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Application of docking methods for metal chelate affinity precipitation of endo-glucanase using pH-response polymer
Colloids and Surfaces B: Biointerfaces 113 (2014) 412–420
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Application of docking methods for metal chelate affinity precipitation of endo-glucanase using pH-response polymer Zhaoyang Ding, Lin Kang, Xuejun Cao ∗ State Key Laboratory of Bioreactor Engineering, Department of Bioengineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 29 August 2013 Accepted 18 September 2013 Available online 26 September 2013 Keywords: Docking Label-free detection Metal chelate affinity precipitation pH-response Endo-glucanase
a b s t r a c t An endo-glucanase could be efficiently purified using metal chelate affinity precipitation by a pHresponse polymer PMMDN with iminodiacetic acid (IDA) and Cu2+ as affinity ligand. In this study, docking method was used to identify the appropriate chelator and the metal ion as ligand by Grid score. The simulation results were compared with the label-free detection data analyzed by ForteBio’s Octet. The ligand IDA-Cu2+ was the final choice. A pH-response polymer PMMDN was polymerized and subsequently coupled with IDA-Cu2+ as the ligand. The pI and recovery of PMMDN and PMMDN -IDA-Cu2+ were 4.50, 99.8% and 4.39, 97.6%, respectively. Optimal adsorption conditions were found to be ligand density of 3.0 mmol/g, pH 5.5 and 1.0 mol/L NaCl. The adsorption isotherm showed the maximum adsorption as 57.62 mg/g polymer and the dissociation constant as 1.08 mg/mL. For the elution of the PMMDN -IDA-Cu2+ with the protein, 0.5 mol/L imidazole containing 1.0 mol/L guanidine hydrochloride was used as the eluent. Under these conditions, electrophoretic purity of endo-glucanase was obtained by only one step, and the elution recoveries were 96.45% (protein) and 93.24% (activity). © 2013 Elsevier B.V. All rights reserved.
1. Introduction Purification of proteins is an important task in bioindustry. With the rapid development of the gene technology, any desire enzyme could be obtained, but the fast and convenient purification method is still a challenge in a large scale. Affinity technology of purification means to separate a specific protein from a complex mixture. Metal chelate affinity chromatography was reported by Porath et al. [1]. Hundreds of papers [2–6] have been published, describing the use of this technique in group separation and selectively purification for target protein from complex samples. Metal chelate affinity precipitation [7–9] was introduced into purification proteins of large-scale process as a more feasible and cost effective tool. Metal chelate affinity technique utilizes the differential affinity force between proteins and immobilized metal ions to purify proteins [10]. Metal chelate affinity precipitation is achieved by a response polymer with immobilized metal ions as ligands, and its transition can be obtained by the changing of surrounding parameters, such as pH, temperature, ionic strength or special ions, organic solvent and opposite charged electrolyte polymer [11–13]. Kumar et al. [14] bound Cu (II) to poly(N-isopropylacrylamide/vinylimidazole) to purification histidine-tagged protein by metal chelate affinity
∗ Corresponding author. Tel.: +86 21 64252695; fax: +86 21 64252695. E-mail address: [email protected] (X. Cao). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.041
precipitation. A thermo-sensitive copolymer consisted of N-vinyl2-caprolactam (NVCL) and methacrylic acid was used by Ling et al. [15] to purify BSA with copper ions as the ligand. Bresolin [16] used poly (ethylene vinyl alcohol) immobilized Zn (II) for purification of anti-TNP IgG1 mouse MAbs from cell culture supernatant. Metal chelate affinity precipitation was applied in purification of not only enzymes [17], but also RNA, DNA [18] and antibody [19]. Docking method is playing an increasingly important role in new drug discovery and design. It has been recognized as a ‘lock-andkey’ model of receptor–ligand interaction [20]. Docking method of molecular simulation has emerged to become a useful part of drug discovery and ligand screening [21,22]. An important use of protein–ligand docking programs is virtual screening, in which large libraries of compounds are docked into a target binding site and scored [23,24]. Docking method was applied to a set of 12 compounds binding to cytochrome P450 by Stjernschantz and Oostenbrink [25]. A successful docking method could search matched spaces effectively and use a scoring function to rank the candidate dockings. DOCK was the first widely used docking program [26,27] and its usefulness of applications was developed. DOCK was used in protein-based drug design, particularly DOCKing algorithms, to be extended or adapted for nucleic acids study [28]. DOCK6 was used to analyze the structural information for brain creatine kinase and its interaction with sodium dodecyl sulfate [29]. In this work, it is applied to the study of interaction between affinity ligand and protein.
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In recent years, label-free detection has been used for study of quantify the kinetics and affinities of protein and ligand interactions. In particular, a label-free platform based on biolayer interferometry has been developed by ForteBio’s Company. ForteBio’s Octet monitors the binding of proteins to other biomolecules directly in real time utilizing disposable fiber-optic tips that address samples from an open shaking micro plate without any micro fluidics [30]. The results of docking could be contrasted by this experiment. As the most important part of cellulase, endo-glucanases (EC 3.2.1.4) cleave the internal glycosidic bonds of cellulosic chains and act synergistically with exo-glucanases and -glucosidases during the enzymatic degradation of cellulose [31]. Endo-glucanases are produced by a broad range of organisms including fungi, bacteria, plants, and insects [32,33]. Endo-glucanases have broad applications in industry [34,35]. For instance, in the biopolishing process, endo-glucanases are much more efficient than any other cellulases in removing color from denim to produce a good stonewashing effect [36]. In addition, endo-glucanases also benefit beer brewing, and improves the quality of animal feed [34]. Consequently, there has been a rapid growth in demand for endo-glucanases. Mitsuhiro Ueda et al. purified endo-glucanase by ion exchange method with DEAE-TOYOPEARL 650 M column [37]. Salting out by ammonium sulfate [33,38], fast protein liquid chromatography [39], affinity chromatography [40,41] or multi-step chromatographic strategy [42], etc. have been applied in purification of endo-glucanase. We reported application of docking methods for metal chelate affinity precipitation of endo-glucanase using pH-response polymer. Docking of different ligands and protein was simulated by Dock 6.4, and subsequently the experiments of ForteBio’s Octet confirmed the docking result. A pH-response polymer PMMDN was polymerized and coupled with the ligand. The adsorption and elution of endo-glucanase on the polymer were investigated. 2. Materials and methods
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distributions. The first step is the calculation of the solvent accessible surface of each receptor, without hydrogen atoms, using a probe radius of 1.4 A´˚ with the program DMS. A negative image of the surface is then generated as a set of overlapping spheres using the programs SPHGEN or sphgen cpp. A subset of spheres near the crystallographic ligand is then selected with the aid of the program sphere selector. Preparation of the docking grids is then performed and follows a two-step procedure. Firstly, a box around the binding site is constructed with the program SHOWBOX. The box includes the selected spheres and a protocol dependent margin. Secondly, the actual grids are computed with the accessory program GRID using 0.3 A´˚ grid spacing, a 9999 A´˚ distance cutoff, and a 4r distance dependent dielectric constant. 2.2.1.3. Docking. After the identification, 16 ligands which consisted of four chelate agents and four metal ions were prepared for docking to the protein. Grid score was used to judge the results of docking. Grid score consists of intermolecular non-bonded van der Waals (VDW) and Coulombic energies (scaled by a distancedependent dielectric) of electrostatic force (ES) between the ligand and receptor [44]. 2.2.2. pH-response polymer polymerization The synthesis procedure of followed Ding’s method [45]. The mixture of specified amount of four monomers (MAA, MMA, DMAEMA and N-MAM), and AIBN as polymerization initiator and absolute ethanol as solvent were poured into a flask with nitrogen maintained for 15 min. The pH-response polymer synthesized by MAA, MMA, DMAEMA and N-MAM was named PMMDN . N-MAM monomer has a hydroxyl group which could be used to connect ligand to the polymer by activation. The reaction was carried out for 24 h at 60 ◦ C and the product was precipitated from the reaction solution and washed three times with acetone and absolute ethanol to remove residual monomers. The solid product was dried under vacuum condition.
2.1. Materials Azobisisobutyronitrile (AIBN), methyl acrylic acid (MAA), methyl methacrylate (MMA), methacrylic acid 2-(dimethylamino) ethylester (DMAEMA), N-methylolacrylamide (N-MAM), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), carboxymethylaspartic acid (CM-Asp), N,N,N -tris(carboxymethyl) ethylenediamine (TED), CuSO4 , ZnSO4 , NiSO4 and MgSO4 were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Endo-glucanase was provided by Novozymes (Denmark). Other chemicals were of analytical reagent grade. 2.2. Methods 2.2.1. Docking 2.2.1.1. Protein and ligand preparation. The endo-glucanase file was downloaded from the Protein Data Bank (PDB) website (www.rcsb.org) (PDB ID code 1WZZ). The structure of the endoglucanase was processed with the DOCK Prepare module in Chimera [43] in these steps: solvent deletion, deletion of alternate positions, hydrogen addition, partial charge assignment and output in Mol2 format. AM1-BCC charges were computed for the endo-glucanase cofactors with ANTECHAMBER. The ligand was protonated and assigned AM1-BCC charges with Chimera as described above [28]. 2.2.1.2. Binding sites identification [27]. A few steps are required to prepare the binding sites prior to running actual DOCK calculations including such as DMS, SPHGEN, sphgen cpp, sphere selector, SHOWBOX and GRID which are available with the standard DOCK
2.2.3. Immobilization of ligand on PMMDN polymer The hydroxyl groups on the copolymer were activated by epichlorohydrine (ECH). 0.01 mol chelator was put into 30 mL Na2 CO3 (1 mol/L) and mixed with 0.5 mL ECH. The reaction was carried out in a shaker at 200 rpm for 24 h at 60 ◦ C. Adjust the pH to 5.5 by adding 1 M HCl and add 0.01 mol metal ions into the solution. The reaction continued in the shaker at 200 rpm for 2 h at 30 ◦ C. Meanwhile, 1 g PMMDN was dissolved in 100 mL NaOH (1 mol/L). All the solutions above were mixed together in the shaker at 40 ◦ C for 2 h. After the reaction finished, adjust the pH to pI of the polymer to precipitate the polymer. Remove impurities in the solution by suction filtration and vacuum drying. The concentration of metal ions was detected by ICP-AES Varian 710ES (Varian, USA). The reaction formula of PMMDN and immobilization of ligand on PMMDN polymer was shown in Fig. 1. The polymer PMMDN immobilized metal ions as ligand was named PMMDN -M. 2.2.4. Affinity precipitation of endo-glucanase 2.2.4.1. Adsorption of endo-glucanase. PMMDN -M was dissolved in aqueous solution up to 8.0% (w/v). Meanwhile 2.5 mL endoglucanase solutions of varied concentration were mixed with 2.5 mL above PMMDN -M solution together. All the samples were kept for 2.0 h at 25.0 ◦ C with constant mixing on a rotation shaker. At last, the complex precipitate was collected by adjusting the pH to 4.35 and centrifugation at 4000 rpm at 25.0 ◦ C for 5.0 min. The adsorption capacity of PMMDN -M was investigated by varying the time, pH (3.0–9.0 buffers), ionic strength (0.0–1.0 mol/L NaCl), ligand density (0.5–4.0 mmol/g) and endo-glucanase concentration (0.5–10.0 mg/mL).
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Fig. 1. The reaction formula of PMMDN and the connection of IDA-Cu2+ .
2.2.4.2. Desorption. The collected precipitate was dissolved in 5.0 mL eluant and precipitated again in the same method as the adsorption step. Suitable elution conditions were chosen.
2.2.4.3. Affinity precipitation of endo-glucanase from crude endoglucanase. Affinity precipitation of endo-glucanase from crude endo-glucanase was carried out in the same process as above. The optimal adsorption and desorption conditions obtained above were
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applied in this experiment. The result was analyzed by SDS polyacrylamide gel electrophoresis. 2.2.4.4. Recycle of affinity polymer. After desorption experiments of the endo-glucanase, the affinity polymer was recovered and regenerated with the suitable eluent. The regenerated polymer was reused in the next cycle of purification experiments. 2.2.5. Analytic methods 2.2.5.1. ForteBio Octet system assay. The interaction between different ligand and endo-glucanase was tested by the ForteBio Octet System (ForteBio Inc., USA) to confirm the Docking results. The PMMDN -M was immobilized onto the biosensors, and then the processes of adsorption and desorption of the endo-glucanase molecule were monitored in parallel. PMMDN -M was biotinylated by adding equivalent biotin for 30.0 min. Then the unconjugated biotin was removed by using PD-10 desalting column (Catalog number 17-0851-01, GE Healthcare). The sensors (Super Streptavidin, SSA) were wetted in dialysis buffer for 15 min prior to use. For binding affinity assay, the sensors were loaded with biotinylated PMMDN -M for 15 min, and then quenched in 10.0 mol/L biotin for 1.0 min. Endo-glucanase was prepared in 2 mol/L. The sensor without loading biotinylated PMMDN -M was used as a control to correct baseline drift. The whole process of experiment was carried out at room temperature and all tests were repeated in triplicate (Fig. 2). 2.2.5.2. Test of the isoelectric point (pI) and recovery of polymer. Determination of zeta potential was used to detect the isoelectric point (pI) of the polymer. Different pH values of the polymer solution (10 ppm) were adjusted and then the zeta potentials were measured using Zetasizer Nano ZEN3600 (Malvern, British). The pH-response polymer could be precipitated and dissolved by adjusting pH of the polymer solution and the recovery was determined by weight of the polymer after pH-precipitation. The recovery was calculated as the ratio of the dried weight of the precipitated polymer by heating to the initial weight. 1.0 g polymer was dissolved in a centrifuge tube, then HCl (0.1 mol/L) was added slowly to adjust pH of the solution to pI. The precipitate was separated from solution by centrifugation at 4000 rpm for 15 min and dried to constant mass. The recovery of the polymer was measured in three trials.
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Table 1 The Grid scores of various ligands with endo-glucanase. No.
Ligand
VDW (kcal/mol)
ES (kcal/mol)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
IDA3 -Mg2+ IDA3 -Cu2+ IDA3 -Zn2+ IDA3 -Ni2+ NTA4 -Mg2+ NTA4 -Cu2+ NTA4 -Zn2+ NTA4 -Ni2+ CM-Asp4 -Mg2+ CM-Asp4 -Cu2+ CM-Asp4 -Zn2+ CM-Asp4 -Ni2+ TED5 -Mg2+ TED5 -Cu2+ TED5 -Zn2+ TED5 -Ni2+
−67.54 −82.35 −76.01 −72.22 −58.51 −72.40 −67.54 −62.98 −59.71 −73.03 −66.85 −61.25 −45.85 −61.52 −55.02 −52.29
−19.25 −24.21 −22.25 −21.34 −17.52 −21.35 −19.52 −18.99 −16.98 −22.01 −19.87 −19.22 −14.25 −19.69 −17.24 −16.70
2.2.5.3. Determination of endo-glucanase activity. The activity of endo-glucanase was measured by using sodium carboxy-methyl cellulose as substrates [46]. One unit of enzyme activity is defined as the amount of enzyme required to produce one mg of reducing sugar per hour under assay conditions. The amount of reducing sugar was determined with the dinitrosalicylic acid method [47]. 2.2.5.4. SDS polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli [48] with 10.0% separating gel to check the purity of the enzyme sample obtained by affinity precipitation. The gel was stained with 0.25% Coomassie Brilliant Blue R-250. 3. Results and discussion 3.1. Docking results 3.1.1. The Grid score of docking The docking results were judged by Grid score including VDW and ES. The generalized force between the ligand and protein could be useful to choose the appropriate ligand. The choice of chelator is of crucial importance for the binding of the metal ion. The ligands consisted of chelators and metal ions, and Table 1 shows 16 different ligands with different chelators and metal ions.
Fig. 2. Immobilization of PMMDN -IDA-Cu2+ on biosensors with subsequent endo-glucanase binding. After biotinylated and binding of PMMDN -IDA-Cu2+ , and then the kinetics of adsorption and desorption between the immobilized PMMDN -IDA-Cu2+ and endo-glucanase are measured.
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Table 2 Elution recovery of different conditions. No.
Elution condition
Elution recovery of protein (%)
Elution recovery of activity (%)
Dissociation ratio of metal ions (%)
1 2 3 4 5 6 7 8 9 10
0.1 mol/L imidazole 0.2 mol/L imidazole 0.5 mol/L imidazole 0.5 mol/L imidazole + 0.1 mol/L guanidine hydrochloride 0.5 mol/L imidazole + 0.5 mol/L guanidine hydrochloride 0.5 mol/L imidazole + 1.0 mol/L guanidine hydrochloride 0.01 mol/LEDTA 0.05 mol/LEDTA 0.1 mol/LEDTA 0.2 mol/LEDTA
49.25 55.43 60.74 72.34 85.21 96.45 66.38 86.28 96.11 98.52
45.35 51.32 61.65 69.78 86.12 93.24 72.96 85.82 97.23 99.33
0.12 0.41 0.65 0.77 0.79 1.21 15.22 42.35 73.71 86.95
According to the results, for the same chelator and different metal ions, the Grid score between ligand and protein is following as the sequence Mg2+ > Ni2+ > Zn2+ > Cu2+ . The higher values of VDW and ES indicated the stronger action between ligand and protein. The Grid scores between ligand and protein are following as the sequence TED5 > NTA4 ≥ CM-Asp4 > IDA3 with the metal ion. The
results showed that IDA3 -Cu2+ gained the lowest Grid score and TED5 -Mg2+ gained the highest score, which meant that IDA3 -Cu2+ should be the most stable ligand with the protein. 3.1.2. Binding experiments The Qke Octet system was employed to analyze the interactions between ligand and protein and testify the results of docking. In this work, we evaluated the affinity of different chelators and metal ions as ligands. Fig. 3a shows the effects of various chelators as the ligands, and the light shift distance decreased as the sequence IDA3 -Cu2+ > CM-Asp4 -Cu2+ ≥ NTA4 -Cu2+ > TED5 -Cu2+ , which meant IDA3 -Cu2+ had higher interaction with endo-glucanase than other three ligands. Different metal ions were also evaluated by this system (Fig. 3b), then the results showed that the IDA3 -Cu2+ had the highest affinity strength and IDA3 -Mg2+ lower than others. The obtained data here could match the docking results well in both chelators and metal ions. To sum up, IDA3 -Cu2+ could gain the highest Grid score and strongest interaction with endo-glucanase due to the terdentate structure of IDA3 and the metal ion, thus IDA3 -Cu2+ was selected as ligand for further research in this work. 3.2. Experimental results 3.2.1. Synthesis of PMMDN -IDA-Cu2+ The isoelectric point (pI) and recovery are two important parameters for pH-response polymer during affinity precipitation. The pI is defined as the pH at which the net charge on a macromolecule is zero, and it could be determined as the pH when the zeta potential is zero.
Fig. 3. Data of affinity interactions between various ligands and endo-glucanase. The vertical and horizontal axes represent the light shift distance (nm) of different ligands and adsorption/desorption time (s), respectively. Various chelators (a) with metal ion settled and various metal ions (b) with chelator settled were bound to the sensors for affinity of endo-glucanase, and all ligands were in the same concentration.
Fig. 4. Zeta potetinals of polymers in different pH.
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Fig. 5. Adsorption conditions of PMMDN -IDA-Cu2+ binding endo-glucanase. (a) Effect of reaction time of PMMDN -IDA-Cu2+ on adsorbing endo-glucanase. Initial endo-glucanase concentration 10.0 mg/mL, T = 30.0 ◦ C, pH = 5.0, affinity ligand density 3.0 mmol/g and 0.1 g polymer. (b) Effect of ligand densities of PMMDN -IDA-Cu2+ on adsorbing endoglucanase. Initial endo-glucanase concentration 10.0 mg/mL, T = 30.0 ◦ C, pH = 5.0 and 0.1 g polymer. (c) Effect of pH of PMMDN -IDA-Cu2+ on binding endo-glucanase. Initial endoglucanase concentration 10.0 mg/mL, T = 30.0 ◦ C, affinity ligand density 3.0 mmol/g and 0.1 g polymer. (d) Effect of ionic strength of PMMDN -IDA-Cu2+ on binding endo-glucanase. Initial endo-glucanase concentration 10.0 mg/mL, T = 30.0 ◦ C, pH = 5.0, affinity ligand density 3.0 mmol/g and 0.1 g polymer. (e) Adsorption isotherm of endo-glucanase binding to the affinity polymer. T = 30.0 ◦ C, pH = 5.0, affinity ligand density 3.0 mmol/g and 0.1 g polymer.
The pI of PMMDN was 4.50 and PMMDN -IDA-Cu2+ was 4.39 as shown in Fig. 4. The pI of polymer is attributed to the ionization of different concentration of COOH from monomer MAA and N (CH3 )2 from monomer DMAEMA. If the ratio of COOH to N (CH3 )2 became higher, more H+ were needed to neutralize the polymer which leading to lower pI [49,50]. With the connection of IDA, more COOH were brought in to the polymer. Hence the pI of PMMDN -IDA-Cu2+ decreased from 4.50 to 4.39. Fortunately, it had no effect on the further study. The recovery of PMMDN was 99.8% and PMMDN -IDA-Cu2+ was 97.6%. 3.2.2. Adsorption results 3.2.2.1. Adsorption conditions. For adsorption of endo-glucanase, several factors (time, ligand densities, pH and the concentration of
NaCl) were investigated. Fig. 5a illustrates the effect of the reaction time on adsorbing endo-glucanase. The adsorption kinetics curve expressed a fast binding rate at the beginning of the adsorption process, then for the adsorption equilibrium was achieved gradually at about 2.0 h. This may be due to the decrease of the endo-glucanase concentration in the solution and increase of the endo-glucanase adsorbed on the polymer with time during adsorption. The ligand density is a significant factor that could influence the adsorption capacity and affinity selectivity. The effect of ligand densities on adsorbing endo-glucanase is shown in Fig. 5b. The maximum adsorption capacity of endo-glucanase was 38.85 mg endo-glucananse/g PMMDN -IDA-Cu2+ in the case of the ligand densities around 3.0 mmol/g. The adsorption capacity increased linearly with the ligand density below 2.5 mmol/g. At higher ligand
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densities, however, a slow decline of the adsorption capacity happened. The reason probably was the steric effects of binding sites blockage. The ligand density of PMMDN -IDA-Cu2+ applied in the following experiments was 3.0 mmol/g. Fig. 5c illustrates the effect of pH on the adsorbing endoglucanase. The charges at the surface of the enzyme changed with varying pH values. The optimal adsorption was achieved at pH 5–6. In alkaline conditions above pH 7 and in acidic conditions below pH 5.5, the adsorption of the enzyme decreased dramatically. The decrease of enzyme adsorption in acidic solution could be explained by the protonation of histidine residues at pH values below 6 and hence decreasing their propensity to coordinate metal ions. PMMDN -IDA-Cu2+ could be precipitated when the pH of the solution is around the pI, hence the precipitated polymer results in a low adsorption capacity. Unfortunately, under alkaline conditions, the copper ions on the polymer would precipitate leading to the reduction in the amount of endo-glucanase absorbed to the polymer. The optimal value of pH for adsorption was pH 5.5. The effect of NaCl concentration on adsorbing endo-glucanase was also studied, and the results are presented in Fig. 5d. An increase of endo-glucanase adsorption occurred in response to higher NaCl concentration. Firstly, the ions could shield more surface charge of the enzyme at higher concentrations, resulting in reduced electrostatic repulsive interactions between charged enzyme groups and the ligands connected to the polymer. Secondly, the rise in ions strength would affect the hydrophobic interaction among the enzyme. 3.2.2.2. Adsorption isotherm. Fig. 5e shows the adsorption isotherm of endo-glucanase binding on the affinity polymer. The curve behaves as Langmuir isotherm. A Langmuir model can be applied to fit experimental data as following: Qm C q= Kd + C where q is the amount of the adsorbed endo-glucanase, C is the concentration of unbound endo-glucanase in solution, Qm is the maximum capacity of the affinity polymer and Kd is the dissociation constant. Simulating the equation with experimental data, we obtained Qm as 57.62 mg/g polymer and Kd as 1.08 mg/mL. 3.2.3. Elution results 3.2.3.1. Elution conditions. The suitable reagents as follows were chosen for elution experiments. Protein amount and activity were both measured in this work. The results of elution recovery were shown in Table 2. The maximal elution recoveries of both protein and activity were obtained by elution conditions No. 6, 9 and 10, and more than 95.0% protein and activity could be eluted. Imidazole is a monodentate molecule with metal ions, resulting in the weak binding of imidazole molecules to metal ions in solution, and therefore No. 1–3 elution conditions could not obtain high elution recovery. With the addition of guanidine hydrochloride, the elution recovery was promoted as in conditions No. 4–6. As a chaotrope, guanidine hydrochloride had the function of propelling the elution of endo-glucanase from the polymer by dissociating hydrogen bonds. EDTA was usually regarded as the strongest competition eluent for its binding with metal ions. The data of No. 7–10 indicated that endo-glucanase could be eluted effectively by EDTA. Nevertheless, the copper ions also are dissociated from the polymer together with the protein according to the dissociation ratio of metal ions (Table 2). With higher EDTA concentration, more copper ions would be separated from the polymer, which was not good for reuse of the polymer. In sum, 0.5 mol/L imidazole with 1.0 mol/L guanidine hydrochloride was selected as the eluent, and the elution recoveries were 96.45% (protein) and 93.24% (activity).
Fig. 6. SDS-PAGE (10.0%) of the purity of endo-glucanase. Lane M, molecular weight standards; lane 1, purified endo-glucanase; lane 2, crude endo-glucanase.
3.2.3.2. SDS-PAGE analysis. As shown in Fig. 6, SDS-PAGE of the purified endo-glucanase showed a single band, corresponding to a molecular weight of about 65.0 kDa. This indicates that the affinity precipitation was a feasible method for the purification of endoglucanase from crude endo-glucanase. Electrophoretic purity of endo-glucanase can be obtained by only one-step purification. It indicated that metal chelate affinity precipitation of endoglucanase by PMMDN -IDA-Cu2+ was more efficient than some other purification methods [37,38,42]. 3.2.4. Reusability of affinity polymer In order to investigate the reusability of the polymer, the adsorption–desorption cycle of endo-glucanase was repeated five times using the same polymer. The results showed that affinity polymers could be repeatedly used in endo-glucanase adsorption without any noticeable reduction with related to the initial adsorption capacities. The adsorption capacity of the polymer decreased only 4% from 42.2 mg/g to 40.5 mg/g after the five repeated cycles. Compared with the other response polymers applied in purification of proteins [51], the polymer shows potential application with high recovery. 4. Conclusion In this study, DOCK 6.4 was applied to analyze the interactions between various ligands and target protein, and ligands were docked into the protein and scored by GRID to screen an appropriate ligand for metal chelate affinity precipitation. Label free detection was also employed to analyze the binding of them, and the results obtained by ForteBio’s Octet could match the docking
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results. The ligand IDA-Cu2+ was the final choice. A pH-response polymer PMMDN was polymerized and subsequently coupled with IDA-Cu2+ as the ligand. The recovery of PMMDN was 99.8% and PMMDN -IDA-Cu2+ was 97.6%. The optimal adsorption conditions were ligand density of 3.0 mmol/g affinity polymer, pH 5.5 and 1.0 mol/L NaCl. The adsorption isotherm showed Qm as 57.62 mg/g polymer and Kd as 1.08 mg/mL. For the elution of the PMMDN -IDACu2+ with the protein, 0.5 mol/L imidazole with 1.0 mol/L guanidine hydrochloride was selected as the eluent. Under these conditions, the elution recoveries were 96.45% (protein) and 93.24% (activity), and electrophoretic purity of endo-glucanase was obtained by only one step. In conclusion, docking method could be applied to screen affinity ligand and PMMDN -IDA-Cu2+ was suitable for purification of endo-glucanase for its good adsorption capacity, high recovery and reusability. Acknowledgement The authors very appreciate financial support from National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204). References [1] J. Porath, J.A.N. Carlsson, I. Olsson, G. Belfrage, Metal chelate affinity chromatography, a new approach to protein fractionation, Nature 258 (1975) 598–599. [2] G.S. Chaga, Twenty-five years of immobilized metal ion affinity chromatography: past, present and future, J. Biochem. Biophys. Methods 49 (2001) 313. [3] S.S. Suh, M.E. Van Dam, G.E. Wuenschell, S. Plunkett, F.H. Arnold, Novel MetalAffinity Protein Separations Protein Purification, vol. 427, American Chemical Society, 1990, pp. 139–149. [4] J. Porath, B. Olin, Immobilized metal affinity adsorption and immobilized metal affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions, Biochemistry 22 (1983) 1621–1630. [5] M. Benelmekki, C. Caparros, E. Xuriguera, S. Lanceros-Mendez, E. RodriguezCarmona, R. Mendoza, J.L. Corchero, L.M. Martinez, Improving the binding capacity of Ni2+ decorated porous magnetic silica spheres for histidine-rich protein separation, Colloids Surf. B: Biointerfaces 101 (2013) 370–375. [6] A. Denizli, S. S¸enel, M.Y. Arıca, Cibacron Blue F3GA and Cu(II) derived poly(2hydroxyethylmethacrylate) membranes for lysozyme adsorption, Colloids Surf. B: Biointerfaces 11 (1998) 113–122. [7] A. Kumar, I.Y. Galaev, B. Mattiasson, Metal chelate affinity precipitation: a new approach to protein purification, Bioseparation 7 (1998) 185–194. [8] I.Y. Galaev, A. Kumar, R. Agarwal, M.N. Gupta, B. Mattiasson, Imidazole—a new ligand for metal affinity precipitation, Appl. Biochem. Biotechnol. 68 (1997) 121–133. [9] A. Kumar, I.Y. Galaev, B. Mattiasson, Isolation and separation of ␣-amylase inhibitors I-1 and I-2 from seeds of ragi (Indian finger millet, Eleusine coracana) by metal chelate affinity precipitation, Bioseparation 7 (1998) 129–136. [10] Z. Wu, L. Ding, H. Chen, L. Yuan, H. Huang, W. Song, Immobilization of proteins on metal ion chelated polymer surfaces, Colloids Surf. B: Biointerfaces 69 (2009) 71–76. [11] A. Kumar, A. Srivastava, I.Y. Galaev, B. Mattiasson, Smart polymers: physical forms and bioengineering applications, Prog. Polym. Sci. 32 (2007) 1205–1237. [12] B. Mattiasson, A. Kumar, A.E. Ivanov, I.Y. Galaev, Metal-chelate affinity precipitation of proteins using responsive polymers, Nat. Protoc. 2 (2007) 213–220. [13] A. Kumar, I. Galaev, B. Mattiasson, Affinity precipitation of proteins using metal chelates, in: M. Zachariou (Ed.), Affinity Chromatography, vol. 421, Humana Press, 2008, pp. 37–52. [14] A. Kumar, M. Kamihira, I.Y. Galaev, S. Iijima, B. Mattiasson, Binding of Cu(II)poly(N-isopropylacrylamide/vinylimidazole) copolymer to histidine-tagged protein: a surface plasmon resonance study, Langmuir 19 (2002) 865–871. [15] Y.-Q. Ling, H.-L. Nie, C. Brandford-White, G.R. Williams, L.-M. Zhu, Metal chelate affinity precipitation: purification of BSA using poly(N-vinylcaprolactamco-methacrylic acid) copolymers, Colloids Surf. B: Biointerfaces 94 (2012) 281–287. [16] I.T.L. Bresolin, M. Borsoi-Ribeiro, W.M.S.C. Tamashiro, E.F.P. Augusto, M.A. Vijayalakshmi, S.M.A. Bueno, Evaluation of immobilized metal-ion affinity chromatography (IMAC) as a technique for IgG1 monoclonal antibodies purification: the effect of chelating ligand and support, Appl. Biochem. Biotechnol. 160 (2010) 2148–2165. [17] A. Kumar, A.A.M. Khalil, I.Y. Galaev, B. Mattiasson, Metal chelate affinity precipitation: purification of (His)6-tagged lactate dehydrogenase using poly(vinylimidazole-co-N-isopropylacrylamide) copolymers, Enzyme Microb. Technol. 33 (2003) 113–117. [18] S. Balan, J. Murphy, I. Galaev, A. Kumar, G. Fox, B. Mattiasson, R. Willson, Metal chelate affinity precipitation of RNA and purification of plasmid DNA, Biotechnol. Lett. 25 (2003) 1111–1116.
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Glossary Mol2: The Mol2 file format is a popular method for specifying chemical structure, including atom types, positions, and bonding AM1-BCC: Semi-empirical (AM1) with bond charge correction (BCC)
ANTECHAMBER: Antechamber is a set of auxiliary programs for molecular mechanic studies DMS: DMS is an open source program written in C for computing the molecular surface of a molecule SPHGEN: Sphgen generates sets of overlapping spheres to describe the shape of a molecule or molecular surface sphgen cpp: Sphgen cpp has the same function as Sphgen sphere selector: Sphere selector filters the output from sphgen selecting all spheres within a user-defined radius of a target molecule SHOWBOX: Showbox is an interactive program for specifying the location and the size of the grids that will be calculated by the program grid GRID: Grid creates the grid files necessary for rapid score evaluation in DOCK
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Application of docking methods for metal chelate affinity precipitation of endo-glucanase using pH-response polymer Zhaoyang Ding, Lin Kang, Xuejun Cao ∗ State Key Laboratory of Bioreactor Engineering, Department of Bioengineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 29 August 2013 Accepted 18 September 2013 Available online 26 September 2013 Keywords: Docking Label-free detection Metal chelate affinity precipitation pH-response Endo-glucanase
a b s t r a c t An endo-glucanase could be efficiently purified using metal chelate affinity precipitation by a pHresponse polymer PMMDN with iminodiacetic acid (IDA) and Cu2+ as affinity ligand. In this study, docking method was used to identify the appropriate chelator and the metal ion as ligand by Grid score. The simulation results were compared with the label-free detection data analyzed by ForteBio’s Octet. The ligand IDA-Cu2+ was the final choice. A pH-response polymer PMMDN was polymerized and subsequently coupled with IDA-Cu2+ as the ligand. The pI and recovery of PMMDN and PMMDN -IDA-Cu2+ were 4.50, 99.8% and 4.39, 97.6%, respectively. Optimal adsorption conditions were found to be ligand density of 3.0 mmol/g, pH 5.5 and 1.0 mol/L NaCl. The adsorption isotherm showed the maximum adsorption as 57.62 mg/g polymer and the dissociation constant as 1.08 mg/mL. For the elution of the PMMDN -IDA-Cu2+ with the protein, 0.5 mol/L imidazole containing 1.0 mol/L guanidine hydrochloride was used as the eluent. Under these conditions, electrophoretic purity of endo-glucanase was obtained by only one step, and the elution recoveries were 96.45% (protein) and 93.24% (activity). © 2013 Elsevier B.V. All rights reserved.
1. Introduction Purification of proteins is an important task in bioindustry. With the rapid development of the gene technology, any desire enzyme could be obtained, but the fast and convenient purification method is still a challenge in a large scale. Affinity technology of purification means to separate a specific protein from a complex mixture. Metal chelate affinity chromatography was reported by Porath et al. [1]. Hundreds of papers [2–6] have been published, describing the use of this technique in group separation and selectively purification for target protein from complex samples. Metal chelate affinity precipitation [7–9] was introduced into purification proteins of large-scale process as a more feasible and cost effective tool. Metal chelate affinity technique utilizes the differential affinity force between proteins and immobilized metal ions to purify proteins [10]. Metal chelate affinity precipitation is achieved by a response polymer with immobilized metal ions as ligands, and its transition can be obtained by the changing of surrounding parameters, such as pH, temperature, ionic strength or special ions, organic solvent and opposite charged electrolyte polymer [11–13]. Kumar et al. [14] bound Cu (II) to poly(N-isopropylacrylamide/vinylimidazole) to purification histidine-tagged protein by metal chelate affinity
∗ Corresponding author. Tel.: +86 21 64252695; fax: +86 21 64252695. E-mail address: [email protected] (X. Cao). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.041
precipitation. A thermo-sensitive copolymer consisted of N-vinyl2-caprolactam (NVCL) and methacrylic acid was used by Ling et al. [15] to purify BSA with copper ions as the ligand. Bresolin [16] used poly (ethylene vinyl alcohol) immobilized Zn (II) for purification of anti-TNP IgG1 mouse MAbs from cell culture supernatant. Metal chelate affinity precipitation was applied in purification of not only enzymes [17], but also RNA, DNA [18] and antibody [19]. Docking method is playing an increasingly important role in new drug discovery and design. It has been recognized as a ‘lock-andkey’ model of receptor–ligand interaction [20]. Docking method of molecular simulation has emerged to become a useful part of drug discovery and ligand screening [21,22]. An important use of protein–ligand docking programs is virtual screening, in which large libraries of compounds are docked into a target binding site and scored [23,24]. Docking method was applied to a set of 12 compounds binding to cytochrome P450 by Stjernschantz and Oostenbrink [25]. A successful docking method could search matched spaces effectively and use a scoring function to rank the candidate dockings. DOCK was the first widely used docking program [26,27] and its usefulness of applications was developed. DOCK was used in protein-based drug design, particularly DOCKing algorithms, to be extended or adapted for nucleic acids study [28]. DOCK6 was used to analyze the structural information for brain creatine kinase and its interaction with sodium dodecyl sulfate [29]. In this work, it is applied to the study of interaction between affinity ligand and protein.
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In recent years, label-free detection has been used for study of quantify the kinetics and affinities of protein and ligand interactions. In particular, a label-free platform based on biolayer interferometry has been developed by ForteBio’s Company. ForteBio’s Octet monitors the binding of proteins to other biomolecules directly in real time utilizing disposable fiber-optic tips that address samples from an open shaking micro plate without any micro fluidics [30]. The results of docking could be contrasted by this experiment. As the most important part of cellulase, endo-glucanases (EC 3.2.1.4) cleave the internal glycosidic bonds of cellulosic chains and act synergistically with exo-glucanases and -glucosidases during the enzymatic degradation of cellulose [31]. Endo-glucanases are produced by a broad range of organisms including fungi, bacteria, plants, and insects [32,33]. Endo-glucanases have broad applications in industry [34,35]. For instance, in the biopolishing process, endo-glucanases are much more efficient than any other cellulases in removing color from denim to produce a good stonewashing effect [36]. In addition, endo-glucanases also benefit beer brewing, and improves the quality of animal feed [34]. Consequently, there has been a rapid growth in demand for endo-glucanases. Mitsuhiro Ueda et al. purified endo-glucanase by ion exchange method with DEAE-TOYOPEARL 650 M column [37]. Salting out by ammonium sulfate [33,38], fast protein liquid chromatography [39], affinity chromatography [40,41] or multi-step chromatographic strategy [42], etc. have been applied in purification of endo-glucanase. We reported application of docking methods for metal chelate affinity precipitation of endo-glucanase using pH-response polymer. Docking of different ligands and protein was simulated by Dock 6.4, and subsequently the experiments of ForteBio’s Octet confirmed the docking result. A pH-response polymer PMMDN was polymerized and coupled with the ligand. The adsorption and elution of endo-glucanase on the polymer were investigated. 2. Materials and methods
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distributions. The first step is the calculation of the solvent accessible surface of each receptor, without hydrogen atoms, using a probe radius of 1.4 A´˚ with the program DMS. A negative image of the surface is then generated as a set of overlapping spheres using the programs SPHGEN or sphgen cpp. A subset of spheres near the crystallographic ligand is then selected with the aid of the program sphere selector. Preparation of the docking grids is then performed and follows a two-step procedure. Firstly, a box around the binding site is constructed with the program SHOWBOX. The box includes the selected spheres and a protocol dependent margin. Secondly, the actual grids are computed with the accessory program GRID using 0.3 A´˚ grid spacing, a 9999 A´˚ distance cutoff, and a 4r distance dependent dielectric constant. 2.2.1.3. Docking. After the identification, 16 ligands which consisted of four chelate agents and four metal ions were prepared for docking to the protein. Grid score was used to judge the results of docking. Grid score consists of intermolecular non-bonded van der Waals (VDW) and Coulombic energies (scaled by a distancedependent dielectric) of electrostatic force (ES) between the ligand and receptor [44]. 2.2.2. pH-response polymer polymerization The synthesis procedure of followed Ding’s method [45]. The mixture of specified amount of four monomers (MAA, MMA, DMAEMA and N-MAM), and AIBN as polymerization initiator and absolute ethanol as solvent were poured into a flask with nitrogen maintained for 15 min. The pH-response polymer synthesized by MAA, MMA, DMAEMA and N-MAM was named PMMDN . N-MAM monomer has a hydroxyl group which could be used to connect ligand to the polymer by activation. The reaction was carried out for 24 h at 60 ◦ C and the product was precipitated from the reaction solution and washed three times with acetone and absolute ethanol to remove residual monomers. The solid product was dried under vacuum condition.
2.1. Materials Azobisisobutyronitrile (AIBN), methyl acrylic acid (MAA), methyl methacrylate (MMA), methacrylic acid 2-(dimethylamino) ethylester (DMAEMA), N-methylolacrylamide (N-MAM), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), carboxymethylaspartic acid (CM-Asp), N,N,N -tris(carboxymethyl) ethylenediamine (TED), CuSO4 , ZnSO4 , NiSO4 and MgSO4 were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Endo-glucanase was provided by Novozymes (Denmark). Other chemicals were of analytical reagent grade. 2.2. Methods 2.2.1. Docking 2.2.1.1. Protein and ligand preparation. The endo-glucanase file was downloaded from the Protein Data Bank (PDB) website (www.rcsb.org) (PDB ID code 1WZZ). The structure of the endoglucanase was processed with the DOCK Prepare module in Chimera [43] in these steps: solvent deletion, deletion of alternate positions, hydrogen addition, partial charge assignment and output in Mol2 format. AM1-BCC charges were computed for the endo-glucanase cofactors with ANTECHAMBER. The ligand was protonated and assigned AM1-BCC charges with Chimera as described above [28]. 2.2.1.2. Binding sites identification [27]. A few steps are required to prepare the binding sites prior to running actual DOCK calculations including such as DMS, SPHGEN, sphgen cpp, sphere selector, SHOWBOX and GRID which are available with the standard DOCK
2.2.3. Immobilization of ligand on PMMDN polymer The hydroxyl groups on the copolymer were activated by epichlorohydrine (ECH). 0.01 mol chelator was put into 30 mL Na2 CO3 (1 mol/L) and mixed with 0.5 mL ECH. The reaction was carried out in a shaker at 200 rpm for 24 h at 60 ◦ C. Adjust the pH to 5.5 by adding 1 M HCl and add 0.01 mol metal ions into the solution. The reaction continued in the shaker at 200 rpm for 2 h at 30 ◦ C. Meanwhile, 1 g PMMDN was dissolved in 100 mL NaOH (1 mol/L). All the solutions above were mixed together in the shaker at 40 ◦ C for 2 h. After the reaction finished, adjust the pH to pI of the polymer to precipitate the polymer. Remove impurities in the solution by suction filtration and vacuum drying. The concentration of metal ions was detected by ICP-AES Varian 710ES (Varian, USA). The reaction formula of PMMDN and immobilization of ligand on PMMDN polymer was shown in Fig. 1. The polymer PMMDN immobilized metal ions as ligand was named PMMDN -M. 2.2.4. Affinity precipitation of endo-glucanase 2.2.4.1. Adsorption of endo-glucanase. PMMDN -M was dissolved in aqueous solution up to 8.0% (w/v). Meanwhile 2.5 mL endoglucanase solutions of varied concentration were mixed with 2.5 mL above PMMDN -M solution together. All the samples were kept for 2.0 h at 25.0 ◦ C with constant mixing on a rotation shaker. At last, the complex precipitate was collected by adjusting the pH to 4.35 and centrifugation at 4000 rpm at 25.0 ◦ C for 5.0 min. The adsorption capacity of PMMDN -M was investigated by varying the time, pH (3.0–9.0 buffers), ionic strength (0.0–1.0 mol/L NaCl), ligand density (0.5–4.0 mmol/g) and endo-glucanase concentration (0.5–10.0 mg/mL).
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Fig. 1. The reaction formula of PMMDN and the connection of IDA-Cu2+ .
2.2.4.2. Desorption. The collected precipitate was dissolved in 5.0 mL eluant and precipitated again in the same method as the adsorption step. Suitable elution conditions were chosen.
2.2.4.3. Affinity precipitation of endo-glucanase from crude endoglucanase. Affinity precipitation of endo-glucanase from crude endo-glucanase was carried out in the same process as above. The optimal adsorption and desorption conditions obtained above were
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applied in this experiment. The result was analyzed by SDS polyacrylamide gel electrophoresis. 2.2.4.4. Recycle of affinity polymer. After desorption experiments of the endo-glucanase, the affinity polymer was recovered and regenerated with the suitable eluent. The regenerated polymer was reused in the next cycle of purification experiments. 2.2.5. Analytic methods 2.2.5.1. ForteBio Octet system assay. The interaction between different ligand and endo-glucanase was tested by the ForteBio Octet System (ForteBio Inc., USA) to confirm the Docking results. The PMMDN -M was immobilized onto the biosensors, and then the processes of adsorption and desorption of the endo-glucanase molecule were monitored in parallel. PMMDN -M was biotinylated by adding equivalent biotin for 30.0 min. Then the unconjugated biotin was removed by using PD-10 desalting column (Catalog number 17-0851-01, GE Healthcare). The sensors (Super Streptavidin, SSA) were wetted in dialysis buffer for 15 min prior to use. For binding affinity assay, the sensors were loaded with biotinylated PMMDN -M for 15 min, and then quenched in 10.0 mol/L biotin for 1.0 min. Endo-glucanase was prepared in 2 mol/L. The sensor without loading biotinylated PMMDN -M was used as a control to correct baseline drift. The whole process of experiment was carried out at room temperature and all tests were repeated in triplicate (Fig. 2). 2.2.5.2. Test of the isoelectric point (pI) and recovery of polymer. Determination of zeta potential was used to detect the isoelectric point (pI) of the polymer. Different pH values of the polymer solution (10 ppm) were adjusted and then the zeta potentials were measured using Zetasizer Nano ZEN3600 (Malvern, British). The pH-response polymer could be precipitated and dissolved by adjusting pH of the polymer solution and the recovery was determined by weight of the polymer after pH-precipitation. The recovery was calculated as the ratio of the dried weight of the precipitated polymer by heating to the initial weight. 1.0 g polymer was dissolved in a centrifuge tube, then HCl (0.1 mol/L) was added slowly to adjust pH of the solution to pI. The precipitate was separated from solution by centrifugation at 4000 rpm for 15 min and dried to constant mass. The recovery of the polymer was measured in three trials.
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Table 1 The Grid scores of various ligands with endo-glucanase. No.
Ligand
VDW (kcal/mol)
ES (kcal/mol)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
IDA3 -Mg2+ IDA3 -Cu2+ IDA3 -Zn2+ IDA3 -Ni2+ NTA4 -Mg2+ NTA4 -Cu2+ NTA4 -Zn2+ NTA4 -Ni2+ CM-Asp4 -Mg2+ CM-Asp4 -Cu2+ CM-Asp4 -Zn2+ CM-Asp4 -Ni2+ TED5 -Mg2+ TED5 -Cu2+ TED5 -Zn2+ TED5 -Ni2+
−67.54 −82.35 −76.01 −72.22 −58.51 −72.40 −67.54 −62.98 −59.71 −73.03 −66.85 −61.25 −45.85 −61.52 −55.02 −52.29
−19.25 −24.21 −22.25 −21.34 −17.52 −21.35 −19.52 −18.99 −16.98 −22.01 −19.87 −19.22 −14.25 −19.69 −17.24 −16.70
2.2.5.3. Determination of endo-glucanase activity. The activity of endo-glucanase was measured by using sodium carboxy-methyl cellulose as substrates [46]. One unit of enzyme activity is defined as the amount of enzyme required to produce one mg of reducing sugar per hour under assay conditions. The amount of reducing sugar was determined with the dinitrosalicylic acid method [47]. 2.2.5.4. SDS polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli [48] with 10.0% separating gel to check the purity of the enzyme sample obtained by affinity precipitation. The gel was stained with 0.25% Coomassie Brilliant Blue R-250. 3. Results and discussion 3.1. Docking results 3.1.1. The Grid score of docking The docking results were judged by Grid score including VDW and ES. The generalized force between the ligand and protein could be useful to choose the appropriate ligand. The choice of chelator is of crucial importance for the binding of the metal ion. The ligands consisted of chelators and metal ions, and Table 1 shows 16 different ligands with different chelators and metal ions.
Fig. 2. Immobilization of PMMDN -IDA-Cu2+ on biosensors with subsequent endo-glucanase binding. After biotinylated and binding of PMMDN -IDA-Cu2+ , and then the kinetics of adsorption and desorption between the immobilized PMMDN -IDA-Cu2+ and endo-glucanase are measured.
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Table 2 Elution recovery of different conditions. No.
Elution condition
Elution recovery of protein (%)
Elution recovery of activity (%)
Dissociation ratio of metal ions (%)
1 2 3 4 5 6 7 8 9 10
0.1 mol/L imidazole 0.2 mol/L imidazole 0.5 mol/L imidazole 0.5 mol/L imidazole + 0.1 mol/L guanidine hydrochloride 0.5 mol/L imidazole + 0.5 mol/L guanidine hydrochloride 0.5 mol/L imidazole + 1.0 mol/L guanidine hydrochloride 0.01 mol/LEDTA 0.05 mol/LEDTA 0.1 mol/LEDTA 0.2 mol/LEDTA
49.25 55.43 60.74 72.34 85.21 96.45 66.38 86.28 96.11 98.52
45.35 51.32 61.65 69.78 86.12 93.24 72.96 85.82 97.23 99.33
0.12 0.41 0.65 0.77 0.79 1.21 15.22 42.35 73.71 86.95
According to the results, for the same chelator and different metal ions, the Grid score between ligand and protein is following as the sequence Mg2+ > Ni2+ > Zn2+ > Cu2+ . The higher values of VDW and ES indicated the stronger action between ligand and protein. The Grid scores between ligand and protein are following as the sequence TED5 > NTA4 ≥ CM-Asp4 > IDA3 with the metal ion. The
results showed that IDA3 -Cu2+ gained the lowest Grid score and TED5 -Mg2+ gained the highest score, which meant that IDA3 -Cu2+ should be the most stable ligand with the protein. 3.1.2. Binding experiments The Qke Octet system was employed to analyze the interactions between ligand and protein and testify the results of docking. In this work, we evaluated the affinity of different chelators and metal ions as ligands. Fig. 3a shows the effects of various chelators as the ligands, and the light shift distance decreased as the sequence IDA3 -Cu2+ > CM-Asp4 -Cu2+ ≥ NTA4 -Cu2+ > TED5 -Cu2+ , which meant IDA3 -Cu2+ had higher interaction with endo-glucanase than other three ligands. Different metal ions were also evaluated by this system (Fig. 3b), then the results showed that the IDA3 -Cu2+ had the highest affinity strength and IDA3 -Mg2+ lower than others. The obtained data here could match the docking results well in both chelators and metal ions. To sum up, IDA3 -Cu2+ could gain the highest Grid score and strongest interaction with endo-glucanase due to the terdentate structure of IDA3 and the metal ion, thus IDA3 -Cu2+ was selected as ligand for further research in this work. 3.2. Experimental results 3.2.1. Synthesis of PMMDN -IDA-Cu2+ The isoelectric point (pI) and recovery are two important parameters for pH-response polymer during affinity precipitation. The pI is defined as the pH at which the net charge on a macromolecule is zero, and it could be determined as the pH when the zeta potential is zero.
Fig. 3. Data of affinity interactions between various ligands and endo-glucanase. The vertical and horizontal axes represent the light shift distance (nm) of different ligands and adsorption/desorption time (s), respectively. Various chelators (a) with metal ion settled and various metal ions (b) with chelator settled were bound to the sensors for affinity of endo-glucanase, and all ligands were in the same concentration.
Fig. 4. Zeta potetinals of polymers in different pH.
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417
Fig. 5. Adsorption conditions of PMMDN -IDA-Cu2+ binding endo-glucanase. (a) Effect of reaction time of PMMDN -IDA-Cu2+ on adsorbing endo-glucanase. Initial endo-glucanase concentration 10.0 mg/mL, T = 30.0 ◦ C, pH = 5.0, affinity ligand density 3.0 mmol/g and 0.1 g polymer. (b) Effect of ligand densities of PMMDN -IDA-Cu2+ on adsorbing endoglucanase. Initial endo-glucanase concentration 10.0 mg/mL, T = 30.0 ◦ C, pH = 5.0 and 0.1 g polymer. (c) Effect of pH of PMMDN -IDA-Cu2+ on binding endo-glucanase. Initial endoglucanase concentration 10.0 mg/mL, T = 30.0 ◦ C, affinity ligand density 3.0 mmol/g and 0.1 g polymer. (d) Effect of ionic strength of PMMDN -IDA-Cu2+ on binding endo-glucanase. Initial endo-glucanase concentration 10.0 mg/mL, T = 30.0 ◦ C, pH = 5.0, affinity ligand density 3.0 mmol/g and 0.1 g polymer. (e) Adsorption isotherm of endo-glucanase binding to the affinity polymer. T = 30.0 ◦ C, pH = 5.0, affinity ligand density 3.0 mmol/g and 0.1 g polymer.
The pI of PMMDN was 4.50 and PMMDN -IDA-Cu2+ was 4.39 as shown in Fig. 4. The pI of polymer is attributed to the ionization of different concentration of COOH from monomer MAA and N (CH3 )2 from monomer DMAEMA. If the ratio of COOH to N (CH3 )2 became higher, more H+ were needed to neutralize the polymer which leading to lower pI [49,50]. With the connection of IDA, more COOH were brought in to the polymer. Hence the pI of PMMDN -IDA-Cu2+ decreased from 4.50 to 4.39. Fortunately, it had no effect on the further study. The recovery of PMMDN was 99.8% and PMMDN -IDA-Cu2+ was 97.6%. 3.2.2. Adsorption results 3.2.2.1. Adsorption conditions. For adsorption of endo-glucanase, several factors (time, ligand densities, pH and the concentration of
NaCl) were investigated. Fig. 5a illustrates the effect of the reaction time on adsorbing endo-glucanase. The adsorption kinetics curve expressed a fast binding rate at the beginning of the adsorption process, then for the adsorption equilibrium was achieved gradually at about 2.0 h. This may be due to the decrease of the endo-glucanase concentration in the solution and increase of the endo-glucanase adsorbed on the polymer with time during adsorption. The ligand density is a significant factor that could influence the adsorption capacity and affinity selectivity. The effect of ligand densities on adsorbing endo-glucanase is shown in Fig. 5b. The maximum adsorption capacity of endo-glucanase was 38.85 mg endo-glucananse/g PMMDN -IDA-Cu2+ in the case of the ligand densities around 3.0 mmol/g. The adsorption capacity increased linearly with the ligand density below 2.5 mmol/g. At higher ligand
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densities, however, a slow decline of the adsorption capacity happened. The reason probably was the steric effects of binding sites blockage. The ligand density of PMMDN -IDA-Cu2+ applied in the following experiments was 3.0 mmol/g. Fig. 5c illustrates the effect of pH on the adsorbing endoglucanase. The charges at the surface of the enzyme changed with varying pH values. The optimal adsorption was achieved at pH 5–6. In alkaline conditions above pH 7 and in acidic conditions below pH 5.5, the adsorption of the enzyme decreased dramatically. The decrease of enzyme adsorption in acidic solution could be explained by the protonation of histidine residues at pH values below 6 and hence decreasing their propensity to coordinate metal ions. PMMDN -IDA-Cu2+ could be precipitated when the pH of the solution is around the pI, hence the precipitated polymer results in a low adsorption capacity. Unfortunately, under alkaline conditions, the copper ions on the polymer would precipitate leading to the reduction in the amount of endo-glucanase absorbed to the polymer. The optimal value of pH for adsorption was pH 5.5. The effect of NaCl concentration on adsorbing endo-glucanase was also studied, and the results are presented in Fig. 5d. An increase of endo-glucanase adsorption occurred in response to higher NaCl concentration. Firstly, the ions could shield more surface charge of the enzyme at higher concentrations, resulting in reduced electrostatic repulsive interactions between charged enzyme groups and the ligands connected to the polymer. Secondly, the rise in ions strength would affect the hydrophobic interaction among the enzyme. 3.2.2.2. Adsorption isotherm. Fig. 5e shows the adsorption isotherm of endo-glucanase binding on the affinity polymer. The curve behaves as Langmuir isotherm. A Langmuir model can be applied to fit experimental data as following: Qm C q= Kd + C where q is the amount of the adsorbed endo-glucanase, C is the concentration of unbound endo-glucanase in solution, Qm is the maximum capacity of the affinity polymer and Kd is the dissociation constant. Simulating the equation with experimental data, we obtained Qm as 57.62 mg/g polymer and Kd as 1.08 mg/mL. 3.2.3. Elution results 3.2.3.1. Elution conditions. The suitable reagents as follows were chosen for elution experiments. Protein amount and activity were both measured in this work. The results of elution recovery were shown in Table 2. The maximal elution recoveries of both protein and activity were obtained by elution conditions No. 6, 9 and 10, and more than 95.0% protein and activity could be eluted. Imidazole is a monodentate molecule with metal ions, resulting in the weak binding of imidazole molecules to metal ions in solution, and therefore No. 1–3 elution conditions could not obtain high elution recovery. With the addition of guanidine hydrochloride, the elution recovery was promoted as in conditions No. 4–6. As a chaotrope, guanidine hydrochloride had the function of propelling the elution of endo-glucanase from the polymer by dissociating hydrogen bonds. EDTA was usually regarded as the strongest competition eluent for its binding with metal ions. The data of No. 7–10 indicated that endo-glucanase could be eluted effectively by EDTA. Nevertheless, the copper ions also are dissociated from the polymer together with the protein according to the dissociation ratio of metal ions (Table 2). With higher EDTA concentration, more copper ions would be separated from the polymer, which was not good for reuse of the polymer. In sum, 0.5 mol/L imidazole with 1.0 mol/L guanidine hydrochloride was selected as the eluent, and the elution recoveries were 96.45% (protein) and 93.24% (activity).
Fig. 6. SDS-PAGE (10.0%) of the purity of endo-glucanase. Lane M, molecular weight standards; lane 1, purified endo-glucanase; lane 2, crude endo-glucanase.
3.2.3.2. SDS-PAGE analysis. As shown in Fig. 6, SDS-PAGE of the purified endo-glucanase showed a single band, corresponding to a molecular weight of about 65.0 kDa. This indicates that the affinity precipitation was a feasible method for the purification of endoglucanase from crude endo-glucanase. Electrophoretic purity of endo-glucanase can be obtained by only one-step purification. It indicated that metal chelate affinity precipitation of endoglucanase by PMMDN -IDA-Cu2+ was more efficient than some other purification methods [37,38,42]. 3.2.4. Reusability of affinity polymer In order to investigate the reusability of the polymer, the adsorption–desorption cycle of endo-glucanase was repeated five times using the same polymer. The results showed that affinity polymers could be repeatedly used in endo-glucanase adsorption without any noticeable reduction with related to the initial adsorption capacities. The adsorption capacity of the polymer decreased only 4% from 42.2 mg/g to 40.5 mg/g after the five repeated cycles. Compared with the other response polymers applied in purification of proteins [51], the polymer shows potential application with high recovery. 4. Conclusion In this study, DOCK 6.4 was applied to analyze the interactions between various ligands and target protein, and ligands were docked into the protein and scored by GRID to screen an appropriate ligand for metal chelate affinity precipitation. Label free detection was also employed to analyze the binding of them, and the results obtained by ForteBio’s Octet could match the docking
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results. The ligand IDA-Cu2+ was the final choice. A pH-response polymer PMMDN was polymerized and subsequently coupled with IDA-Cu2+ as the ligand. The recovery of PMMDN was 99.8% and PMMDN -IDA-Cu2+ was 97.6%. The optimal adsorption conditions were ligand density of 3.0 mmol/g affinity polymer, pH 5.5 and 1.0 mol/L NaCl. The adsorption isotherm showed Qm as 57.62 mg/g polymer and Kd as 1.08 mg/mL. For the elution of the PMMDN -IDACu2+ with the protein, 0.5 mol/L imidazole with 1.0 mol/L guanidine hydrochloride was selected as the eluent. Under these conditions, the elution recoveries were 96.45% (protein) and 93.24% (activity), and electrophoretic purity of endo-glucanase was obtained by only one step. In conclusion, docking method could be applied to screen affinity ligand and PMMDN -IDA-Cu2+ was suitable for purification of endo-glucanase for its good adsorption capacity, high recovery and reusability. Acknowledgement The authors very appreciate financial support from National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204). References [1] J. Porath, J.A.N. Carlsson, I. Olsson, G. Belfrage, Metal chelate affinity chromatography, a new approach to protein fractionation, Nature 258 (1975) 598–599. [2] G.S. Chaga, Twenty-five years of immobilized metal ion affinity chromatography: past, present and future, J. Biochem. Biophys. Methods 49 (2001) 313. [3] S.S. Suh, M.E. Van Dam, G.E. Wuenschell, S. Plunkett, F.H. Arnold, Novel MetalAffinity Protein Separations Protein Purification, vol. 427, American Chemical Society, 1990, pp. 139–149. [4] J. Porath, B. Olin, Immobilized metal affinity adsorption and immobilized metal affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions, Biochemistry 22 (1983) 1621–1630. [5] M. Benelmekki, C. Caparros, E. Xuriguera, S. Lanceros-Mendez, E. RodriguezCarmona, R. Mendoza, J.L. Corchero, L.M. Martinez, Improving the binding capacity of Ni2+ decorated porous magnetic silica spheres for histidine-rich protein separation, Colloids Surf. B: Biointerfaces 101 (2013) 370–375. [6] A. Denizli, S. S¸enel, M.Y. Arıca, Cibacron Blue F3GA and Cu(II) derived poly(2hydroxyethylmethacrylate) membranes for lysozyme adsorption, Colloids Surf. B: Biointerfaces 11 (1998) 113–122. [7] A. Kumar, I.Y. Galaev, B. Mattiasson, Metal chelate affinity precipitation: a new approach to protein purification, Bioseparation 7 (1998) 185–194. [8] I.Y. Galaev, A. Kumar, R. Agarwal, M.N. Gupta, B. Mattiasson, Imidazole—a new ligand for metal affinity precipitation, Appl. Biochem. Biotechnol. 68 (1997) 121–133. [9] A. Kumar, I.Y. Galaev, B. Mattiasson, Isolation and separation of ␣-amylase inhibitors I-1 and I-2 from seeds of ragi (Indian finger millet, Eleusine coracana) by metal chelate affinity precipitation, Bioseparation 7 (1998) 129–136. [10] Z. Wu, L. Ding, H. Chen, L. Yuan, H. Huang, W. Song, Immobilization of proteins on metal ion chelated polymer surfaces, Colloids Surf. B: Biointerfaces 69 (2009) 71–76. [11] A. Kumar, A. Srivastava, I.Y. Galaev, B. Mattiasson, Smart polymers: physical forms and bioengineering applications, Prog. Polym. Sci. 32 (2007) 1205–1237. [12] B. Mattiasson, A. Kumar, A.E. Ivanov, I.Y. Galaev, Metal-chelate affinity precipitation of proteins using responsive polymers, Nat. Protoc. 2 (2007) 213–220. [13] A. Kumar, I. Galaev, B. Mattiasson, Affinity precipitation of proteins using metal chelates, in: M. Zachariou (Ed.), Affinity Chromatography, vol. 421, Humana Press, 2008, pp. 37–52. [14] A. Kumar, M. Kamihira, I.Y. Galaev, S. Iijima, B. Mattiasson, Binding of Cu(II)poly(N-isopropylacrylamide/vinylimidazole) copolymer to histidine-tagged protein: a surface plasmon resonance study, Langmuir 19 (2002) 865–871. [15] Y.-Q. Ling, H.-L. Nie, C. Brandford-White, G.R. Williams, L.-M. Zhu, Metal chelate affinity precipitation: purification of BSA using poly(N-vinylcaprolactamco-methacrylic acid) copolymers, Colloids Surf. B: Biointerfaces 94 (2012) 281–287. [16] I.T.L. Bresolin, M. Borsoi-Ribeiro, W.M.S.C. Tamashiro, E.F.P. Augusto, M.A. Vijayalakshmi, S.M.A. Bueno, Evaluation of immobilized metal-ion affinity chromatography (IMAC) as a technique for IgG1 monoclonal antibodies purification: the effect of chelating ligand and support, Appl. Biochem. Biotechnol. 160 (2010) 2148–2165. [17] A. Kumar, A.A.M. Khalil, I.Y. Galaev, B. Mattiasson, Metal chelate affinity precipitation: purification of (His)6-tagged lactate dehydrogenase using poly(vinylimidazole-co-N-isopropylacrylamide) copolymers, Enzyme Microb. Technol. 33 (2003) 113–117. [18] S. Balan, J. Murphy, I. Galaev, A. Kumar, G. Fox, B. Mattiasson, R. Willson, Metal chelate affinity precipitation of RNA and purification of plasmid DNA, Biotechnol. Lett. 25 (2003) 1111–1116.
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Glossary Mol2: The Mol2 file format is a popular method for specifying chemical structure, including atom types, positions, and bonding AM1-BCC: Semi-empirical (AM1) with bond charge correction (BCC)
ANTECHAMBER: Antechamber is a set of auxiliary programs for molecular mechanic studies DMS: DMS is an open source program written in C for computing the molecular surface of a molecule SPHGEN: Sphgen generates sets of overlapping spheres to describe the shape of a molecule or molecular surface sphgen cpp: Sphgen cpp has the same function as Sphgen sphere selector: Sphere selector filters the output from sphgen selecting all spheres within a user-defined radius of a target molecule SHOWBOX: Showbox is an interactive program for specifying the location and the size of the grids that will be calculated by the program grid GRID: Grid creates the grid files necessary for rapid score evaluation in DOCK