Protein Purification Applications Research Articles

Microscopically segregated ligand distribution in co-assembled peptide-amphiphile nanofibers.

Peptide amphiphiles (PAs) self-assemble into cylindrical nanofibers with applications in protein purification, tissue engineering, and regenerative medicine. For these applications, functionalized PAs are often co-assembled with oppositely charged filler PAs. Finding the conditions at which these fibers are homogeneously mixed or segregated is crucial for the required application. We co-assemble negative C12VVEE fillers and positive C12VVKK-OEG4-Z33 ligands, which are important for antibody purifications. Our results show that the ligands tend to cluster and locally segregate in the fiber surfaces. The Z33s are overall neutral and form large aggregates in bulk solution due to short range attractions. However, full segregation of the C12VVKK-OEG4-Z33 is not observed in the cylindrical surface due to the electrostatic penalty of forming large domains of similarly charged molecules. This is commensurate with previous theoretical predictions, showing that the competition between short-range attractive interactions and long-range electrostatic repulsions leads to pattern formation in cylindrical surfaces. This work offers valuable insight into the design of functionalized nanofibers for various biomedical and chemical applications.

Improvement of Selectivity and Antifouling Properties of Chitosan-modified Polyvinylidene Fluoride (PVDF) Membrane for Protein Filtration

<p>Protein with high purity is currently a valuable commercial commodity, prized for its high value and application in specialized functions. PVDF-based membrane technology has found widespread use in protein purification processes. However, the issue of fouling on the membrane surface frequently hampers the performance of PVDF membranes. The present study aims to enhance PVDF membranes' selectivity and antifouling properties through chitosan modification, thereby optimizing protein filtration. PVDF and PVDF/Chitosan membranes were fabricated using the phase inversion technique, followed by a comprehensive characterization of their surface properties, thermal attributes, and performance in bovine serum albumin (BSA) protein filtration. The collected data revealed notable increases in membrane hydrophilicity, porosity, and pure water flux due to the incorporation of chitosan. The membrane protein rejection capabilities were significantly enhanced to levels exceeding 90% with the introduction of 0.5% chitosan. Moreover, as the chitosan concentration increased, the membrane exhibited superior antifouling characteristics, with the 0.5% chitosan concentration yielding the highest Flux Recovery Ratio (FRR) value of 85.35%. Considering the significant improvement selectivity and antifouling properties in the combined system, the chitosan-modified PVDF membrane holds substantial promise in protein purification applications.</p>

Improvement of Selectivity and Antifouling Properties of Chitosan-modified Polyvinylidene Fluoride (PVDF) Membrane for Protein Filtration

<p>Protein with high purity is currently a valuable commercial commodity, prized for its high value and application in specialized functions. PVDF-based membrane technology has found widespread use in protein purification processes. However, the issue of fouling on the membrane surface frequently hampers the performance of PVDF membranes. The present study aims to enhance PVDF membranes' selectivity and antifouling properties through chitosan modification, thereby optimizing protein filtration. PVDF and PVDF/Chitosan membranes were fabricated using the phase inversion technique, followed by a comprehensive characterization of their surface properties, thermal attributes, and performance in bovine serum albumin (BSA) protein filtration. The collected data revealed notable increases in membrane hydrophilicity, porosity, and pure water flux due to the incorporation of chitosan. The membrane protein rejection capabilities were significantly enhanced to levels exceeding 90% with the introduction of 0.5% chitosan. Moreover, as the chitosan concentration increased, the membrane exhibited superior antifouling characteristics, with the 0.5% chitosan concentration yielding the highest Flux Recovery Ratio (FRR) value of 85.35%. Considering the significant improvement selectivity and antifouling properties in the combined system, the chitosan-modified PVDF membrane holds substantial promise in protein purification applications.</p><p> </p><p><img src="/public/site/images/fitria/rsz_72110.png" alt="" /></p>

Adaptation of the binding domain of Lactobacillus acidophilus S-layer protein as a molecular tag for affinity chromatography development.

The S-layer proteins are a class of self-assembling proteins that form bi-dimensional lattices named S-Layer on the cell surface of bacteria and archaea. The protein SlpA, which is the major constituent of the Lactobacillus acidophilus S-layer, contains in its C-terminus region (SlpA284 - 444), a protein domain (named here as SLAPTAG) responsible for the association of SlpA to the bacterial surface. SLAPTAG was adapted for the development of a novel affinity chromatography method: the SLAPTAG-based affinity chromatography (SAC). Proteins with different molecular weights or biochemical functions were fused in-frame to the SLAPTAG and efficiently purified by a Bacillus subtilis-derived affinity matrix (named Bio-Matrix or BM). Different binding and elution conditions were evaluated to establish an optimized protocol. The binding equilibrium between SLAPTAG and BM was reached after a few minutes of incubation at 4°C, with an apparent dissociation constant (KD) of 4.3μM. A reporter protein (H6-GFP-SLAPTAG) was used to compare SAC protein purification efficiency against commercial immobilized metal affinity chromatography. No differences in protein purification performance were observed between the two methods. The stability and reusability of the BM were evaluated, and it was found that the matrix remained stable for more than a year. BM could be reused up to five times without a significant loss in performance. Additionally, the recovery of bound SLAP-tagged proteins was explored using proteolysis with a SLAP-tagged version of the HRV-3c protease (SLAPASE). This released the untagged GFP while the cut SLAPTAG and the SLAPASE were retained in the BM. As an alternative, iron nanoparticles were linked to the BM, resulting in BMmag. The BMmag was successfully adapted for a magnetic SAC, a technique with potential applications in high-throughput protein production and purification. The SAC protocol can be adapted as a universal tool for the purification of recombinant proteins. Furthermore, the SAC protocol utilizes simple and low-cost reagents, making it suitable for in-house protein purification systems in laboratories worldwide. This enables the production of pure recombinant proteins for research, diagnosis, and the food industry.

Effect of the Competing Ligand on Ni-NTA/Histag Strength Revealed by Click Chemistry-Based Force Spectroscopy.

The specific interaction between Ni-nitrilotriacetic acid and the six-histidine tag may be one of the most important coordination bonds utilized in biological research because of its wide application for recombinant protein purification. The complex stability is critical for target protein binding. Thus, measurement of the mechanical stability of the system was attempted soon after the invention of atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) two decades ago. Moreover, the competing ligand imidazole and protons are the two critical factors for target protein elution. However, the mechanochemistry between the system and the imidazole/proton has not been determined. Here, an AFM-SMFS system using strain-promoted alkyne-azide cycloaddition and Cu-free click chemistry was used to characterize the system. Consequently, the destabilizing effect of the imidazole and proton on the interaction was revealed quantitatively, leading to a 3-fold increase in the bond dissociation rate.

Selective Immobilization of His-Tagged Enzyme on Ni-Chelated Ion Exchange Resin and Its Application in Protein Purification.

Ion exchange resins are suitable as carriers for immobilized enzymes because of their stable physicochemical properties, appropriate particle size and pore structure, and lower loss in continuous operation. In this paper, we report the application of the Ni-chelated ion exchange resin in the immobilization of His-tagged enzyme and protein purification. Acrylic weak acid cation exchange resin (D113H) was selected from four cationic macroporous resins that could chelate the transition metal ion Ni. The maximum adsorption capacity of Ni was ~198 mg/g. Phosphomannose isomerase (PMI) can be successfully immobilized on Ni-chelated D113H from crude enzyme solution through chelation of transition metal ions with the His-tag on the enzyme. The maximum amount of immobilized PMI on the resin was ~143 mg/g. Notably, the immobilized enzyme showed excellent reusability and maintained 92% of its initial activity with 10 cycles of catalytic reaction. In addition, PMI was successfully purified using an affinity chromatography column prepared by Ni-chelated D113H, which showed the potential for the immobilization and purification process to be realized in one step.

Synthesis of reverse-selective nanoporous ultrafiltration membranes using dual phase separations of ionic liquid and Poly(ethylene glycol) from the gelating urea-linked covalent network

Many membranes with various pore structures have been studied for size-based selective filtration and separation. However, one exhibiting reverse size selectivity has rarely been reported. Here, we present a facile synthesis of a hierarchically nanoporous covalent framework (NCF) membranes using concurrent phase separation and gelation of the mixtures of a urea-linked network (UN) sol, poly (ethylene glycol) (PEG), and ionic liquid (IL). Subsequent solvent extraction of the dual porogens, i.e., PEG and IL, from the ternary blend gave a hierarchically nanoporous structure consisting of a continuous macropore running across a continuous mesoporous framework. The structure formation was accounted for by the primary separation of the IL-rich phase, followed by the secondary separation of the PEG domain from the continuous UN skeleton. The porosity and pore distribution of the membrane was varied by adjusting the composition of the PEG/IL/UN mixture. The hierarchical nanoporosity provided a reverse size selectivity for the ultrafiltration of proteins and polymers, promising potential applications in membrane-based protein purification or chromatography.

Adsorption Performance of a Multimodal Anion-Exchange Chromatography Membrane: Effect of Liquid Phase Composition and Separation Mode.

Membrane chromatography is a modern, high-throughput separation method that finds important applications in therapeutic protein purification. Multimodal, salt-tolerant membranes are the most recent innovation in chromatographic membrane adsorbents. Due to the complex structure of their ligands and the bimodal texture of their carriers, their adsorption properties have not been sufficiently investigated. This work deals with the equilibrium and kinetic properties of a multimodal anion-exchange chromatography membrane, Sartobind STIC. Single- and two-component adsorption experiments were carried out with bovine serum albumin (BSA) and salmon DNA as model target and impurity components. The effect of the Hofmeister series ions and ionic strength on the BSA/DNA adsorption was investigated in micromembrane flow experiments. A significant difference was observed between the effects of monovalent and polyvalent ions when strong kosmotropic salts with polyvalent anions acted as strong displacers of BSA. On the contrary, DNA binding was rather high at elevated ionic strength, independent of the salt type. Two-component micromembrane experiments confirmed very high selectivity of DNA binding at a rather low sodium sulfate feed content and at pH 8. The strength of binding was examined in more than a dozen different desorption experiments. While BSA was desorbed relatively easily using high salt concentrations independent of buffer type and pH, while DNA was desorbed only in a very limited measure under any conditions. Separation experiments in a laboratory membrane module were carried out for the feed containing 1 g/L of BSA, 0.3 g/L of DNA, and 0.15 M of sodium sulfate. The negative flow-through mode was found to be more advantageous than the bind-elute mode, as BSA was obtained with 99% purity and a 97% yield. Membrane reuse was investigated in three adsorption-desorption-regeneration cycles.

Biomolecular Complexation on the "Wrong Side": A Case Study of the Influence of Salts and Sugars on the Interactions between Bovine Serum Albumin and Sodium Polystyrene Sulfonate.

In the protein purification, drug delivery, food industry, and biotechnological applications involving protein–polyelectrolyte complexation, proper selection of co-solutes and solution conditions plays a crucial role. The onset of (bio)macromolecular complexation occurs even on the so-called “wrong side” of the protein isoionic point where both the protein and the polyelectrolyte are net like-charged. To gain mechanistic insights into the modulatory role of salts (NaCl, NaBr, and NaI) and sugars (sucrose and sucralose) in protein–polyelectrolyte complexation under such conditions, interaction between bovine serum albumin (BSA) and sodium polystyrene sulfonate (NaPSS) at pH = 8.0 was studied by a combination of isothermal titration calorimetry, fluorescence spectroscopy, circular dichroism, and thermodynamic modeling. The BSA–NaPSS complexation proceeds by two binding processes (first, formation of intrapolymer complexes and then formation of interpolymer complexes), both driven by favorable electrostatic interactions between the negatively charged sulfonic groups (−SO3–) of NaPSS and positively charged patches on the BSA surface. Two such positive patches were identified, each responsible for one of the two binding processes. The presence of salts screened both short-range attractive and long-range repulsive electrostatic interactions between both macromolecules, resulting in a nonmonotonic dependence of the binding affinity on the total ionic strength for both binding processes. In addition, distinct anion-specific effects were observed (NaCl < NaBr < NaI). The effect of sugars was less pronounced: sucrose had no effect on the complexation, but its chlorinated analogue, sucralose, promoted it slightly due to the screening of long-range repulsive electrostatic interactions between BSA and NaPSS. Although short-range non-electrostatic interactions are frequently mentioned in the literature in relation to BSA or NaPSS, we found that the main driving force of complexation on the “wrong side” are electrostatic interactions.

Amino acid-immobilized copper ion-modified carbon-based adsorbent for selective adsorption of bovine hemoglobin

We prepared an amino acid-immobilized copper ion-modified carbon-based adsorbent (C@TA@P@A-Cu) for selective bovine hemoglobin (BHb) adsorption in biological samples. Carbon nanoparticles were used as the matrix, and copper ions were attached to the amino acid-modified carbon nanoparticles as metal chelate complexes via immobilized metal affinity. BSA, Lyz, OVA, and HRP were chosen as reference proteins for further study. Furthermore, the synthesis conditions of adsorbents, SPE conditions, selectivity, competitivity, reproducibility, and reusability were extensively investigated. The results showed that the maximum adsorption capacity of C@TA@P@A-Cu microspheres for BHb under optimal conditions was 673.0 mg g–1. The addition of a TiO2 layer with an increased specific surface area of the adsorbent and the addition of poly-l-lysine (PLL) inhibited the adsorbent's binding ability to non-BHb proteins, but chelated Cu2+ increased the adsorbent's specific binding ability to BHb. Furthermore, after six adsorption-desorption cycles, the adsorbent has satisfactory reusability with no significant change in adsorption capacity. Furthermore, C@TA@P@A-Cu was successfully used to identify BHb from real blood samples, as confirmed by SDS-PAGE, and it is expected to have potential applications in protein purification and disease diagnosis.

Facile Fabrication of Multi-Hydrogen Bond Self-Assembly Poly(MAAc-co-MAAm) Hydrogel Modified PVDF Ultrafiltration Membrane to Enhance Anti-Fouling Property.

In this work, a facile preparation method was proposed to reduce natural organics fouling of hydrophobic membrane via UV grafting polymerization with methacrylic acid (MAAc) and methyl acrylamide (MAAm) as hydrophilic monomers, followed by multihydrogen bond self-assembly. The resulting poly(vinylidene fluoride)-membranes were characterized with respect to monomer ratio, chemical structure and morphology, surface potential, and water contact angle, as well as water flux and organic foulants ultrafiltration property. The results indicated that the optimal membrane modified with a poly(MAAc-co-MAAm) polymer gel layer derived from a 1:1 monomer ratio exhibited superior hydrophilicity and excellent gel layer stability, even after ultrasonic treatment or soaking in acid or alkaline aqueous solution. The initial water contact angle of modified membranes was only 36.6° ± 2.9, and dropped to 0° within 13 s. Moreover, flux recovery rates (FRR) of modified membranes tested by bovine serum albumin (BSA), humic acid (HA), and sodium alginate (SA) solution, respectively, were all above 90% after one-cycle filtration (2 h), significantly higher than that of the pure membrane (70–76%). The total fouling rates (Rt) of the pure membrane for three foulants were as high as 47.8–56.2%, while the Rt values for modified membranes were less than 30.8%. Where Rt of BSA dynamic filtration was merely 10.7%. The membrane designed through grafting a thin-layer hydrophilic hydrogel possessed a robust antifouling property and stability, which offers new insights for applications in pure water treatment or protein purification.

Diffusion of Protein Molecules through Microporous Nanofibrous Polyacrylonitrile Membranes.

Porous nanofibrous membranes have ultrahigh specific surface areas and could be broadly employed in protein purification, enzyme immobilization, and biosensors with enhanced selectivity, sensitivity, and efficiency. However, large biomolecules, such as proteins, have hindered diffusion behavior in the micro-porous media, significantly reducing the benefits provided by the nanofibrous membranes. The study of protein diffusion in polyacrylonitrile (PAN) nanofibrous membranes produced under varied humidity and polymer concentration of electrospinning revealed that heterogeneous structures of the nanofibrous membranes possess much smaller effective pore sizes than the measured pore sizes, which significantly affects the diffusion of large molecules through the system though sizes of proteins and pH conditions also have great impacts. Only when the measured membrane pore size is at least 1000 times higher than the protein size, the diffusion behavior of the protein is predictable in the system. The results provide insights into the design and applications of proper nanofibrous materials for improved applications in protein purification and immobilizations.

Tetrasulfonate calix[4]arene modified large pore mesoporous silica microspheres: Synthesis, characterization, and application in protein separation

Effective protein adsorption by solid matrices from complex biological samples has attracted attention for broad application in biomedical field. Immobilization of calixarenes to solid supports is an essential process for their application in protein separation and purification. Silica is the most widely used support material in calixarene immobilization. With high concentration of polymer microspheres as templates, the large pore mesoporous silica microspheres with controllable, uniform size and structure were successfully synthesized and the resulting large pore mesoporous silica microspheres were modified with water-soluble tetrasulfonate calix[4]arene of unique hollow cavity-shaped structure. The tetrasulfonate calix[4]arene modified large pore mesoporous silica microspheres (SCLX4@LPMS) were characterized by diverse analytical techniques and their protein adsorption performance were also investigated. The obtained SCLX4@LPMS gave rise to an adsorption efficiency of >90% for cytochrome c and lysozyme within a wide pH range of 3.0–10.0 and possessed remarkably high adsorption capacity of cytochrome c (363.64 mg g−1) and lysozyme (166.11 mg g−1). The retained cytochrome c and lysozyme can be readily eluted by using phosphate buffer solution containing NaCl as a stripping reagent with the recoveries of 81% and 86% after 5 times enrichment, respectively. The SCLX4@LPMS microspheres have been applied for the selective adsorption of proteins in real samples and had the application potential in protein adsorption, drug delivery, biosensors, and other biomedical fields.

High-Throughput Screening of Dye-Ligands for Chromatography.

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. The choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices serve as a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose the best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lie in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

A robust affinity chromatography system based on ceramic monoliths coated with poly(amino acid)‐based polymeric constructs

AbstractTraditional chromatographic separation systems are disadvantaged by low flow rates, a high pressure drop across the column, low capacity and poor reusability. Searching for more efficient separation systems we introduced the use of a ceramic monolith as robust support in bioseparations. A coating consisting of l‐asparagine as ligand, poly(l‐lysine) as spacer arm and a commercial poly(ethylene acrylic acid) film forming copolymer network (Michem 4983‐40R) was developed as a coating for these ceramic monoliths. Poly(l‐lysine) was synthesized by ring‐opening polymerization of ε‐trifluoroacetyl‐l‐lysine N‐carboxyanhydride and coupled to a commercial film‐forming poly(ethylene acrylic acid) network. This construct was then ‘decorated’ with l‐asparagine via the terminal amino functional groups of poly(L‐lysine) and coated onto the ceramic monolith to selectively bind l‐asparaginase. Adsorption/elution experiments showed reversible binding between l‐asparagine and l‐asparaginase, and the subsequent release of l‐asparaginase, and between 83% and 94% of the active enzyme was recovered by elution with d‐asparagine and NaCl solutions. The functional activity of the eluted l‐asparaginase was verified by a Nessler's assay. While traditional separation processes (adsorption and elution) using gel bead packings take many hours, the ceramic monolith system achieves the same of level of separation in about 1 h. This new system served as a proof of concept for its application in protein separation and purification. This work paves the way to a better understanding of the use of ceramic monoliths as stationary phase coated with a stable polymer construct for more robust and efficient supports in affinity chromatography. © 2020 Society of Industrial Chemistry

Synthesis and characterization of chitosan mini-spheres with immobilized dye as affinity ligand for the purification of lactoperoxidase and lactoferrin from dairy whey

Lactoperoxidase (LP) and lactoferrin (LF) are two interesting proteins present in dairy whey due to their antioxidant, anti-inflammatory, antibacterial, antiviral and antifungal properties. LP and LF are traditionally purified using cation-exchange chromatography, but since the concentration of these proteins in sweet whey are very low (0.03–0.06 g/L for LP and around 0.08–0.20 g/L for LF), clarification, diafiltration, concentration and/or depletion of others whey proteins prior to the ion-exchange chromatography is needed to obtain good adsorption yields. After their elution from the matrix, the overall process results in variable yields. Due to their similar physico-chemical properties, a high cross-contamination is usually present, especially for the LP fraction, where part of the LF is co-eluted.The development of novel supports with immobilized affinity and/or multimodal ligands aims to the direct adsorption of the target proteins even at low concentration and without the need of a pre-treatment of the source. Herein, a new, low-cost support material using chitosan mini-spheres with Orange R-HE triazine dye as immobilized ligand (CMS-Orange R-HE) was synthesized, physico-mechanically characterized and used for the purification of LP and LF by a one-step direct adsorption from dairy whey and a two-step elution process, with minor cross-contamination. CMS-Orange R-HE matrix showed a compression modulus of 47.6 ± 9.8 kPa, predominantly elastic deformation and an open-porous structure with mesopores around 5–20 nm and without ink-bottle pores, ideal for protein purification applications. The affinity between LP and LF for CMS-Orange R-HE matrix was characterized using the Langmuir isotherm model (Kd = 0.48 ± 0.07 mg/mL and 0.31 ± 0.09 mg/mL; qmax = 77.5 ± 4.0 mg/g and 56.0 ± 2.1 mg/g, respectively). Adsorption studies showed that LP interaction with CMS-Orange R-HE was partially influenced by the presence of ionic strength (p < 0.05), whereas LF adsorption was not. These differences of both electrostatic and hydrophobic interactions between LP and LF with the CMS-Orange R-HE matrix were used to develop a differential elution process using Response Surface Methodology (RSM). By one-step direct adsorption from dairy whey and two differential elution steps –by using the CMS-Orange R-HE matrix- LP and LF were obtained with good yields (≈70% for LP and ≈60% for LF), high purity and with minor cross-contamination between them. The strategy presented here, could be applied in other cases of cross-contamination between two proteins, being especially interesting for its application in multimodal chromatography.

Synthesis of Magnetic Hydrogels for Target Delivery of Doxorubicin

Magnetic hydrogels are the stimuli responsive polymeric networks with potential applications in protein purification, cell separation and target drug delivery. In the present investigation, efforts are made on synthesis and characterization of magnetic hydrogels from poly(N-isopropylacrylamide) for onwards application in target delivery of an anticancer drug Doxorubicin. Formation of reaction products was ascertained through diversified spectral methods. Ultraviolet spectra reveal in vitro biocompatibility of Doxorubicin that imparts sustained delivery up to 72 hrs at pH 7.2 ± 0.2 under ambient conditions. The present investigation provides an inexpensive method of synthesis of magnetic hydrogels suitable for magnetically derived delivery of anticancer drugs.

Adsorption-selectivity customization and competitive adsorption of tryptamine-based mixed-mode chromatography

Mixed-mode chromatography (MMC) shows great promise for applications in protein purification. Therefore, novel MMC ligands are always desired. However, achieving high adsorption selectivity of ligands is a major challenge due to competitive adsorption. Herein, MMC with tryptamine (4 F F-TA) exhibited equal adsorption capacity for bovine serum albumin (BSA) and bovine immunoglobulin (bIgG), affording a unique competitive adsorption process, but non ideal purity. Molecular imprinting technology (MIT) was adopted for the customized adsorption selectivity of MMC, and BSA and bIgG were used as imprinting templates. Static and dynamic binding performances under competitive adsorption before and after imprinting were investigated at different pH values, salt concentrations, and BSA/bIgG mass ratios. The results showed that the adsorption capacity of the target protein remained similar level but competitive protein adsorption dropped significantly (below 40 mg/g resin). This indicated that the adsorption selectivity of 4 F F-TA could be customized by changing the template protein used during imprinting. In addition, the pH-dependence and salt tolerance of MMC were unchanged after imprinting. The binding behaviors of BSA and bIgG in column chromatographic separation further confirmed the improved in adsorption selectivity. The purity of BSA increased from 55.4% to 78.2% for 4 F F-TA@BSA and that of bIgG from 40.7% to 71.2% for 4 F F-TA@bIgG. This study indicated that MIT is an effective strategy to change and improve the adsorption selectivity of MMC.

Macro- and Microphase Separated Protein-Polyelectrolyte Complexes: Design Parameters and Current Progress.

Protein-containing polyelectrolyte complexes (PECs) are a diverse class of materials, composed of two or more oppositely charged polyelectrolytes that condense and phase separate near overall charge neutrality. Such phase-separation can take on a variety of morphologies from macrophase separated liquid condensates, to solid precipitates, to monodispersed spherical micelles. In this review, we present an overview of recent advances in protein-containing PECs, with an overall goal of defining relevant design parameters for macro- and microphase separated PECs. For both classes of PECs, the influence of protein characteristics, such as surface charge and patchiness, co-polyelectrolyte characteristics, such as charge density and structure, and overall solution characteristics, such as salt concentration and pH, are considered. After overall design features are established, potential applications in food processing, biosensing, drug delivery, and protein purification are discussed and recent characterization techniques for protein-containing PECs are highlighted.

Manipulation of Grafting Location via Photografting To Fabricate High-Performance Ethylene Vinyl Alcohol Copolymer Membrane for Protein Separation.

Ethylene vinyl alcohol copolymer (EVAL) membrane has great potential for applications in protein separation and purification, but the uncontrollable distribution of grafting location when membranes are modified by the grafting method limits the membrane performance. Herein, an effective strategy for controlling the distribution of grafting location was designed to fabricate a high-performance EVAL membrane via photografting. The UV intensity through the membranes was weakened when the local concentration of the photoinitiator benzophenone (BP) on the topside of the membrane increased; thus, the grafting location inside the EVAL membrane changed from homogenous to asymmetric distribution based on the UV absorbability of BP. The grafting inside the membrane pores can be promoted when the loose and porous surface of the EVAL membrane was used as the UV-facing side. More importantly, the varied distribution of grafting location played different roles on improving the membrane performance. For protein binding, the limited convection in the membrane bed was avoided, and the desorption efficiency could be improved when the grafting location enriched inside the membrane pores. For protein filtration, the antifouling properties of the EVAL membrane were enhanced when the grafting location enriched on the topside. This research offers a novel approach to achieve controllable grafting location distribution of membranes and provides a perspective to design the high-performance EVAL membranes for protein separation.