A Biomimetic Polymer-Based Method for Selective Capture of C

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Biological and Medical Applications of Materials and Interfaces

A Biomimetic Polymer-Based Method for Selective Capture of C-Reactive Protein in Biological Fluids Qiqin Wang, Hanying Jin, Donghai Xia, Huikai Shao, Kun Peng, Xiao Liu, Hao Huang, Qiaoxuan Zhang, Jialiang Guo, Yuqiang Wang, Jacques Crommen, Ning Gan, and Zhengjin Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15581 • Publication Date (Web): 09 Nov 2018 Downloaded from http://pubs.acs.org on November 10, 2018

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ACS Applied Materials & Interfaces

A Biomimetic Polymer-Based Method for Selective Capture of CReactive Protein in Biological Fluids Qiqin Wanga, b‡, Hanying Jina,‡, Donghai Xiaa, Huikai Shaoa, Kun Penga, Xiao Liua, Hao Huanga, Qiaoxuan Zhangc, Jialiang Guoa, Yuqiang Wanga, Jacques Crommena,d, Ning Gane*, Zhengjin Jianga,b*

a

Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University,

Guangzhou 510632, China. b

Department of Pharmacy and Guangdong Province Key Laboratory of

Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou 510632, China c

Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou

University of Chinese Medicine, Guangzhou 510120, China. d

Laboratory of Analytical Pharmaceutical Chemistry, Department of Pharmaceutical

Sciences, CIRM, University of Liege, CHU B36, B-4000 Liege, Belgium e

Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo,

315211, China ‡

These authors contributed equally to this work.

*Corresponding authors: E-mail: [email protected] (Prof. Ning Gan), [email protected] (Prof. Zhengjin Jiang)

Keywords: zwitterionic phosphorylcholine, cell membrane biomimetic polymer, Creactive protein, selective enrichment, biological fluids

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ABSTRACT Selective capturing and purification of C-reactive protein (CRP) from complex biological fluids plays a pivotal role in studying biological activities of CRP in various diseases. However, obvious non-specific adsorption of proteins was observed on current affinity sorbents, and thus additional purification steps were often required which could compromise the recovery of the target protein and/or introduce new impurities. In this study, inspired by the highly specific interaction between CRP and cell membrane, an excellent anti-biofouling compound 2-(methacryloyloxy) ethyl phosphorylcholine and a highly hydrophilic crosslinker N,N'-methylenebisacrylamide were employed to fabricate a novel cell membrane biomimetic polymer for selective capture of CRP in the presence of calcium ions. Based on the polymer described above, a facile enrichment approach was established after systematic optimization of washing and elution conditions. With its favorable properties, such as good porosity, weak electrostatic interaction, high hydrophilicity, and biocompatibility, the novel biomimetic polymer exhibits good specificity, selectivity, recovery (near 100%), purity (95%) and lower nonspecific protein adsorption for CRP in comparison with commercial Immobilized p-Aminophenyl Phosphoryl Choline Gel and other purification materials. Furthermore, the structural integrity and functionality of CRP in the elution fraction were well preserved and confirmed by circular dichroism spectroscopy, fluorescence spectroscopy, and immunoturbidimetric assay. Finally, the biomimetic polymer was successfully applied to the selective enrichment of CRP from sera of patients with inflammation and rats. The proposed novel enrichment approach based on the versatile biomimetic polymer can be used for effective CRP purification, which will benefit the in-depth study of its biological roles.


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1. INTRODUCTION Human C-reactive protein (CRP), a homopentameric protein (MW ~ 115 kDa), is produced by the liver in response to tissue injury and inflammation.1 Its circulation level sharply increases by up to 1000-fold compared to normal conditions within 2448 h of injury.2 The interaction between CRP and phosphorylcholine (PC) receptor can activate classical complement pathways in damaged tissue, leading to an innate immune cascade.2-4 CRP also has many activities that are similar to those of immunoglobulin (IgG), including the ability to promote agglutination, phagocytosis, bacterial capsular swelling, and precipitation of polycationic and polyanionic compounds.5,6 Many large-scale prospective studies also demonstrated that CRP serves as an important biomarker for a number of diseases, such as infection, inflammation, cardiovascular disease, atherosclerosis and its sequels, etc.4,7-10 Furthermore, CRP is a promising therapeutic target for cardioprotection in acute myocardial infarction and neuroprotection in stroke.11 However, some of its putative biological roles in health and disease are controversial due to the inadequate use of contaminated human CRP or commercial recombinant CRP from E. coli.12,13 To study the mechanism of action of CRP in depth in various diseases and more precisely analyze the signaling pathways, high purity CRP with intact structure and functionality is urgently required. Different methods for purification of CRP have been reported, such as preparative zone

electrophoresis

in

starch

block,

non-specific

and

specific

affinity

chromatography, magnetic separation, thin-film molecularly imprinted polymer (MIP) method, as well as lecithin precipitation and negative affinity on gel filtration chromatography.14-16 Among these approaches, affinity chromatography turns out to be a versatile method for selective purification of CRP from human serum or ascites,



which relies on the interaction between CRP and specific ligands (PC, Cpolysaccharide (CPS), or phosphoethanolamine (PE)) in the presence of calcium ions.14 However, significant non-specific adsorption of proteins including IgG, human serum albumin (HSA), and immunoglobulin M (IgM), was observed in the EDTAeluate.17,18 Additional purification steps (ion exchange chromatography, sizeexclusion chromatography) are therefore employed to improve the purity of the eluted CRP, and thus might lead to a decrease in the recovery of the target protein or the introduction of new impurities.17,19,20 Thus, it is imperative to develop novel affinity materials to meet the challenges related to CRP purification. In recent years, 2-(methacryloyloxy) ethyl phosphorylcholine (MPC) polymers with a bioinspired biomembrane structure, have received increasing attention and have been widely used to construct non-biofouling surfaces in various biomedical applications.21-25 MPC grafted biomedical devices have already been shown to suppress unfavorable biological reactions even when they are in contact with living organisms, and MPC is now clinically used on the surfaces of intravascular stents, intravascular guide wires, soft contact lenses and the oxygenator (artificial lung) under the authorization of the FDA.26-28 Furthermore, as successful biomaterials, MPC polymers are also useful in other fields, such as biosensors,29 biochips,30 bioimaging tools,31 and for the delivery of biomolecules.32 These outstanding performances of MPC polymers could be attributed to their excellent biocompatibility and hydrophilicity that can prevent both protein adsorption and cell adhesion.21 On the basis of the above, MPC can be considered as having a great potential as a candidate affinity ligand for effective purification of CRP in the presence of calcium ions. However, most reported MPC materials for biomedical applications are linear polymers, which are soluble in water. In this study, as shown in Scheme 1, a novel cell


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membrane biomimetic polymer with high hydrophilicity and anti-nonspecific protein adsorption interface was prepared by employing MPC as affinity ligand and hydrophilic N,N'-methylenebisacrylamide (MBA) as crosslinker via thermally initiated free-radical polymerization strategy. The specific binding ability of the resulting biomimetic polymer was evaluated using a series of proteins as probes (including CRP, IgG, HSA, β-lactoglobulin (β-Lg), lysozyme (Lyz), cytochrome c (Cyt c) and myohemoglobin (Mb)) in the presence of calcium ions. The static binding capacity and long-term reusability of the biomimetic polymer were also tested. Finally, the purification of CRP from different biological fluids was performed using this biomimetic polymer and compared with that obtained with other materials. The purity of the collected fractions was investigated using SDS-PAGE and MALDI-TOF MS, while the structural integrity and functionality of the eluted CRP were confirmed by circular

dichroism

spectroscopy

(CD),

fluorescence

spectroscopy,

and

immunoturbidimetric assay.

Scheme 1. Schematic representation of the enrichment strategy based on the cell membrane biomimetic polymer for highly selective capture of CRP in biological fluids.



2. EXPERIMENTAL SECTION 2.1 Fabrication of the cell membrane biomimetic polymer. The biomimetic polymer was fabricated in a 200-μL pipette through a thermally initiated free-radical polymerization. In brief, the end of pipette was first sealed prior to the copolymerization of a mixture containing the affinity ligand (MPC), the crosslinker (MBA), the binary porogenic mixture (IPA and THF) and the initiator (AIBN, 1 wt% with respect to the total amount of affinity ligand and crosslinker). According to the selected composition given in Table S1, the polymerization mixture was ultrasonically blended into a homogenous solution in a 2-mL vial and then 120 μL solution was filled into the pipette. After sealing its other end with a syringe, the pipette was submerged into a water bath at 60oC for 12 h. Finally, the biomimetic polymer was washed with MeOH using the syringe pump to remove residues. The bulk polymer in the pipette was dried and cut for SEM, EDS, and pore size distribution analysis. The biomimetic polymer was also crushed into small pieces, Soxhlet extracted with MeOH for 24 h, and then dried under vacuum at 60°C for 6 h. These polymer pieces were successively used for FT-IR, specific surface area, and water contact angle analysis. 2.2 Specificity and selectivity of the cell membrane biomimetic polymer. To demonstrate the recognition specificity and selectivity of the biomimetic polymer, a mixture of CRP, IgG, HSA, β-Lg, Lyz, Cyt c, and Mb in buffer A (10 mM Tris, 140 mM NaCl, 2 mM CaCl2, pH 8.0) was chosen as probe. Firstly, polymer was activated using 600 µL buffer A. Then, 100 μL of protein mixture (concentration of every protein: 0.1 mg/mL) was loaded and washed with 1920 µL buffer A at a flow rate of 2 mL/min to exclude non-retained proteins. Buffer B (10 mM Tris, 140 mM NaCl, 2 mM EDTA-2Na, pH 8.0) was finally applied at the same flow rate to elute the


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proteins retained on the biomimetic polymer. Different fractions were successively collected for purity and recovery tests by SDS-PAGE and immunoturbidimetric assay. 2.3 Comparison of the cell membrane biomimetic polymer with commercial CRP purification material. In order to compare the purification efficiency of the biomimetic polymer with that of a commercial CRP purification material, the biomimetic polymer and the Immobilized p-Aminophenyl Phosphoryl Choline Gel (Thermo Scientific, Shanghai, China.) were employed for target protein purification in human serum sample spiked with CRP (0.35 mg/mL) solution. The purification of CRP from a spiked human serum sample was carried out on the biomimetic polymer according to the protocol described in section “Specificity and selectivity of the cell membrane biomimetic polymer”. For the application of the commercial CRP purification material, pAminophenyl PC based agarose beads (120 μL) were first equilibrated with buffer A in a 1.5-mL EP tube and then mixed with a spiked human serum sample. After 1 h of incubation, the mixture was successively centrifuged with buffer A (1920 μL) and buffer B (720 μL). The obtained supernatant solutions were analyzed by immunoturbidimetric assay and SDS-PAGE for testing the recovery and purity of CRP, respectively. 2.4 Enrichment and purification of CRP in different biological fluids. Similar to the specificity experiment, 100 μL of serum from a patient with inflammation or rat without dilution were first loaded on the cell membrane biomimetic polymer at 2 mL/min, and then immediately washed by buffer A (1920 μL) and eluted by buffer B (720 μL) at the same flow rate. All fractions were collected for quantitative analysis and purity determination by immunoturbidimetric assay and SDS-PAGE, respectively. The washing and elution fractions were also subjected to



MALDI-TOF MS analysis for further purity verification. The structural integrity of the obtained CRP was identified by CD and fluorescence spectra (Experimental details are shown in Supporting Information). 3. RESULTS AND DISCUSSION 3.1 Fabrication and characterization of the cell membrane biomimetic polymer. High recovery yields are desirable properties for protein purification materials. In order to obtain a versatile cell membrane biomimetic polymer with high performance, several key polymerization factors including the amount of affinity ligand, the weight ratios and the composition of the porogenic mixture were systematically optimized, and the recovery of CRP was chosen as evaluation criterion. The influence of the proportion of affinity ligand on the binding capacity and recovery of the biomimetic polymer was first investigated. As shown in Table S1, when the weight ratio of MPC in the monomer mixture was decreased from 75% to 67.5% (w/wtotal monomers), no significant change in the recovery of CRP was observed (varying from 103.2% (M1) to 99.3% (M3)). The effect of the proportion of porogenic solvents (IPA and THF) on CRP recovery was then investigated by varying IPA weight content from 65% (M4) to 55% (M5). IPA/THF (60/40, w/w) was finally chosen for further experiments because the polymer M2 exhibited the highest CRP recovery. The influence of the porogen content in the polymerization mixture was also studied. As seen from Table S1, the recovery of CRP increased from 78.5% (M6) to 105.3% (M7) with decreasing porogen content. Finally, M2 was considered as the optimum biomimetic polymer for the purification of CRP in biological fluids. Ideal bio-separation materials must be hydrophilic, possess chemical and mechanical resistance, and adequate porosity.33,34 Therefore, the morphological and physical properties of the biomimetic polymer were thoroughly characterized. EDS


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data revealed the existence of P element in the polymer, which demonstrated that MPC was successfully incorporated into the polymeric matrix (Figure 1a, b).35 FT-IR spectra provided a direct evidence for successful preparation of the biomimetic polymer (Figure 1c). Compared to Poly MBA, all characteristic peaks of PC group, including P=O (1239 cm-1), P-O-alkyl (1069 cm-1), and N+(CH3)3 (961 cm-1) stretches, can be identified in the FT-IR spectrum of the biomimetic polymer.36 SEM images showed that spherical units agglomerate into large clusters interdispersed by largepore channels (Figure 1d, e). Moreover, the pore size distribution of the biomimetic polymer was measured by mercury intrusion porosimetry. As depicted in Figure 1f, large amounts of macropores (> 3 μm) and good porosity (78%) were observed, which permits rapid flow through the polymer under low backpressure. According to BET method, the specific surface area of the biomimetic polymer was determined to be 6.4 m2/g in dry state, which is favorable for protein capture. Furthermore, the measured water contact angle is 39.6o (Figure S1), which is far less than 90o, indicating that the polymer has good hydrophilicity.

Figure 1. EDS (a, b), FT-IR spectra(c), SEM (d, e) and Pore size distribution curve (f) of the cell membrane biomimetic polymer.



3.2 Optimization of the enrichment protocol. The adsorption of CRP onto the cell membrane biomimetic polymer is a complex process in which several driving forces could be involved, such as hydrophobic, electrostatic interactions, and chelate effect between PC group and CRP in the presence of calcium ions.14 In order to obtain excellent recovery and high purity, a solution consisting of 10 mM Tris, 140 mM NaCl, 2 mM CaCl2, pH 8.0, was selected as buffer A for the loading and washing of CRP samples according to a previous report.11 Furthermore, the volume of the washing buffer, and the composition and volume of the elution buffer were systemically optimized. 3.3 Effect of the washing buffer volume. As is well-known, non-specific adsorption remains a major challenge for the enrichment of proteins from biosamples and has a serious detrimental effect on the purity, quality, and bioactivity of the target protein.37,38 The washing step plays an essential role in removing non-specific adsorption of proteins from the affinity sorbent. Therefore, complex biological fluids including serum from a patient with inflammation were selected as test samples. After loading 100 μL of serum into the biomimetic polymer, the effect of washing buffer (buffer A, 10 mM Tris, 140 mM NaCl, 2 mM CaCl2, pH 8.0) volumes on the CRP purity was evaluated. 720 µL of buffer B (10 mM Tris, 140 mM NaCl, 2 mM EDTA-2Na, pH 8.0) were used for CRP elution. As depicted in Figure S2, after washing with 120 μL of buffer A, lots of highly abundant serum proteins (e.g. IgG, HSA) were observed on the SDS-PAGE image of the corresponding elution fraction (El-1). However, the purity of CRP was further enhanced from 2.5% (El-1) to 95% (El-6) by increasing the washing buffer volume from 120 μL to 1920 μL. Taking into consideration the purity of the eluted CRP, 1920 μL of washing buffer was used for further enrichment experiments.


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3.4 Effect of the composition and volume of elution buffer. Other critical factors that affect the recovery are the composition and volume of elution buffer applied to release CRP from the cell membrane biomimetic polymer. Considering that the biomimetic polymer combines a zwitterionic character and hydrophilic properties, the influence of NaCl concentration (50-400 mM) and pH (4.0-9.0) in the elution buffer on the recovery of CRP was investigated. As depicted in Figure S3, no significant difference was observed in the NaCl concentration range studied and thus this concentration plays a marginal role on the recovery of CRP. 140 mM NaCl was finally selected to be consistent with the NaCl concentration in the washing buffer. However, it is interesting to note that the recovery of CRP was almost 0% when the elution buffer pH was between 4.0 to 6.0. The CRP recovery was dramatically increased from 9% to 114% by increasing the pH of the elution buffer from 6.0 to 9.0 (Figure S4). These results indicate that the pH can strongly influence the interactions between CRP and the affinity sorbent. pH 8.0 was found to be the most suitable with respect to recovery and stability of CRP. To find out proper volumes without loss of recovery, the effect of a series of elution buffer volumes (60960 μL) was also compared. Excellent recovery was obtained when 720 μL of a buffer made of 10 mM Tris, 140 mM NaCl, 2 mM EDTA-2Na (pH 8.0) was used as elution solvent (buffer B) (Figure S5). Based on the above, a facile enrichment approach based on the biomimetic polymer was established for the selective purification of CRP from complex samples. 3.5 Specificity, selectivity, and static binding capacity of the cell membrane biomimetic polymer. To demonstrate the recognition specificity and selectivity of the biomimetic polymer towards human CRP, a mixture of IgG, HSA, β-Lg, Lyz, Cyt c, Mb, and CRP,



was chosen as probe. As can be seen from the SDS-PAGE image, six out of the seven test proteins (HSA, IgG, Mb, β-Lg, Cyt c, Lyz) were observed in the washing fraction (Figure 2a), while only CRP was detected in the elution fraction. Furthermore, the recovery of CRP was almost 100%. These results indicate that CRP can be selectively captured on the biomimetic polymer. The superior performance of the novel cell membrane biomimetic polymer could be attributed to that it not only maintains the specific selectivity of traditional PC materials to CRP, but also combines the advantages of hydrophilic macroporous polymers. According to the crystal structure of CRP-PC complex (Figure 2b), the PC moiety on the polymer surface could be bound in a shallow surface pocket on each subunit of CRP, interacting with the two CRP-bound calcium ions via the phosphate group and with Glu81 of CRP via the quaternary ammonium group39,40. The hydrophilic skeleton of the cell membrane biomimetic polymer can suppress hydrophobic interactions, and then eliminate nonspecific adsorption of interfering proteins. Besides, Lund et al.41 reported that the sepharose particles with higher porosity exhibited superior binding capacity due to the increased accessible surface area. However, sepharose and agarose present certain shortcomings such as gel compressibility, poor pore diffusion, mass transfer limitations, and some non-specific adsorption.34 The properties of macroporous polymers, such as an excellent morphology, high porosity and permeability, fast mass transfer rate, have been proved to be beneficial for protein purification.24,25,42


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Figure 2. (a) Specificity and selectivity of the cell membrane biomimetic polymer for CRP using SDS-PAGE analysis (reducing conditions). Lanes: M, protein marker; El, elution fraction; WF, washing fraction; LF, loading fraction; F, feedstock. (b) Crystal structure of CRP-PC complex (PDB ID: 1b09)

39

. The molecular operating

environment 2015 software was used to generate the ribbon diagram of the x-ray crystal structure of CRP-PC complex. The binding site of CRP subunit and PC was shown. In the docking diagram of CRP-PC complex, four red balls represent the oxygen of phosphate group of PC moiety; three green balls represent the three methyl groups of choline. As shown in Figure S6, the isotherm for CRP adsorption to the biomimetic polymer was also determined. The adsorption data were fitted to a Langmuir model (Eq. (1)) and the adsorption equilibrium constants could be deduced. The maximum static binding capacity of this affinity material and the dissociation constant Kd were determined to be 53.5 mg/g of dried biomimetic polymer and 0.00123 mg/mL (8.2 × 10-9 mol/L), respectively. 3.6 Long-term stability, reusability, and reproducibility. Stability and reusability are of great importance for the practical application of affinity materials. The cell membrane biomimetic polymer provided reproducible



results without any loss of CRP recovery (106% ± 4.0%) even after 100 cycles of continuous use (Figure 3a). Furthermore, it is worth mentioning that CRP recoveries were kept between 93% and 116% after 105 injections over 6 months (Figure 3b), which confirms the high robustness and long-term stability of the biomimetic polymer. As shown in Table S2, the stability and reproducibility of most CRP enrichment materials have not been reported. The O-(4-nitrophenylphosphoryl) choline based MIP polymer can be used only once, and the commercial CRP enrichment material (Immobilized p-Aminophenyl PC Gel) was claimed to be reusable for only several times (Instructions). Clearly, the cell biomimetic polymer has much better or comparable long-term stability and reusability in comparison with these materials. The batch to batch reproducibility of CRP recovery was also tested. Five batches of the biomimetic polymer were prepared and all enrichment experiments were performed using standard CRP solution (0.05 mg/mL) as test sample. The average recovery and RSD value were 102% and 8%, respectively. These results demonstrate that the biomimetic polymer exhibits good reproducibility, reusability, and long-term stability.

Figure 3. Reusability (a) and long-term stability (b) of the cell membrane biomimetic polymer. 3.7 Comparison with a commercial CRP enrichment sorbent and other purification


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materials. Compared to the testing of model protein samples as mentioned above, CRP enrichment from biological fluids is substantially more challenging, because of the higher complexity and wider dynamic range of protein concentrations. The applicability of the cell membrane biomimetic polymer was tested, the commercially available Immobilized p-Aminophenyl Phosphoryl Choline Gel being employed for comparison. Human serum spiked with CRP (0.35 mg/mL) was selected as test sample. To evaluate the enrichment of CRP, all purification experiments were performed under the optimized conditions mentioned above. Furthermore, the influence of incubation time for the human serum sample on the adsorbent was further studied and compared. As shown in Table S3 and Figure 4, the purity of CRP was significantly improved from 70% to 85% after 1 h of incubation on the commercial PC modified sorbent. This is the reason why a 1h of incubation is usually required in the CRP enrichment protocol using the commercial PC modified sorbent.43 Unfortunately, nonspecific adsorption of serum proteins was detected in the eluate (Figure 4). The anti-protein fouling capacity of the commercial PC modified sorbent was also found to be questionable in previous studies.44,45 However, the biomimetic polymer exhibited higher recovery (> 99%) and purity (> 93%) than the commercial PC modified sorbent either with or without incubation. Furthermore, no significant change of CRP recovery was observed before or after incubation (Table S3), which suggests a fast binding rate between CRP and the affinity sorbent in the presence of calcium ions. It may be attributed to that the biomimetic polymer possesses high porosity and permeability, pore interconnections as well as fast mass transfer. In particular, no obvious interfering protein was observed in the SDS-PAGE image of the eluate after purification without incubation. The low propensity for nonspecific



protein adsorption could attribute to the weak electrostatic interaction (the ζ-potential value is -0.693 mV), high hydrophilicity (the water contact angle is 39.6o) and biocompatibility of the biomimetic polymer (Scheme 1). The whole processing time was also decreased to 30 min at the flow rate of 2 mL/min with the biomimetic polymer, which is shorter than with the commercial PC modified sorbent for the selective purification of CRP from complex matrices. Finally, the effect of CRP concentration (from 0.032 to 0.429 mg/mL) in the spiked human serum samples on recovery was evaluated using the biomimetic polymer. Good recoveries (from 90.0% to 115.5%) were achieved over the studied concentration range (Table S4). The superior specificity, selectivity, lower nonspecific protein adsorption, and shorter processing time of the biomimetic polymer were further evidenced through the comparison between our affinity material and other CRP purification materials used in previous studies (Table 1). All the above results indicate that the biomimetic polymer has a high potential for selective capture of low concentrations of CRP even in the presence of large amounts of interfering proteins.

Figure 4. Comparison of the biomimetic polymer and a commercial CRP enrichment sorbent for CRP purification without (a) or with (b) 1 h of incubation using SDSPAGE analysis (reducing conditions). Lanes: M, protein marker; S, standard CRP; F,


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feedstock; LF, loading fraction; WF, washing fraction; El, elution fraction. It was reported that the CRP obtained using Sepharose-CPS and Sepharose-pnitrophenyl PC failed to react with antisera and anti-whole human serum, respectively (Table S2). Therefore, the structural integrity of the obtained CRP on the cell membrane biomimetic polymer was then confirmed by CD and fluorescence spectroscopy. The CD spectrum is an effective tool for studying protein secondary structure, such as α-helix and β-sheet, in the far UV region.50 Figure 5a shows the CD spectra of standard CRP and purified CRP with a negative signal at 215 nm (typical of β-structured protein). No significant change was observed between the CD spectra of standard CRP and CRP obtained after enrichment, demonstrating that the secondary structure of CRP was well preserved during the purification process. As depicted in Figure 5b, the fluorescence emission spectra of standard CRP and purified CRP exhibit a maximum emission of around 350 nm (typical for tryptophan (Trp) residues exposed to water), which indicates that the tertiary association of regions containing Trp residues may be almost identical in proteins of the pentraxin family.51,52 All these results suggest that the structure of the obtained CRP is intact and consistent with standard CRP. The ability of the obtained CRP to bind to antibodies was also verified by immunoturbidimetric assay.

Figure 5. CD spectra (a) and fluorescence emission spectra (b) of standard CRP and



purified CRP. 3.8 Application to the CRP purification in different biological fluids. In order to test the potential of the cell membrane biomimetic polymer for selective enrichment of low abundance CRP, a serum without dilution (91 mg/mL of total protein containing 0.05 mg/mL of CRP) from a patient with inflammation was employed as test sample. Figure 6a shows that the loading and washing fractions contain a lot of interfering proteins while the elution fraction comprises only CRP (revealed as the 23 kDa band corresponding to CRP protomer). The purity of CRP was calculated to be 95% based on the SDS-PAGE image using the Image J software and increased about 1900-fold compared with the initial purity (~ 0.05%)53 The washing and elution fractions were further analyzed by MALDI-TOF MS (Figure 6b). The MS analysis revealed that the washing fraction was composed of interfering proteins with different molecular weights (e.g. HSA). A series of characteristic peaks were observed in the MS spectrum of the elution fraction and successively assigned to protomeric CRP with two charge states, protomeric, dimeric, trimeric, tetrameric, and pentameric CRP, which is consistent with the MS spectrum of standard CRP.54


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Figure 6. Purification of CRP from serum of a patient with inflammation and rat on the cell membrane biomimetic polymer. (a, c) SDS-PAGE analysis (reducing conditions) of the feedstock, loading, washing and elution fractions. Lanes: M, protein marker; S, standard CRP; F, feedstock; LF, loading fraction; WF, washing fraction; El, elution fraction. (b, d) MALDI-TOF MS spectra of standard CRP, washing fraction and elution fraction.


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Table 1. Comparison between the cell membrane biomimetic polymer and other affinity materials for the purification of CRP from different complex media (Ascitic fluid46,47, Serum from patient with inflammation48,49, Standard CRP solution15, Spiked human serum16,this work ). Affinity materials

Protocols

Recovery (%)

Purity (%)

Refs

Sepharose-CPS

Two steps (Affinity chromatography with Bio-Gel purification)

44.0

Obvious nonspecific protein adsorption

(46)

Sepharose-p-nitrophenyl PC

Four steps (p-nitrophenyl PC modified Sepharose affinity resin, DE-52 column, pnitrophenyl PC modified Sepharose column, Sephadex G-200 column)

30.0-35.0

64.2 a

(47)

Sepharose-PE

Affinity purification with gradient elution up to 12 h

70.0

95.4 b

(48)

p-Aminophenyl PC modified polymer

Affinity purification with 12 h incubation between CRP and sorbent

82.3

95.0 c

(49)

O-(-4-nitrophenylphosphoryl) choline MIP polymer

Affinity purification with complex operation and long operation time (more than 17 h)

/

Obvious nonspecificity (HSA)

(15)

3-(4)-Vinylbenzyl-12-PC dodecanoate modified magnetic nanoparticles

Affinity purification with 0.5 h incubation

/

91

(16)

Commercial Immobilized pAminophenyl PC Gel

Affinity purification with 1 h incubation

88.4

85

This work

Cell membrane biomimetic polymer

Affinity purification without incubation

102.6

95

This work

a, b, c

The purity was calculated based on the SDS-PAGE images found in literature using the Image J software.


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Inspired by aforementioned results, the cell membrane biomimetic polymer was further applied to CRP purification from a more complex sample, a rat serum with a total protein concentration of 185 mg/mL. SDS-PAGE (Figure 6c) and MALDI-TOF MS (Figure 6d) analyses suggested that the washing fraction could contain i.e. rat albumin, globulin, while the elution fraction comprised only rat CRP (revealed by SDS-PAGE as 25 kDa band or by MS as m/z 12670 for rat protomeric CRP 2+, 25455 for rat protomeric CRP+, 50904 for rat CRP dimer+, 75851 for rat CRP trimer+). The purity of rat CRP was finally calculated to be 95% by the Image J software. These results confirm the excellent performance of the biomimetic polymer for CRP purification from complex biological fluids. 4. CONCLUSIONS In the current study, a facile CRP enrichment approach was established based on the novel cell membrane biomimetic polymer with CRP recognition group and antinonspecific protein adsorption interface. Due to the high specificity of the PC moiety to CRP in the presence of calcium ions and its superior properties (weak electrostatic interaction, high mass transfer rate, hydrophilicity and biocompatibility), the biomimetic polymer exhibited higher specificity, selectivity, recovery, purity, and lower nonspecific protein adsorption in comparison with the commercial Immobilized p-Aminophenyl Phosphoryl Choline Gel and other purification materials. Notably, high purity (95%) has not been achieved at the expense of recovery, which remained stably above 99% on the biomimetic polymer without incubation. The structural integrity and functionality of CRP were also well preserved during the purification process. Finally, the versatile biomimetic polymer was successfully applied to the selective enrichment of CRP from different biological fluids, such as a serum from a patient with inflammation and rat serum. All results indicate that the novel enrichment



technology based on the cell membrane biomimetic polymer has a great potential as an alternative to traditional methods for single-step purification of CRP from complex biological media. ASSOCIATED CONTENT Supporting Information. Brief statement in non-sentence format listing the contents of the material supplied as Supporting Information. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Zhengjin Jiang), [email protected] (Prof. Ning Gan). ORCID Zhengjin Jiang: 0000-0002-5612-4331 Author Contributions ‡

Qiqin Wang and Hanying Jin contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grant: 81703460, 81673391), and the China Postdoctoral Science Foundation (Grants: 2016M602610, 2017T100662). There are no conflict of interest to declare. REFERENCE (1) Braig, D.; Nero, T. L.; Koch, H. G.; Kaiser, B.; Wang, X.; Thiele, J. R.; Morton, C. J.; Zeller, J.; Kiefer, J.; Potempa, L. A.; Mellett, N. A.; Miles, L. A.; Du, X.; Meikle, P. J.; Huber-Lang, M.; Stark, G. B.; Parker, M. W.; Peter, K.; Eisenhardt, S. U.


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A Biomimetic Polymer-Based Method for Selective Capture of C

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