Selective Separation Of Proteins Research Articles

Exploiting Interactions between Polyoxometalates and Proteins for Applications in (Bio)chemistry and Medicine

AbstractThe specific interactions of anionic metal‐oxo clusters, known as polyoxometalates (POMs), with proteins can be leveraged for a wide range of analytical and biomedical applications. For example, POMs have been developed as selective catalysts that can induce protein modifications and have also been shown to facilitate protein crystallization, both of which are instrumental in the structural characterization of proteins. POMs can also be used for selective protein separation and enzyme inhibition, which makes them promising therapeutic agents. Hence, understanding POM‐protein interactions is essential for the development of POM‐based materials and their implementation in several fields. In this Review we summarize in detail the key insights that have been gained so far on POM‐protein interactions. Emphasis is also given to hybrid POMs functionalized with organic ligands to prompt further research in this direction owing to the promising recent results on tuning POM‐protein interactions through POM functionalization.

Exploiting Interactions between Polyoxometalates and Proteins for Applications in (Bio)chemistry and Medicine.

The specific interactions of anionic metal-oxo clusters, known as polyoxometalates (POMs), with proteins can be leveraged for a wide range of analytical and biomedical applications. For example, POMs have been developed as selective catalysts that can induce protein modifications and have also been shown to facilitate protein crystallization, both of which are instrumental in the structural characterization of proteins. POMs can also be used for selective protein separation and enzyme inhibition, which makes them promising therapeutic agents. Hence, understanding POM-protein interactions is essential for the development of POM-based materials and their implementation in several fields. In this Review we summarize in detail the key insights that have been gained so far on POM-protein interactions. Emphasis is also given to hybrid POMs functionalized with organic ligands to prompt further research in this direction owing to the promising recent results on tuning POM-protein interactions through POM functionalization.

Diffusion toward a nanoforest of absorbing pillars.

Spiky coatings (also known as nanoforests or Fakir-like surfaces) have found many applications in chemical physics, material sciences, and biotechnology, such as superhydrophobic materials, filtration and sensing systems, and selective protein separation, to name but a few. In this paper, we provide a systematic study of steady-state diffusion toward a periodic array of absorbing cylindrical pillars protruding from a flat base. We approximate a periodic cell of this system by a circular tube containing a single pillar, derive an exact solution of the underlying Laplace equation, and deduce a simple yet exact representation for the total flux of particles onto the pillar. The dependence of this flux on the geometric parameters of the model is thoroughly analyzed. In particular, we investigate several asymptotic regimes, such as a thin pillar limit, a disk-like pillar, and an infinitely long pillar. Our study sheds light onto the trapping efficiency of spiky coatings and reveals the roles of pillar anisotropy and diffusional screening.

Characterization and interaction mechanism of selective protein separation by epsilon-polylysine: The role of hydrophobic attraction

ε-polylysine (EPL) is a nature-occurred cationic polypeptide. Despite its antimicrobial effectiveness being deactivated by phase separation from proteins, this article proposes a novel strategy via selective phase separation (SPS) for protein purification at neutral pH conditions. Since a step-by-step phase separation in the presence of EPL was observed in skim milk, a mixed model comprised of β-casein (BCN), β-lactoglobulin (BLG) and bovine serum albumin (BSA) is expected to elucidate this phenomenon where an SPS in terms of the order: BCN > BLG > BSA was presented. To investigate the phase separation mechanism, we first established phase diagrams for different EPL-protein systems and speculated that the pH and ionic strength dependence of EPL-protein complexation are mainly driven by electrostatic attraction (EA) and hydrophobic attraction (HA). Moreover, although the interaction process is enthalpically driven as the ΔH is highly negative characterized by isothermal titration calorimetry (ITC), we found that the phase separation is a kinetic process following the “nucleation and growth” mechanism. By altering the environmental conditions (1 M NaCl and 8 M urea), Dynamic light scattering (DLS) measurement and molecular dynamics simulation (MDs) indirectly illustrated that the mechanism of phase separation of the EPL-protein system consists of a two-stage process at which the initial complexes (clusters) and subsequent precipitations are driven by EA and HA, respectively. Since the irreversible HA drives the phase separation to fall into the primary energy potential well to minimize the free energy of the system, we proposed that the priority of precipitation arising from HA plays a major role in selectivity. This hypothesis suggests that “hydrophobic patches” from proteins rather than “electrostatic patches” could regulate the SPS via EPL, which provides a novel strategy for protein purification. • ε-polylysine (EPL) could precipitate proteins step-by-step selectively. • We focused on the phase separation driven by hydrophobic attraction (HA). • The irreversible HA could drive the system to fall into the primary energy well. • The priority of phase separation arising from HA plays a major role in selectivity. • We proposed a potential strategy for protein purification via EPL precipitation.

Hydrazone bond-oriented molecularly imprinted nanocomposites for the selective separation of protein via the well-defined recognition sites.

The development of hydrazone bond-oriented epitope imprinting strategy is reported to synthesize the polymeric binders for the selective recognition of a protein-β2-microglobulin through either its N- or C-terminal epitope. The dynamic reversibility of hydrazone bond facilitated not only the oriented assembly of the template peptide hydrazides onto the substrate but also the efficient removal of them from the imprinted cavities. The well-defined surface imprinted layer was successfully constructed through the precise control over the polymerization of silicate esters. Binding performance of the C-terminal peptide imprinted nanocomposite was significantly improved after tuning the non-covalent interactions using the sequence-matching aromatic co-monomers. The dissociation constant (Kd) between the optimized nanocomposite and epitope peptide was 0.5µmol L-1. The nanomaterial was utilized for the selective extraction and determination of β2-microglobulin from human urine by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and HPLC-UV with satisfied recoveries of 93.1-112.3% in a concentration range 1.0-50.0μg⋅mL-1.

Selective Protein Separation Based on Charge Anisotropy by Spherical Polyelectrolyte Brushes.

Protein purification is of vital importance in the food industry, drug discovery, and other related fields. Among many separation methods, polyelectrolyte (PE)-based phase separation was developed and recognized as a low-cost purification technique. In this work, spherical polyelectrolyte brushes (SPBs) with a high specific surface area were utilized to study the protein accessibility and selective protein binding on highly charged nanoparticles (NPs) as well as the selective phase separation of proteins. The correlation between charge anisotropy, protein binding, and phase separation was investigated on various protein systems including those proteins with similar isoelectric points (pI) such as bovine serum albumin (BSA) and β-lactoglobulin (BLG), proteins with similar molecular weights such as BSA and hemoglobin (HB), and even protein variants (BLG-A and -B) with a tiny difference of amino acids. The nonspecific electrostatic interaction studied by turbidimetric titrations and isothermal calorimetry titration (ITC) indicates a specific binding between proteins and SPBs arising from the charge anisotropy of proteins. An optimized output based on selective protein binding on SPBs could be correlated for efficient protein separation through tuning external conditions including pH and ionic strength. These findings, therefore, proved that phase separation based on selective protein adsorption by SPBs was an efficient alternative for protein separation compared with the traditional practice.

High-flux anti-fouling nanofibrous composite ultrafiltration membranes containing negatively charged water channels

High-flux nanofibrous composite ultrafiltration (UF) membranes consist of nanocellulose integrated polyacrylonitrile (PAN) as the barrier layer and electrospun PAN nanofibers deposited on a polyethylene terephthalate (PET) non-woven mat as the supporting layer were fabricated by solution coating approach. The chemical composition of nanofibrous composite membranes was investigated with ATR-FTIR, TGA, and EDX measurements, while the top and cross-sectional morphology of the membranes were observed via SEM and AFM scans. An interpenetrating nanofiber-polymer network was created in the barrier layer which drastically enhanced the mechanical strength of the composite membrane. The hydrophilicity of the nanofibrous composite membranes increased with nanocellulose component evidenced by water contact angle measurements, which could enhance the anti-fouling properties of the membranes. The as-prepared membranes were investigated for protein separation and wastewater treatment. The ultrafiltration performance of nanofibrous composite membranes with different composition ratios of nanocellulose to PAN was demonstrated using a cross-flow filtration system, the highest pure water permeability of 1508 L/(m2h·MPa) was achieved with a coating solution of 0.10 wt% of nanocellulose and 0.05 wt% of PAN where it created optimized water channels in the barrier layer of the composite membranes. The molecular weight cut-off (MWCO) of these membranes was determined with dextran as a marker, where the maximum pore size of the membranes varied in the range of 20.8–46.0 nm. Moreover, proteins and polypeptides with different molecular weights were employed to challenge the nanofiber composite membrane performance. High permeation flux of 147.1 L/m2h and high rejection ratio of 98.4% were accomplished; meanwhile, the high selectivity against protein and polypeptides was attained, indicating the membrane's applicability for the selective separation. The anti-fouling properties of the nanofibrous composite membranes were also intensely investigated and the filtration efficiency, in terms of permeation flux and rejection ratio, was evaluated and compared with that of commercially available UF membranes (UN100, US050, and UE050) that possess similar membrane pore size. The permeation flux of the nanofibrous composite membrane was 2–3 times higher than that of commercial membranes, due to the formation of water channels in the barrier layer, while the rejection ratio remained the same as 99.6%, indicating that the nanofibrous composite membrane could be a good candidate for wastewater treatment, in addition to the highly selective separation of proteins.

Integration of mixed-mode chromatography and molecular imprinting technology for double recognition and selective separation of proteins

Abstract Adsorption selectivity is one of the most important parameters for chromatography. As a promising technology for protein purification, mixed-mode chromatography (MMC) has unique advantages; however, its selectivity needs to be improved further. In this study, one strategy for improving selectivity was tested. We combined MMC with molecular imprinting technology (MIT) to gain double-recognition capabilities. Molecularly imprinted polymer (MIP) enabled the enhancement of the selectivity of MMC. Cross-linked 4% agarose beads (4FF) with tryptamine (Try) as ligands were used to prepare the double-recognition resin (4FF-Try/MIPs), with bovine serum albumin (BSA) as the imprinting template protein. After removing the template protein from the imprinted layer, 3D cavities with double-recognition abilities toward BSA were obtained, owing to the combination of hydrophobic interaction and molecularly imprinted spatial matched cavities. The static and dynamic adsorption behaviors of BSA, bovine immunoglobulin G (bIgG), and bovine hemoglobin (BHb) on resins were investigated. The results showed 4FF-Try/MIPs retained the typical adsorption properties of pH dependence and salt tolerance of MMC. High and similar dynamic binding capacities for BSA were obtained for both 4FF-Try and 4FF-Try/MIPs. However, 4FF-Try/MIPs showed lower dynamic binding capacity for the competitive impurity bIgG. When 4FF-Try/MIPs was used to separate BSA from bovine serum, the purity reached 75.1%, which was higher than 4FF-Try by ∼20%. We conclude that MIT could be applied to enhance selectivity of MMC with double recognition capabilities, and this might be a straightforward strategy for improving selectivity in other modes of chromatography.

Preparation and evaluation of Fe $$_{3}$$ 3 O $$_{4}$$ 4 nanoparticles incorporated molecularly imprinted polymers for protein separation

Protein imprinting is still a challenge due to the low binding kinetics and poor binding selectivity. In this study, a facile method of the preparation of magnetic molecularly imprinted polymers (MIPs) for selective protein separation was reported. Carboxyl group functionalized Fe $$_{3}$$ O $$_{4}$$ nanoparticles (NPs) were synthesized using a solvothermal method. After pre-assembly of caroxyl group functionalized Fe $$_{3}$$ O $$_{4}$$ NPs and template protein lysozyme (Lyz) to form Lyz–Fe $$_{3}$$ O $$_{4}$$ complex, magnetic MIPs were synthesized by a sol–gel process of 3-aminopropyltriethoxylsilane and tetraethyl silicate with Lyz–Fe $$_{3}$$ O $$_{4}$$ complex incorporated. Then Fe $$_{3}$$ O $$_{4}$$ –MIPs particles with magnetic response could be collected by simple crush of bulk polymers. This preparation process avoid the need of high dilution of monomer for anti-agglomeration in the surface imprinting, and large amount of solvent is spared. The morphology and structure property of the prepared magnetic NPs were characterized by transmission electronic microscopy, Fourier transform infrared spectroscopy, and vibrating sample magnetometer. Binding experiments were carried out to evaluate Fe $$_{3}$$ O $$_{4}$$ –MIPs particles’ binding performance and selectivity. And results showed fast binding kinetics, high binding capacity, and favorable specific recognition behavior toward template protein, which is due to the role of carboxyl group functionalized Fe $$_{3}$$ O $$_{4}$$ NPs as both magnetic source and importantly as co-functional monomer incorporated in the polysiloxane imprinting system. Real egg white sample tests demonstrate good separation effect. This report provides a possibility of the selective separation of protein in complex matrix.

Preparation of protein imprinted polymer beads by Pickering emulsion polymerization.

We present a new method for preparation of protein-specific polymer beads based on surface molecular imprinting in Pickering emulsion. In the first step, adult human hemoglobin (Hb) was adsorbed on silica nanoparticles. The protein-coated silica particles were then used to stabilize an oil-in-water emulsion (Pickering emulsion) composed of cross-linking monomer in the oil phase. After free radical polymerization of the oil phase, the protein-silica particles were removed to leave Hb-imprinted sites on the polymer surface. The protein-imprinted polymer microspheres were characterized by scanning electron microscopy and their selectivity was investigated by protein binding analysis. The new synthetic method based on Pickering emulsion polymerization produced easily accessible Hb binding sites on the surface of spherical polymer particles, which are useful for protein separation, purification and analysis.

Atom transfer radical polymerization of diverse functional SBA-15 for selective separation of proteins

Poor detection of low-abundant proteins is a common problem in most current protein identification schemes. This is attributed partially to the existence of high-abundant protein. Therefore, selective removal of specific proteins prior to analysis is in great demand. In response these needs, we previously described two kinds of ion exchange groups functionalized SBA-15 synthesized by direct silylanization reaction. Herein, we designed and synthesized strong anion, cation and zwitterion functionalized SBA-15 via atom transfer radical polymerization (ATRP). The previous and current materials were characterized and theirs adsorption abilities for some model proteins (bovine serum albumin (BSA), hemoglobin (Hb), myoglobin (Mb) and lysozyme (Lys)) were evaluated. The results demonstrated that different functionalized SBA-15 is capable to selective removal of certain proteins in protein mixture. It also indicated that this sample preparation technique could be potentially applied to reduce the complexity on protein identification for biological samples with a wide dynamic range.

Facile Fabrication of Hydrophilic Nanofibrous Membranes with an Immobilized Metal–Chelate Affinity Complex for Selective Protein Separation

In this study, we report a facile approach to fabricate functionalized poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membranes as immobilized metal affinity membranes for selective protein separation. Hydrophilic PVA-co-PE nanofibrous membranes with controlled fiber sizes were prepared via a melt extrusion process. A chelating group, iminodiacetic acid (IDA), was covalently attached to cyanuric acid activated membrane surfaces to form coordinative complexes with metal ions. The prepared membranes were applied to recover a model protein, lysozyme, under various conditions, and a high lysozyme adsorption capacity of 199 mg/g membrane was found under the defined optimum conditions. Smaller fiber size with a higher immobilized metal ion density on membrane surfaces showed greater lysozyme adsorption capacity. The lysozyme adsorption capacity remained consistent during five repeated cycles of adsorption-elution operations, and up to 95% of adsorbed lysozyme was efficiently eluted by using a phosphate buffer containing 0.5 M NaCl and 0.5 M imidazole as an elution media. The successful separation of lysozyme with high purity from fresh chicken egg white was achieved by using the present affinity membrane. These remarkable features, such as high capacity and selectivity, easy regeneration, as well as reliable reusability, demonstrated the great potential of the metal-chelate affinity complex immobilized nanofibrous membranes for selective protein separation.

Functionalized chitosan derived novel positively charged organic–inorganic hybrid ultrafiltration membranes for protein separation

Positively charged organic–inorganic hybrid ultrafiltration membranes for selective protein separation were fabricated from blends of PVA, functionalized600 dpi in TIF format)??> chitosan and tetraethylorthosilicate.

Development of functionalized mesoporous silica for adsorption and separation of dairy proteins

A class of mesoporous silica material, FDU-12, was synthesized with different pore sizes and amine functionalized to determine its potential to differentially adsorb and separate dairy proteins. The adsorption behavior of the dairy proteins α-lactalbumin and β-lactoglobulin on the amino surface functionalised and unfunctionalised material was examined. It was shown that the pore size of unfunctionalised FDU-12 is an important factor in the protein adsorption capacity. Amine functionalization of FDU-12 significantly increased protein adsorption for larger pore size materials, with a marked difference in the rate of uptake between the two proteins. The kinetics of adsorption were investigated as a function of temperature and were modeled using pseudo second order kinetics and an intraparticle diffusion model, showing that intraparticle protein diffusion can be rate limiting. These results indicate that functionalised FDU-12 has potential for the selective separation of proteins with similar size and isoelectric point, under dynamic processing conditions.

Choline-like ionic liquid-based aqueous two-phase extraction of selected proteins

In this paper, an ionic liquid-based aqueous two-phase system (ATPs) has been developed for the effective extraction of bovine serum albumin (BSA). Compared with the effects of three different ionic liquids used in an aqueous two-phase system, a choline-like ionic liquid was selected as the suitable solution for bovine serum albumin. Extraction efficiencies of BSA were found to be dependant on the temperature, time, IL mass and the salt content. Based on single-factor experiments, these significant operating parameters of the ILs-ATP were respectively optimized. Experimental results show that 92.03–100.03% of the proteins could be extracted into the ionic liquid-rich phase in a single-step extraction. Repeatability, precision and stability experiments were investigated and the RSDs are lower than 2.3%, 1.7% and 4.7% respectively. Experiments indicated that intermolecular forces, the micelle phenomenon and the salting-out effect play important roles in the transfer process, and the aggregation phenomenon was the main driving force for the separation. Conformation of the protein was not affected after extraction into the upper ionic liquid-rich phase according to the determination by UV-visible (UV-vis), fluorescence spectra, dynamic light scattering (DLS), Fourier transform infrared (FT-IR) spectroscopy and TEM. It is suggested that the method of choline-like ILs-based ATPs for the selective separation of protein would have potential applications in bio-analysis and bio-separation.

Selective Protein Separation Using Siliceous Materials with a Trimethoxysilane-Containing Glycopolymer

A copolymer with α-D-mannose (Man) and trimethoxysilane (TMS) units was synthesized for immobilization on siliceous matrices such as a sensor cell and membrane. Immobilization of the trimethoxysilane-containing copolymer on the matrices was readily performed by incubation at high heat. The recognition of lectin by poly(Man-r-TMS) was evaluated by measurement with a quartz crystal microbalance (QCM) and adsorption on an affinity membrane, QCM results showed that the mannose-binding protein, concanavalin A, was specifically bound on a poly(Man-r-TMS)-immobilized cell with a higher binding constant than bovine serum albumin. The amount of concanavalin A adsorbed during permeation through a poly(Man-r-TMS)-immobilized membrane was higher than that through an unmodified membrane. Moreover, the concanavalin A adsorbed onto the poly(Man-r-TMS)-immobilized membrane was recoverable by permeation of a mannose derivative at high concentration.

Mobile phase modifier effects in multimodal cation exchange chromatography

This study examines protein adsorption behavior and the effects of mobile phase modifiers in multimodal chromatographic systems. Chromatography results with a diverse protein library indicate that multimodal and ion exchange resins have markedly different protein binding behavior and selectivity. NMR results corroborate the stronger binding observed for the multimodal system and provide insight into the structural basis for the observed binding behavior. Protein-binding affinity and selectivity in multimodal and ion exchange systems are then examined using a variety of mobile phase modifiers. Arginine and guanidine are found to have dramatic effects on protein adsorption, yielding changes in selectivity in both chromatographic systems. While sodium caprylate leads to slightly weaker chromatographic retention for most proteins, certain proteins exhibit significant losses in retention in both systems. The presence of a competitive binding mechanism between the multimodal ligand and sodium caprylate for binding to ubiquitin is confirmed using STD NMR. Polyol mobile phase modifiers are shown to result in increased retention for weakly bound proteins and decreased retention for strongly bound proteins, indicating that the overall retention behavior is determined by a balance between changes in electrostatic and hydrophobic interactions. This work provides an improved understanding of protein adsorption and mobile phase modifier effects in multimodal chromatographic systems and sets the stage for future work to develop more selective protein separation systems.

Integration of carboxyl modified magnetic particles and aqueous two-phase extraction for selective separation of proteins

Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation.

Selective separation of protein and saccharides by ionic liquids aqueous two-phase systems

In the present work, it was found that aqueous solution of a hydrophilic ionic liquid (IL), 1-butyl-3-methylimidazolium dicyanamide ([C4mim][N(CN)2]), could be separated into an aqueous two-phase system (ATPS) by inorganic salts such as K2HPO4 and K3PO4. The top phase is IL-rich, while the bottom phase is phosphate-rich. It was shown that 82.7%–100% bovine serum albumin (BSA) could be enriched into the top phase and almost quantitative saccharides (arabinose, glucose, sucrose, raffinose or dextran) were preferentially extracted into the bottom phase in a single-step extraction by [C4mim][N(CN)2] + K2HPO4 ATPS. The extraction efficiency of BSA from the aqueous saccharide solutions was influenced by the molecular structure of saccharides. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were combined to investigate the microstructure of the IL-rich top phase and the possible mechanism for the selective separation. It is suggested that the formation of the IL aggregate and the IL aggregate-BSA complex plays a significant role in the separation of BSA from aqueous saccharide solutions. This is the first example for the selective separation by ILs-based ATPSs. It is expected that these findings would have potential applications in bio-analysis, separation, and IL recycle.

Active and biomimetic nanofilters for selective protein separation

Selective protein channels in cell and nuclear membranes act as gateways to control the passage of molecules across. The selectivity of these channels stems from attractive potentials of the binding sites in the transmembrane proteins. These channels can filter out small volume of solutions with high precision. Motivated from this phenomenon, we report biomimetic facilitated transport modality to selectively separate a target molecule from a mixture of molecules. The attractive potential is generated by specific antibodies immobilized inside 15 nm diameter polycarbonate nanochannels. Two proteins with similar physicochemical properties (Bovine Serum Albumin 66 kDa, and Human Hemoglobin 65 kDa) are chosen as model molecules. The protein molecules are mixed in ratios of 1:1, 1:20 and 1:40 (Hb:BSA), and separation of molecules is demonstrated. The selectivity of membrane can be switched from Hb to BSA by changing the immobilized antibody inside the membrane channels. This approach can be used to selectively enrich any target molecule from a complex sample to enhance signal-to-noise ratio for early disease diagnosis.