). The frequency change ∆f and dissipation factor change ∆D were recorded for each step at a third overtone. During the treatment with PSBMA, decrease in ∆f represented a mass increase induced by the deposition of PSBMA, while increase in ∆D indicated more water content due to the high hydration capacity of PSBMA. The mass of the PSBMA layer in the water was 2.7 µg cm-2 and its thickness was 22.5 nm as calculated by fitting with the Voigt model. After being soaked in a solution containing Co2+ ions, the initial surrounding solution water was replaced with 2.5 mg/mL CoCl2 solution. Since the viscosity and density of the surrounding solution was changed, the frequency and the dissipation of film would correspondingly regulate.48-49 Additionally, Co2+ ions could bound to catechol groups as a crosslinking agent in the film and thereby the density of the film was increasing, leading to a sharp decrease of both ∆f and ∆D (Figure 2a). Subsequent washing of the film in water caused both ∆f and ∆D to increase, but then leveled off at values 12


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that were still less than the initial values, indicating that the Co2+ ions had been adsorbed onto the TA/PSBMA film. Moreover, the dry net masses of the TA film, PSBMA layer and CoII were determined by offline QCM in air, and were about 8.9, 0.5 and 0.8 µg cm-2, respectively (Figure 2b).

Figure 3. XPS spectra of modified quartz slides. a) TA. b) TA/PSBMA. c) TA/PSBMA/CoII. d) Surface chemical compositions of TA/PSBMA/CoII films. Insets are the high resolution Co2p XPS spectra. The chemical compositions of the resulting TA, TA/PSBMA and TA/PSBMA/CoII films were determined by XPS (Figure 3). After coating with PSBMA, the surface showed the representative signal of N1s at ∼401.5 eV and S2p at 167.1 (Figure 3b), indicating the 13



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presence of PSBMA on the TA films. After introducing the coablt, Figure 3c shows the representative signals of Co2p at ∼783 eV with a peak separation of ∼15-16 eV,50-51 indicating the presence of CoII species on the films. Furthermore, the ratio of TA:CoII was 1:3 determined by XPS (Figure 3c and d). Compared to the 1:1 ratio of NTA to the bound metal ion, TA was able to bind more CoII ions than NTA. The static water contact-angle test result showed that TA/PSBMA coating possessed a low water contact-angle (below 5°) (Figure 2c), which was much smaller than those of TA (51o) and bare silicon surface (~83o) due to the superhydrophilic property of PSBMA.41, 52 Little change in water contact angle occurred after the chelation of CoII ions. Moreover, in contrast to the 276 nm absorption peak of TA film, TA/PSBMA/CoII film displayed a red-shift in its absorption, which peaked at 317 nm (Figure 2d), showing that the CoII ions were incorporated into the film. In addition, PSBMA coating had no effect on the chelation of CoII ions since both TA/CoII and TA/PSBMA/CoII films had the same UV absorbance (Figure 2d). Furthermore, there were no significance difference in morphology and roughness between TA/PSBMA/CoII films and TA/PSBMA films (Figure S3).

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3.2 Oriented immobilization of His-tagged proteins on TA/PSBMA/CoII/III

Figure 4. Binding behavior of BSA (400 µg/mL) and His-tagged chitinase (400 µg/mL) as determined by QCM-D. a) and b) TA/PSBMA/CoII modified-crystals. c) TA/CoII modified-crystals. d) The hydrated mass of the amount of bound protein as calculated by fitting with the Voigt model. Data are the means ± SDs from three samples. Binding of His-tagged chitinase to the TA/PSBMA/CoII film could become saturated at a protein concentration of 400 µg mL-1 (Figure S4). Moreover, seven layers of TA coating would ensure the film has a high binding capacity for the protein to be immobilized (Figure S6). Thus a protein concentration 400 µg mL-1 and a TA film with 7 layers of coating were used for all subsequent experiments. PSBMA coating has an excellent protein resistance property.41’53 To investigate the 15



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specificity of TA/PSBMA/CoII film for His-tagged protein, the binding between the film and non-His tagged protein, such as BSA, was evaluated on QCM-D. (Figure 4). The QCM-D frequency of TA/PABMA/CoII film was almost the same before and after BSA injection (Figure 4a, b), demonstrating that little BSA was bound to the film. In contrast, the amount of BSA bound to the TA/CoII film surface was about 0.6 µg cm-2 (Figure 4c and d). Therefore, the PSBMA layer could prevent a non-His-tagged protein from binding to the film, while displaying high degree of specificity toward a His-tagged protein (Figure 4b). Furthermore, the mass of His-tagged proteins adhered to the TA/PSBMA/CoII film was almost the same as that adhered to the TA/CoII film (Figure 4d), suggesting that PSBMA did not affect the amount of His-tagged proteins interacting with to the film surface. This implied that the high affinity between CoII ion and His-tagged protein could overcome antifouling performance of PSBMA to the protein (Figure 5b). The fluorescence experiment can also verify that the TA/PSBMA/CoII coating can specifically capture the His-tagged proteins (Figure S5). In addition, TA/PSBMA/CoII film could effectively capture the His-tagged protein in a cell lysate (Figure S7). Thus TA/PSBMA/CoII film could be used in the direct immobilization of His-tagged protein from a mixture of proteins without the need for pretreatment, such as an enrichment step. Since TA film could bind a large amount of CoII/CoIII ions, and these ions could further enhance the amount of His-tagged protein being immobilized, the hydrated mass of the immobilized His-tagged chitinase (~44300 Da) could reach 4.0 µg cm-2 (Figure 4d). Moreover, the binding of His-tagged chitinase to TA/PSBMA/CoII films could be modified on different substrates, such as the quartz, glass, polyamide and polyethersulfone (Figure 5a).

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Figure 5. a) A digital photo of TA/PSBMA/CoII/His-tagged chitinase films on the quartz, glass, polyamide and polyethersulfone substrates with a ruler. b) Diagram showing the capture of His-tagged protein by the TA/PSBMA/CoII film. CoII ions can bind to the His-tag of a protein, but this binding is not stable in the presence of a competitively binding agent such as imidazole. However, CoIII can be regarded as a stable and inert ion for binding His-tagged proteins.28, 30 H2O2 was used as an oxidant to oxidize CoII ions. H2O2 had little influence on PSBMA compositions (Table S2) and film thickness (Figure S8a), but some phenolic hydroxyl groups of TA could be oxidized to quinones by the hydrogen peroxide (Figure S9). After CoII was oxidized to CoIII by treatment with 3 mM H2O2, the satellite peaks of Co2p1/2 and Co2p2/3 on the surface of TA/PSBMA/CoIII were missing (Figure S8b). In addition, for the TA/PSBMA/CoIII, the binding energy difference between Co2p1/2 and Co2p2/3 (Table S1) was 16.0 eV, while for the TA/PSBMA/CoII, the binding energy difference was 17



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15.1 eV. Therefore, after H2O2 oxidization, Co ions were changed to an oxidation state, according to previous reports that the missing satellite peaks and an energy separation of 15.0 eV for CoIII.50, 54-55 Co2p2/3 can be ascribed to Co-O coordination bond (779.9 - 781.5 eV) and Co salt (784.9 eV), and the Co-O coordination bond can be further ascribed to two peaks CoII-O and CoIII-O.56 For the oxidized TA/PSBMA/CoIII coating, a higher content of CoIII-O was observed (~ 94% of Co-O coordination bond, Figure 6b), compared to the TA/PSBMA/CoII coating (~ 11% of Co-O coordination bond, Figure 6a), showing a successful oxidization of CoII by treatment with H2O2.

Figure 6. CoII oxidation of TA/PSBMA/CoII coatings by treatment with 3 mM H2O2, and the stability of immobilized His-tagged protein after washing with 300 mM imidazole. a) High resolution Co2p XPS spectra of TA/PSBMA/CoII coatings on silicon wafer. b) High resolution 18


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Co2p XPS spectra of TA/PSBMA/CoIII coatings on silicon wafer, after oxidation of TA/PSBMA/CoII coatings by H2O2 (3 mM) for 60 min. c) QCM-D curve of TA/PSBMA/CoII/His-tagged chitinase washed with 300 mM imidazole for overnight. d) QCM-D curve of TA/PSBMA/CoIII/His-tagged chitinase washed with 300 mM imidazole for overnight. The stability of the His-tagged chitinase bound to the CoII-treated and CoIII-treated TA/PSBMA films was detected by QCM-D. When both films were washed with 300 mM imidazole, the protein dissociated from the TA/PSBMA/CoII film within 3 h (Figure 6c), but remained bound in the case of TA/PSBMA/CoIII film, even when the film was flushed with imidazole for more than 12 h (Figure 6d). The frequency of protein-modified TA/PSBMA/CoIII film decreased upon washing with imidazole because of the changes in the viscosity of the medium, consistent with previous report.29 Furthermore, on the TA/PSBMA/CoIII film, the fluorescence intensity of FITC-labeled His-tagged chitinase hardly changed after washing with imidazole (Figure S10), consistent with the result obtained from QCM-D analysis, both of which showed that the binding between His-tagged chitinase and CoIII ion was kinetically inert, ensuring that the protein was not easily dislodged by imidazole. 3.3 Activity of immobilized chitinase His-tagged chitinase was used as a model protein to investigate the activity of an immobilized protein (Figure 7a). The activity of immobilized His-tagged chitinase was normalized such that the activity of an equal mole of free His-tagged chitinase (1410 U/g) was considered as 100%. The activity of chitinase immobilized on polystyrene tissue culture 19



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plate was only 25%. This activity increased to 70% for His-tagged chitinase immobilized on TA/CoII film without PSBMA, because TA could interact with the protein via H-bonding, which may result in the enzyme being immobilized in a random orientation, with unexpected denaturation or blocking of the enzyme active site.7 However, on the TA/PSBMA/CoII film, the activity of the immobilized chinitase was about 95% that of the free enzyme. This could be attributed to the fact that the nonfouling PSBMA layer could reduce the interaction between TA and the immobilized protein, while CoII ion only reacted with the His-tag in the N-terminal domain, ensuring high activity for the correctly oriented protein. Among the various

kinds

of

antifouling

polymers

such

as

polyethylene

glycol

(PEG)57,

poly(2-oxazoline)s (POx)58, and poly(vinyl pyrrolidone) (PVP)59 that were examined and which could be coated onto TA film surface, only PSBMA-coated film could yield the best activity for the immobilized chitinase (Figure S11) This may be because PSBMA and TA could form complexes via electrostatic interaction, which would be stronger than the complexes formed between other polymers and TA (Figure S12). PSBMA and the immobilized protein would interact with TA on the same site, so the stronger interaction between TA and PSBMA could contribute to reduced interaction between TA and the immobilized protein.41 Besides, the oxidation process of CoII to CoIII had little influence on the activity of the immobilized chitinase (p > 0.05) (Figure 7a), consistent with the finding reported by Wegner and coworkers.29. Therefore, TA/PSBMA/CoII

and III

film could yield

immobilized protein with a level of activity as high as that of the correspondingly free protein.

20


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Figure 7. a) Activity of immobilized His-tagged chitinase. b) Effect of papain on the activity of immobilized chitinase. The activity of free His-tagged chitinase (1410 U/g) was considered as 100%. Data are the mean ± SD from three samples. Significance was tested by one-way ANOVA test, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

We next investigated the effects of several factors, including proteolytic digestion, high temperature, storage time and repeated use on the stability of the immobilized chitinase. After incubating with 0.1 mg mL−1 papain for 2 hours at 37 oC, the chitinase immobilized on the TA/CoII retained only about 36% of the activity of free chitinase (Figure 7b), while the chitinase immobilized on the TA/PSBMA/CoII film retained more than 95% of the activity under the same condition, indicating that the protein-repelling property of the PSBMA coating may effectively reduce the contact between papain and the immobilized chitinase.

21



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Figure 8. Effect of various factors on the stability and activity of immobilized His-tagged chitinase. a) Thermal stability (60 oC), b) storage stability (4 oC) and c) repeated uses. The initial activity of immobilized His-tagged chitinase was considered as 100%. Data are the mean ± SD from three samples.

The thermal stability of the immobilized chitinase was examined at 60 °C. The free enzyme lost almost all of its activity within 100 min of incubation at 60 oC (Figure 8a), while the enzyme immobilized on TA/PSBMA/CoII film retained 99% of its initial activity under the same condition, demonstrating that the immobilized enzyme had better thermal stability. As for the stability of the enzyme over time, the immobilized enzyme retained more than 90% of the initial activity after storage in PBS (pH 7.4) at 4 °C at for 30 d, while the free enzyme only 22


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retained about 60% of its initial activity under the same storage condition (Figure 8b). This indicated that the immobilized enzyme was again, more superior to the free enzyme in term of stability at low temperature during storage. The practical use of the immobilized enzyme was also demonstrated by subjecting it to repeated uses. The enzyme was able to retain 84% of the initial activity after 8 regenerations following its repeated use (Figure 8c). Considering the excellent reusability, thermal stability, and other properties of the immobilized enzymes, this method of protein immobilization would be more suitable for practical applications.

4. CONCLUSION In summary, a novel approach for the oriented immobilization of His-tagged proteins was described, which involved the use of TA/PSBMA/CoIII film. As a chelator, one mole of TA could chelate three times the amount of CoII/III ions, and the abundant CoII/III ions would improve binding capability between TA and His-tagged protein. For the His-tagged chitinase, the binding capacity reached ~4.0 µg/cm2. In addition, the use of PSBMA as an antifouling coating not only reduced the interaction between TA and the immobilized protein for maintaining high activity, but also for inhibiting the binding of non-His-tagged protein and proteolytic digestion of the immobilized His-tagged protein. Moreover, the proposed oriented immobilization strategy offered several attractive advantages, such as high activity, excellent thermal stability, long storage time, and the benefit of being reusable. The TA/PSBMA/CoIII film described here should enable the capture of proteins from cell lysates, thus circumventing the need for laborious protein purification. To the best of our knowledge, using TA as the ionic chelator to immobilize His-tagged proteins has not yet been reported. We believe that 23



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this low-cost and green protein-oriented immobilization strategy may serve as a versatile platform for a range of applications, such as biomaterials, biocatalysis, sensors, and drug delivery. ASSOCIATED CONTENT Supporting Information Chemical structures of TA and PSBMA, UV-vis absorption spectra of Fe-TA films, SEM and AFM images (insets) of the modified-films, the effect of the number of TA films on proteins capture, the fluorescence microscope images, SDS-PAGE of bound protein eluted from the modified-films, QCM-D curve, XPS spectra and data, the fluorescence intensity of FITC-labeled His-tagged chitinase after washing with imidazole, the effect of different outmost polymer layer on the activity of chitinase, and the corresponding transmittance of the formed complexes between TA and different polymers (PDF) AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected]. Phone: (86) 411 84706125, Fax: (86) 411 84706125

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors acknowledge the financial support of the National Natural Science Foundation of China (Projects Nos. 51773026, 51303019), the Fundamental Research Funds for the National 24


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Key Technologies R&D Program (2016YFC1103002) and the Fundamental Research Funds for the Central Universities (DUT17LAB06). The authors thank Dr. Alan K. Chang for editing the language of the manuscript.

REFERENCE (1) Massad-Ivanir, N.; Shtenberg, G.; Zeidman, T.; Segal, E., Construction and Characterization of Porous SiO2/Hydrogel Hybrids as Optical Biosensors for Rapid Detection of Bacteria. Adv Funct Mater 2010, 20 (14), 2269-2277. (2) Slowing, I. I.; Trewyn, B. G.; Lin, V. S. Y., Mesoporous silica Nanoparticles for Intracellular Delivery of Membrane-Impermeable Proteins. J Am Chem Soc 2007, 129 (28), 8845-8849. (3) Cao, Z. Q.; Yu, Q. M.; Xue, H.; Cheng, G.; Jiang, S. Y., Nanoparticles for Drug Delivery Prepared from Amphiphilic PLGA Zwitterionic Block Copolymers with Sharp Contrast in Polarity between Two Blocks. Angew Chem Int Edit 2010, 49 (22), 3771-3776. (4) Han, H.; Zhou, Y.; Li, S.; Wang, Y.; Kong, X. Z., Immobilization of Lipase from Pseudomonas fluorescens on Porous Polyurea and Its Application in Kinetic Resolution of Racemic 1-Phenylethanol. ACS Appl Mater Interfaces 2016, 8 (39), 25714-25724. (5) Shieh, F. K.; Wang, S. C.; Yen, C. I.; Wu, C. C.; Dutta, S.; Chou, L. Y.; Morabito, J. V.; Hu, P.; Hsu, M. H.; Wu, K. C. W.; Tsung, C. K., Imparting Functionality to Biocatalysts via Embedding Enzymes into Nanoporous Materials by a de Novo Approach: Size-Selective Sheltering of Catalase in Metal-Organic Framework Microcrystals. J Am Chem Soc 2015, 137 (13), 4276-4279. 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(6) Rusmini, F.; Zhong, Z. Y.; Feijen, J., Protein Immobilization Strategies for Protein Biochips. Biomacromolecules 2007, 8 (6), 1775-1789. (7) Liu, Y.; Yu, J., Oriented Immobilization of Proteins on Solid Supports for Use in Biosensors and Biochips: a review. Microchimica Acta 2015, 183 (1), 1-19. (8) Zhang, Y.; Zhang, J. Y.; Huang, X. L.; Zhou, X. J.; Wu, H. X.; Guo, S. W., Assembly of Graphene Oxide-Enzyme Conjugates through Hydrophobic Interaction. Small 2012, 8 (1), 154-159. (9) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M., Protein Nanoarrays Generated by Dip-Pen Nanolithography. Science 2002, 295 (5560), 1702-1705. (10) Phizicky, E.; Bastiaens, P. I. H.; Zhu, H.; Snyder, M.; Fields, S., Protein Analysis on a Proteomic Scale. Nature 2003, 422 (6928), 208-215. (11) Camarero, J. A., Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports. Biopolymers 2008, 90 (3), 450-458. (12) Palla, K. S.; Hurlburt, T. J.; Buyanin, A. M.; Somorjai, G. A.; Francis, M. B., Site-Selective Oxidative Coupling Reactions for the Attachment of Enzymes to Glass Surfaces through DNA-Directed Immobilization. J Am Chem Soc 2017, 139 (5), 1967-1974. (13) van Berkel, S. S.; van Eldijk, M. B.; van Hest, J. C. M., Staudinger Ligation as a Method for Bioconjugation. Angew Chem Int Edit 2011, 50 (38), 8806-8827. (14) Zhang, L.; Vila, N.; Klein, T.; Kohring, G. W.; Mazurenko, I.; Walcarius, A.; Etienne, M., Immobilization of Cysteine-Tagged Proteins on Electrode Surfaces by Thiol-Ene Click Chemistry. Acs Applied Materials & Interfaces 2016, 8 (27), 17591-17598. 26


Page 26 of 41

Page 27 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(15) Finetti, C.; Sola, L.; Pezzullo, M.; Prosperi, D.; Colombo, M.; Riva, B.; Avvakumova, S.; Morasso, C.; Picciolini, S.; Chiari, M., Click Chemistry Immobilization of Antibodies on Polymer Coated Gold Nanoparticles. Langmuir 2016, 32 (29), 7435-7441. (16) Abad, J. M.; Velez, M.; Santamaria, C.; Guisan, J. M.; Matheus, P. R.; Vazquez, L.; Gazaryan, I.; Gorton, L.; Gibson, T.; Fernandez, V. M., Immobilization of peroxidase glycoprotein on gold electrodes modified with mixed epoxy-boronic acid monolayers. J Am Chem Soc 2002, 124 (43), 12845-12853. (17) Mateo, C.; Grazu, V.; Palomo, J. M.; Lopez-Gallego, F.; Fernandez-Lafuente, R.; Guisan, J. M., Immobilization of Enzymes on Heterofunctional Epoxy Supports. Nat Protoc 2007, 2 (5), 1022-1033. (18) Lu, T. C.; Chen, X. S.; Shi, Q.; Wang, Y.; Zhang, P. B.; Jing, X. B., The Immobilization of Proteins on Biodegradable Fibers via Biotin-Streptavidin Bridges. Acta Biomater 2008, 4 (6), 1770-1777. (19) Niemeyer, C. M., Semisynthetic DNA-Protein Conjugates for Biosensing and Nanofabrication. Angew Chem Int Edit 2010, 49 (7), 1200-1216. (20) Redeker, E. S.; Ta, D. T.; Cortens, D.; Billen, B.; Guedens, W.; Adriaensens, P., Protein Engineering For Directed Immobilization. Bioconjugate Chem 2013, 24 (11), 1761-1777. (21) Blanco-Canosa, J. B.; Wu, M.; Susumu, K.; Petryayeva, E.; Jennings, T. L.; Dawson, P. E.; Algar, W. R.; Medintz, I. L., Recent Progress in the Bioconjugation of Quantum Dots. Coordin Chem Rev 2014, 263, 101-137. (22) Hochuli, E.; Bannwarth, W.; Dobeli, H.; Gentz, R.; Stuber, D., Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 41

Bio-Technol 1988, 6 (11), 1321-1325. (23) Kang, E.; Park, J. W.; McClellan, S. J.; Kim, J. M.; Holland, D. P.; Lee, G. U.; Franses, E. I.; Park, K.; Thompson, D. H., Specific Adsorption of Histidine-Tagged Proteins on Silica Surfaces Modified with Ni2+/NTA-Derivatized Poly(ethylene glycol). Langmuir 2007, 23 (11), 6281-6288. (24) Hu, Q. I. N.; Liu, Y. Q.; Li, N.; Cheng, C.; Xu, S.; Wang, N.; Qin, W. E. I.; Tang, B. Z., Ni–NTA-Coated Nanowire Materials for Protein Enrichment and the Application in a Medcal Device Used for Blood Glucose Degradation. Nano 2013, 08 (03), 1350029. (25) Benjamin, C. J.; Wright, K. J.; Hyun, S. H.; Krynski, K.; Yu, G.; Bajaj, R.; Guo, F.; Stauffacher, C. V.; Jiang, W.; Thompson, D. H., Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates. Langmuir 2016, 32 (2), 551-9. (26) Xia, S.; Cartron, M.; Morby, J.; Bryant, D. A.; Hunter, C. N.; Leggett, G. J., Fabrication of Nanometer- and Micrometer-Scale Protein Structures by Site-Specific Immobilization of

Histidine-Tagged

Proteins

to

Aminosiloxane

Films

with

Photoremovable

Protein-Resistant Protecting Groups. Langmuir 2016, 32 (7), 1818-27. (27) Postupalenko, V.; Desplancq, D.; Orlov, I.; Arntz, Y.; Spehner, D.; Mely, Y.; Klaholz, B. P.; Schultz,

P.; Weiss,

E.; Zuber,

G.,

Protein

Delivery System

Containing a

Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer. Angew Chem Int Edit 2015, 54 (36), 10583-10586. (28) Shao, S.; Geng, J.; Ah Yi, H.; Gogia, S.; Neelamegham, S.; Jacobs, A.; Lovell, J. F., Functionalization of cobalt porphyrin-phospholipid bilayers with his-tagged ligands and 28


Page 29 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


antigens. Nat Chem 2015, 7 (5), 438-46. (29) Wegner, S. V.; Schenk, F. C.; Spatz, J. P., Cobalt(III)-Mediated Permanent and Stable Immobilization of Histidine-Tagged Proteins on NTA-Functionalized Surfaces. Chemistry 2016, 22 (9), 3156-62. (30) Wegner, S. V.; Spatz, J. P., Cobalt(III) as a Stable and Inert Mediator Ion between NTA and His6-tagged Proteins. Angew Chem Int Ed Engl 2013, 52 (29), 7593-6. (31) Quideau, S.; Deffieux, D.; Douat-Casassus, C.; Pouysegu, L., Plant Polyphenols: Chemical Properties, Biological Activities, and Synthesis. Angew Chem Int Edit 2011, 50 (3), 586-621. (32) Ejima, H.; Richardson, J. J.; Liang, K.; Best, J. P.; van Koeverden, M. P.; Such, G. K.; Cui, J.; Caruso, F., One-Step Assembly of Coordination Complexes for Versatile Film and Particle Engineering. Science 2013, 341 (6142), 154-7. (33) Sileika, T. S.; Barrett, D. G.; Zhang, R.; Lau, K. H. A.; Messersmith, P. B., Colorless Multifunctional Coatings Inspired by Polyphenols Found in Tea, Chocolate, and Wine. Angew Chem Int Edit 2013, 52 (41), 10766-10770. (34) Barrett, D. G.; Sileika, T. S.; Messersmith, P. B., Molecular Diversity in Phenolic and Polyphenolic Precursors of Tannin-Inspired Nanocoatings. Chem Commun 2014, 50 (55), 7265-7268. (35) Bentley, W. E.; Payne, G. F., Nature's Other Self-Assemblers. Science 2013, 341 (6142), 136-137. (36) Wei, J.; Liang, Y.; Hu, Y. X.; Kong, B. A.; Simon, G. P.; Zhang, J.; Jiang, S. P.; Wang, H. T.,

A

Versatile

Iron-Tannin-Framework

Ink

Coating

29


Strategy

to

Fabricate


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomass-Derived Iron Carbide/Fe-N-Carbon Catalysts for Efficient Oxygen Reduction. Angew Chem Int Edit 2016, 55 (4), 1355-1359. (37) Yang, S. J.; Antonietti, M.; Fechler, N., Self-Assembly of Metal Phenolic Mesocrystals and Morphosynthetic Transformation toward Hierarchically Porous Carbons. J Am Chem Soc 2015, 137 (25), 8269-8273. (38) Guo, J. L.; Ping, Y.; Ejima, H.; Alt, K.; Meissner, M.; Richardson, J. J.; Yan, Y.; Peter, K.; von Elverfeldt, D.; Hagemeyer, C. E.; Caruso, F., Engineering Multifunctional Capsules through the Assembly of Metal-Phenolic Networks. Angew Chem Int Edit 2014, 53 (22), 5546-5551. (39) Oh, H. I.; Hoff, J. E.; Armstrong, G. S.; Haff, L. A., Hydrophobic Interaction in Tannin-Protein Complexes. J Agr Food Chem 1980, 28 (2), 394-398. (40) Siebert, K. J.; Troukhanova, N. V.; Lynn, P. Y., Nature of Polyphenol-Protein Interactions. J Agr Food Chem 1996, 44 (1), 80-85. (41) Ren, P. F.; Yang, H. C.; Liang, H. Q.; Xu, X. L.; Wan, L. S.; Xu, Z. K., Highly Stable, Protein-Resistant Surfaces via the Layer-by-Layer Assembly of Poly(sulfobetaine methacrylate) and Tannic Acid. Langmuir 2015, 31 (21), 5851-8. (42) Ejima, H.; Richardson, J. J.; Liang, K.; Best, J. P.; van Koeverden, M. P.; Such, G. K.; Cui, J. W.; Caruso, F., One-Step Assembly of Coordination Complexes for Versatile Film and Particle Engineering. Science 2013, 341 (6142), 154-157. (43) Yang, L. W.; Han, L. L.; Ren, J.; Wei, H. L.; Jia, L. Y., Coating Process and Stability of Metal-Polyphenol Film. Colloid Surface A 2015, 484, 197-205. (44) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B., Viscoelastic Acoustic Response of 30


Page 30 of 41

Page 31 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Layered Polymer Films at Fluid-Solid Interfaces: Continuum mechanics approach. Phys Scripta 1999, 59 (5), 391-396. (45) Sauerbrey, G. Verwendung von Schwingquarzen zur Wägung dünner Schichten und zur Mikrowägung Z. Phys. 1959, 155, 206-222 (46) Mccreath, K. J.; Gooday, G. W., A Rapid and Sensitive Microassay for Determination of Chitinolytic Activity. J Microbiol Meth 1992, 14 (4), 229-237. (47) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B., Mussel-inspired surface chemistry for multifunctional coatings. Science 2007, 318 (5849), 426-430. (48) Parlak, Z.; Biet, C.; Zauscher, S., Decoupling Mass Adsorption from Fluid Viscosity and Density in Quartz Crystal Microbalance Measurements Using Normalized Conductance Modeling. Meas Sci Technol 2013, 24 (8). (49) Lundgren, S. M.; Persson, K.; Kronberg, B.; Claesson, P. M., Adsorption of Fatty Acids from Alkane Solution Studied with Quartz Crystal Microbalance. Tribol Lett 2006, 22 (1), 15-20. (50) Borodko, Y. G.; Vetchinkin, S. I.; Zimont, S. L.; Ivleva, I. N.; Shulga, Y. M., Nature of Satellites in X-Ray Photoelectron-Spectra Xps of Paramagnetic Cobalt(Ii) Compounds. Chem Phys Lett 1976, 42 (2), 264-267. (51) Tait, A. M.; Busch, D. H., Metal-Promoted Ligand Synthesis, Preparation, and Characterization of Cobalt(II) and Cobalt(III) Complexes with a Highly Unsaturated Tetraaza Macrocyclic Ligand. Inorg Chem 1976, 15 (1), 197-203. (52) Zhang, C.; Li, H. N.; Du, Y.; Ma, M. Q.; Xu, Z. K., CuSO4/H2O2-Triggered Polydopamine/Poly(sulfobetaine methacrylate) Coatings for Antifouling Membrane 31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Surfaces. Langmuir 2017, 33 (5), 1210-1216. (53) Lalani, R.; Liu, L., Electrospun Zwitterionic Poly(Sulfobetaine Methacrylate) for Nonadherent, Superabsorbent, and Antimicrobial Wound Dressing Applications. Biomacromolecules 2012, 13 (6), 1853-1863. (54) Briggs, D.; Gibson, V. A., Direct Observation of Multiplet Splitting in 2p Photoelectron Peaks of Cobalt Complexes. Chem Phys Lett 1974, 25 (4), 493-496. (55) Atzei, D.; Rossi, A.; Sadun, C., Synthesis and Characterization of a Cobalt(III) Complex with 1-(D-3-mercapto-2-methylpropionyl)-L-proline. Spectrochim Acta A 2000, 56 (10), 1875-1886. (56) Wei, J.; Liang, Y.; Hu, Y. X.; Kong, B.; Zhang, J.; Gu, Q. F.; Tong, Y. P.; Wang, X. B.; Jiang, S. P.; Wang, H. T., Hydrothermal Synthesis of Metal-Polyphenol Coordination Crystals and Their Derived Metal/N-doped Carbon Composites for Oxygen Electrocatalysis. Angew Chem Int Edit 2016, 55 (40), 12470-12474. (57) Kim, S.; Gim, T.; Kang, S. M., Versatile, Tannic acid-Mediated Surface PEGylation for Marine Antifouling Applications. ACS Appl Mater Interfaces 2015, 7 (12), 6412-6. (58) Sundaramurthy, A.; Vergaelen, M.; Maji, S.; Auzely-Velty, R.; Zhang, Z.; De Geest, B. G.; Hoogenboom, R., Hydrogen bonded multilayer films based on poly(2-oxazoline)s and tannic acid. Adv Healthc Mater 2014, 3 (12), 2040-7. (59) Serrano, A.; Sterner, O.; Mieszkin, S.; Zurcher, S.; Tosatti, S.; Callow, M. E.; Callow, J. A.; Spencer, N. D., Nonfouling Response of Hydrophilic Uncharged Polymers. Adv Funct Mater 2013, 23 (46), 5706-5718.

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For Table of Contents Use Only

Facile Oriented Immobilization of Histidine-Tagged Proteins on Nonfouling Cobalt Polyphenolic Self-Assembly Surfaces

Lulu Han, Qi Liu, Liwei Yang, Tong Ye, Zhien He and Lingyun Jia*

33



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Figure 1. Scheme of the assembly of TA/PSBMA/CoII film. a) TA film was prepared through the coordination interaction between TA and FeIII ions. b) Zwitterionic poly(sulfobetaine methacrylate) (PSBMA) was assembled on the TA film. c) CoII ions were chelated to the TA film. 272x122mm (300 x 300 DPI)


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Figure 2. Characterization of TA/PSBMA/CoII film. a) Real-time QCM-D data of frequency and dissipation shifts of TA/PSBMA/CoII film. b) Dry mass of TA, PSBMA and cobalt ions calculated by the Sauerbrey equation. c) Static water contact-angle. d) UV-Vis absorption spectra. Data are the means ± SDs of three samples. 272x178mm (300 x 300 DPI)



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Figure 3. XPS spectra of modified quartz slides. a) TA. b) TA/PSBMA. c) TA/PSBMA/CoII. d) Surface chemical compositions of TA/PSBMA/CoII films. Insets are the high resolution Co2p XPS spectra. 225x170mm (300 x 300 DPI)


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Figure 4. Binding behavior of BSA (400 µg/mL) and His-tagged chitinase (400 µg/mL) as determined by QCM-D. a) and b) TA/PSBMA/CoII modified-crystals. c) TA/CoII modified-crystals. d) The hydrated mass of the amount of bound protein as calculated by fitting with the Voigt model. Data are the means ± SDs from three samples. 277x194mm (300 x 300 DPI)



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Figure 5. a) A digital photo of TA/PSBMA/CoII/His-tagged chitinase films on the quartz, glass, polyamide and polyethersulfone substrates with a ruler. b) Diagram showing the capture of His-tagged protein by the TA/PSBMA/CoII film. 417x218mm (300 x 300 DPI)


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Figure 6. CoII oxidation of TA/PSBMA/CoII coatings by treatment with 3 mM H2O2, and the stability of immobilized His-tagged protein after washing with 300 mM imidazole. a) High resolution Co2p XPS spectra of TA/PSBMA/CoII coatings on silicon wafer. b) High resolution Co2p XPS spectra of TA/PSBMA/CoIII coatings on silicon wafer, after oxidation of TA/PSBMA/CoII coatings by H2O2 (3 mM) for 60 min. c) QCM-D curve of TA/PSBMA/CoII/His-tagged chitinase washed with 300 mM imidazole for overnight. d) QCM-D curve of TA/PSBMA/CoIII/His-tagged chitinase washed with 300 mM imidazole for overnight. 243x175mm (300 x 300 DPI)



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Figure 7. a) Activity of immobilized His-tagged chitinase. b) Effect of papain on the activity of immobilized chitinase. The activity of free His-tagged chitinase (1410 U/g) was considered as 100%. Data are the mean ± SD from three samples. Significance was tested by one-way ANOVA test, *: p < 0.05, **: p < 0.01, ***: p < 0.001. 275x115mm (300 x 300 DPI)


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Figure 8. Effect of various factors on the stability and activity of immobilized His-tagged chitinase. a) Thermal stability (60 oC), b) storage stability (4 oC) and c) repeated uses. The initial activity of immobilized His-tagged chitinase was considered as 100%. Data are the mean ± SD from three samples. 259x192mm (300 x 300 DPI)


Facile Oriented Immobilization of Histidine-Tagged ... - ACS Publications

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