Separation of proteins by charged ultrafiltration membranes

The separation of a protein mixture by charged ultrafiltration membranes was studied. A negatively charged polymer was obtained by sulfonation of polysulfone, and a positively charged polymer was synthesized by chloromethylation of polysulfone and then by quaternization of the amino group. Then, the negatively and positively charged ultrafiltration membranes were cast from solutions of charged polymer/NMP(or DMF)/lithium nitrate. The molecular weight cut-off of the membranes were controlled by the changing casting conditions.

Single protein solutions were ultrafiltrated at the isoelectric point and at another pH level by the use of charged membranes. At the isoelectric point, rejection of the protein was low, while it was high at the pH level which gave the protein the same sign of charge as that of the membrane.

A protein mixture of myoglobin and cytochrome C was separated by the charged ultrafiltration membranes at the isoelectric point of one of the proteins. At the isoelectric point of cytochrome C, myoglobin has a negative charge. Thus myoglobin was rejected with a rejection of about 80% by the negatively charged membrane. At the same time, cytochrome C permeated completely through the membrane. Conversely, at the isoelectric point of myoglobin, cytochrome C has a positive charge and thus it was rejected with a rejection of about 20% by the positively charged membrane. The rejection of myoglobin here was almost zero.     

Simulation of high-concentration self-interactions for monoclonal antibodies from well-behaved to poorly-behaved systems

Attractive self-interactions of therapeutic proteins are linked to problematic solution behaviors at high protein concentrations such as reversible or irreversible aggregation, high viscosity, opalescence, phase separation, and low solubility. Prediction of attractive self-interactions early in development can improve the processes of formulation development and candidate selection. To that end, a coarse-grained model with explicit representation of charged sites was used to accurately predict a broad range of protein self-interactions at high protein concentrations (up to 160 mg/ml) for multiple monoclonal antibodies and formulations, including strong electrostatic attractions, with static light scattering measurements at low protein concentrations as the only experimental input. In addition, Mayer-weighted electrostatic energies for charged residues from these simulations can contribute to understanding of electrostatic interactions and guide the development of protein variants.     

Alternating charge clusters of side chains: new surface structural invariants observed in calf eye lens gamma-crystallins

A detailed stereochemical analysis of the oppositely chargedside chains of amino acid residues on the surface of calf eyelens protein gamma-crystallin B has been carried out. The refinedstructural data of very high quality obtained at 1.47 Åresolution have been used. Charge–charge interactionswere considered to be valuable for all the charged oxygen andnitrogen atoms situated at distances, d, between 2.4 and 7.0Å. This means we consider short contact ion pairs as thosewith interchange distances 2.4 < d 4.0 Å and distantcontact ion pairs as those with distances 4.0 < d 7.0 Å.Hydrogen bonding of the charged atomic groups with the structuralwater molecules also has been considered. We have not lookedat the side groups of histidines which are charged only partiallyat neutral pH. Five clusters of charged side chains which werelarge enough were observed. The clusters are comprised of fourto six residues which compose 543% of the total charged residuesin the protein. The clusters contain from eight to 12 chargedatoms and look like the bent chains of oppositely charged atoms.All clusters are of plane geometry and their maximal lineardimensions are from 11 to 18 Å. The root mean square deviationsof charged atoms from the cluster plane varied from 0.63 to0.86 Å for four clusters and was only 1.85 Å forthe largest cluster. All clusters include a number of watermolecules situated on the cluster boundary and grouped nearthe cluster plane. It was shown that the amino acid sequencepositions of charged residues are conservative for all the proteinsof the gamma-crystallin family of vertebrates including fish,frog, mouse, rat, calf and human. The cluster properties werediscussed both in their functional aspect for gamma-crystallinsand in other aspects common for globular proteins. As a result,the alternating charge clusters should be considered as newlyrecognized surface structural invariants. The importance ofthe charged side chain clusters is claimed for the updated conceptof the protein surface.     

Carboxybetaine,sulfobetaine, and cationic block copolymer coatings: A comparison of the surface properties and antibiofouling behavior

Two different zwitterionic block copolymers (BCs) and a cationic BC were synthesized from the same BC precursor, which consisted of a polystyrene (PS) block and a poly[N‐(3‐dimethylamino‐1‐propyl)acrylamide] block. The zwitterionic BCs contained the dimethylammonioacetate (carboxybetaine) and dimethylammoniopropyl sulfonate (sulfobetaine) groups. Thin films cast from these polymers were investigated for surface wettability, surface charge, and protein adsorption. Surface‐energy parameters calculated with advancing contact angle (θ_a) and receding contact angle (θ_r) of different probe liquids showed that it was θ_r and not θ_a that was representative of the polar/ionic groups in the near‐surface regions of the coatings. Electrophoretic mobility was used to characterize the influence of pH on the net surface charge. In aqueous dispersions, the carboxybetaine polymer showed an ampholyte behavior with an isoelectric point of 6, whereas the sulfobetaine polymer was found to be anionic at all pH values between 2 and 10. Protein adsorption on the carboxybetaine BC was relatively independent of the net charges on the protein or the polymer, but the negatively charged sulfobetaine polymer showed a higher adsorption of positively charged protein molecules. Regardless of the net protein charge, both zwitterionic coatings adsorbed less protein compared to the PS and poly(2,3,4,5,6‐pentafluorostyrene) controls. The sulfobetaine and cationic BCs adsorbed higher amounts of oppositely charged protein molecules than like‐charged protein molecules. However, the adsorption of oppositely charged protein was much higher on the cationic surface than on the sulfobetaine surface. The zwitterionic BCs, particularly the carboxybetaine polymer, from this article are expected to function as stable, low‐fouling surface modifiers in different biological environments. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Tunable multifunctional tissue engineering scaffolds composed of three‐component polyampholyte polymers

Resistance to nonspecific protein adsorption and the capability to provide targeted bioactive signals are essential qualities for implantable biomaterials. The development of materials that combine these multifunctional characteristics and tunable mechanical properties has been a target in the tissue engineering field over the last decade. This study is the first to demonstrate that polyampholyte hydrogels prepared with equimolar quantities of positively charged and negatively charged monomer subunits from multiple monomer compositions have great potential to address these needs. The hydrogels were synthesized with positively charged [2‐(acryloyloxy)ethyl] trimethylammonium chloride and different monomer ratios of the negatively charged 2‐carboxyethyl acrylate and 3‐sulfopropyl methacrylate monomers. The physical and chemical properties of the hydrogels were fully characterized, including swelling, hydration, mechanical strength, and chemical composition, and the fouling resistance of the hydrogels was demonstrated using enzyme‐linked immunosorbent assays. Additionally, the capability of the hydrogels to facilitate protein conjugation via EDC/NHS conjugation chemistry was assessed. The results clearly demonstrate that the polyampholyte hydrogels have a range of tunable mechanical strength based on the monomer subunits, while maintaining their excellent nonfouling properties. Additionally, high levels of conjugated protein were achieved for all of the monomer combinations investigated. Therefore, the broadly applicable multifunctional properties of polyampholyte hydrogels and their tunable mechanical properties clearly demonstrate the potential of these materials for tissue engineering. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43985.     

Spatial sign-alternating charge clusters in globular proteins

Large sign-alternating charge clusters formed by the chargedside groups of amino acid residues and N- and C-terminal groupswere found in the majority of considered globular proteins,namely 235 in a total of 274 protein structures, i.e. 85.8%.The clusters were determined by the criteria proposed earlier:charged groups were included in the cluster if their chargedN and O atoms were located at distances between 2.4 and 7.0Å. The set of selected proteins consisted of known non-homologousprotein structures from the Protein Data Bank with a resolutionless than or equal to 2.5 Å and pair sequence similarityless than 25%. Molecular masses of the proteins were from 5.5to 91.5 kDa and protein chain length from 50 to 830 residues.The distribution of charged groups on the protein surface betweenisolated charged groups, small clusters with two and three groups,and large clusters with four or more groups were found to beapproximately similar making 33, 35 and 32% of the total amountof protein charged groups, respectively. The large sign-alternatingcharge clusters with four or more charged groups were studiedin greater detail. The amount of such clusters depends on theprotein chain length. The small proteins contain 1–3 clusterswhile the large proteins display 4–6 or more clusters.On average, 1.5 clusters per each 100 residues were observed.In contrast with this, the size of a cluster, i.e. the numberof charged groups inside a cluster, does not depend on the proteinmolecular mass, and large clusters are observed for proteinsfrom a range of molecular masses. Clusters consisting of fourto six charged groups occur most frequently, although extralarge clusters are also often revealed. We can conclude thatsign-alternating charge clusters are a common feature of theprotein surface of globular protein. They are suggested to playa general functional role as a local polar factor of proteinsurface.     

One‐Step Immobilization of Protein‐Encapsulated Core/Shell Particles onto Nanofibers

A novel method is described to functionalize nanofibers to form a nanocomposite with core/shell particles in order to control protein release. The nanocomposite is produced by electrically neutralizing negatively charged poly(lactic acid) nanofibers with positively charged poly[(lactic acid)‐co‐(glycolic acid)] particles via a one‐step electrohydrodynamic jetting process. The protein‐encapsulated core/shell particles exhibited no significant initial burst release or denaturation. The protein release profile was controlled by porosity and protein/polymer interactions. The method may be promising to engineer intelligent scaffolds that can fulfill the needs of biomimetic materials.

     

PAN ultrafiltration membranes grafted with natural amino acids for improving antifouling property

In this report, antifouling polyacrylonitrile (PAN) ultrafiltration membranes were prepared from blends of PAN/polyglycidyl methacrylate (PGMA) via phase inversion method followed by the grafting of natural amino acids through epoxy ring-opening reaction. The grafted PAN membranes possessed highly stable hydrophilic surfaces as a result of the grafting of amino acids, which was adequately demonstrated in attenuated total reflectance–Fourier transform infrared spectroscopy (ATR/FTIR), X-ray photoelectron spectroscopy (XPS), and contact angle measurements. The results of tensile strength and scanning electron microscopy (SEM) images further proved that the surface modification had little effect on their mechanical properties, surface, and cross-sectional morphologies. Meanwhile, remarkable resistance against bovine serum albumin (BSA) and lysozyme (Lyz) fouling was observed for the neutral amino acid-based PAN membranes due to the formation of zwitterionic hydration layer on the membrane surface, while PAN membranes grafted with charged amino acids were able to prohibit the approach of like charged proteins with reduced deposition and provide the driving force for oppositely charged protein adsorption. Furthermore, the ultrafiltration and antifouling performance of PAN membranes were investigated by BSA filtration experiments. Compared with the pristine PAN membrane, all the modified PAN membranes exhibited higher pure water flux, better flux recovery ratio, lower rejection, less total permeation resistance, and preferable stability, having potential applications in protein separation and purification.     

Modified Polyethersulfone (PES) Ultrafiltration Membranes for Enhanced Filtration of Whey Proteins

Abstract

Application of membrane technology to whey protein separation is an interesting development that has seen growth in recent years. In particular, modification of existing membranes to impart charge properties on the membrane surface or in the pores has been shown to improve membrane selectivity, product purity, and throughput of protein solutions. This paper focuses on exploring the effects of membrane charge and solution pH on filtration of the major whey proteins α‐lactalbumin (14.1 kDa) and β‐lactoglobulin (18.4 kDa) using functionalized PES membranes. The membranes have an open pore structure containing charged sulfonated grafted polymer chains that allows for greater protein retention. The modified membranes were synthesized by polymerization of styrene in the membrane pores followed by sulfuric acid treatment of the resulting polystyrene grafts. The charged membrane gave a calculated selectivity of five times better than the raw membrane at pH 7.2 based on data from single protein transmission experiments. The enhanced selectivity of the tailor‐made membrane was due to increased retention of β‐lactoglobulin due to a reduction in molecular sieving combined with electrostatic repulsion between negatively charged β‐lactoglobulin and the negatively charged membrane.     

Functionalisation of Inorganic Material Surfaces with Staphylococcus Protein A: A Molecular Dynamics Study

Staphylococcus protein A (SpA) is found in the cell wall of Staphylococcus aureus bacteria. Its ability to bind to the constant Fc regions of antibodies means it is useful for antibody extraction, and further integration with inorganic materials can lead to the development of diagnostics and therapeutics. We have investigated the adsorption of SpA on inorganic surface models such as experimentally relevant negatively charged silica, as well as positively charged and neutral surfaces, by use of fully atomistic molecular dynamics simulations. We have found that SpA, which is itself negatively charged at pH7, is able to adsorb on all our surface models. However, adsorption on charged surfaces is more specific in terms of protein orientation compared to a neutral Au (111) surface, while the protein structure is generally well maintained in all cases. The results indicate that SpA adsorption is optimal on the siloxide-rich silica surface, which is negative at pH7 since this keeps the Fc binding regions free to interact with other species in solution. Due to the dominant role of electrostatics, the results are transferable to other inorganic materials and pave the way for new diagnostic and therapeutic designs where SpA might be used to conjugate antibodies to nanoparticles.     

Model studies on the mechanism of endotoxin adsorption on flat-sheet microfiltration membrane adsorbers

The adsorption of endotoxin from E.coli O 111:B₄ and bovine serum albumin on membrane adsorbers with the ligands polymyxin B, L-histidine, deoxycholate, poly(L-lysine), poly(ethyleneimine) and diethylaminoethyl was studied under equilibrium and dynamic conditions. Apparent association constants varied between 30 and 30 000 mL/mg for endotoxin, whereas with BSA only approximately 1 mL/mg was found on average. With all ligands a reduction of the removal efficiency was experienced in the presence of BSA, which was used as model protein. Detailed analysis of the influence of BSA on the association constants as well as the equilibrium and dynamic capacities of endotoxin adsorption demonstrated that endotoxin and BSA molecules recognize distinct binding centres on the membrane adsorbers. Competition for binding sites plays a less important role than assumed so far. It is concluded that interactions of endotoxin with protein molecules in solutions are limiting endotoxin clearance from protein solutions. With net-positively charged proteins, electrostatic interactions yield protein-endotoxin complexes. Net-negatively charged proteins attract endotoxin by the mediation of calcium ions that are derived from endotoxin micelles and vesicles. In the latter case, endotoxin clearance can be significantly improved by the addition of EDTA.     

Physical and mechanical properties of thermosensitive xanthan/collagen-inspired protein composite hydrogels

Functionalization of xanthan hydrogels is of interest for biomaterial applications. The authors report characterization of electrostatic complexation of xanthan with a recombinant collagen-inspired triblock protein polymer. This polymer has one charged polylysine end-block that can bind to xanthan by electrostatic interactions, and another end-block that can self-assemble into thermosensitive collagen-like triple helices; the end-blocks are connected by a neutral, hydrophilic, mostly inert random coil. The protein modifies the xanthan/protein composite hydrogels in three ways: (a) a significant increase in storage modulus, (b) thermosensitivity, and (c) a two-step strain softening in nonlinear rheology.     

Improving the Oxidative Stability of Krill Oil-in-Water Emulsions

A positively charged protein (fish gelatin) or a negatively charged protein species (heat-treated milk protein–carbohydrate mixture) was added to a primary krill oil (KO) emulsion stabilized by the phospholipids inherent in KO, with the aim of improving the oxidative stability of KO-in-water emulsions at pH 8.0 (10 % KO). The positively charged fish gelatin deposited on the primary interface of the oil droplets in the primary KO-in-water emulsion improved the oxidative stability of the KO-in-water emulsion as evidenced by the higher eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) remaining and lower propanal produced after accelerated oxidation (40 °C, 25 days). The addition of the negatively charged heat-treated milk protein–carbohydrate mixture containing Maillard reaction products (MRP) to the bulk phase of the emulsion also enhanced the oxidative stability of the KO-in-water emulsion. The addition of MRP to the aqueous phase of phospholipids stabilized emulsion droplets offered more protection to EPA and DHA of the KO emulsions compared to the formation of an additional layer at the interface of the KO emulsion droplet. This suggests that interventions based on addition of antioxidant species to the formulation were more effective for arresting oxidation than increasing the thickness of the droplet interface. The addition of proteins into KO containing emulsion formulations is a promising strategy for protecting omega-3 marine phospholipids against oxidation.     

Modeling and Experimental Data for the Reverse Micellar Extraction of Proteins Using a New Surfactant

ABSTRACT

In this study the reverse micellar extraction of α-chymotrypsin using bis(2,4,4-trimethylpentyl) sodium dithiophosphinate as the surfactant has been investigated. Experiments were performed to determine the effects of pH and salt concentration on the reverse micellar extraction and backward extraction of α-chymotrypsin in isooctane/decanol mixtures. The results showed that both pH and salt concentration influence the reverse micellar extraction of α-chymotrypsin. The pH affects the extraction of the protein through changing the sign and number of the charges carried by protein molecules and thereby affects their interactions with the charged head groups of the surfactant molecules. It was found that at high pH values the hydrogen ions compete with positively charged protein molecules and exchange with the counterions of the surfactant molecules, and as a result destroy the reverse micelles. This effect was exploited for the backward extraction of protein molecules from the reverse micellar phase. A model has been developed to correlate the experimental data of reverse micellar extraction of proteins. In the model the reverse micelles were treated as noninteractive ion-exchange sites. The activity coefficient of the protein molecules in reverse micelles was represented by the model proposed by Pitzer. It has been shown that the model can correlate the experimental data obtained in this work and those reported in other studies.     

Compaction of DNA in solutions of highly charged proteins carrying the same charge as DNA

The theory of DNA compaction in solutions of highly charged proteins carrying charge of the same sign as DNA is developed. It is shown that the introduction of a negatively charged protein may induce the collapse of DNA that occurs as a first-order phase transition. The concentration of protein in the vicinity of DNA practically coincides with the concentration of protein in solution on the whole, and the introduction of protein into a solution is equivalent to the effective worsening of solvent quality. The higher the absolute value of the protein charge, the more pronounced this worsening. The higher the charge of the protein, the smaller its content that causes the compaction of DNA. The properties of the transition depend on the effective charge of DNA and on the concentration of a low-molecular-mass salt. An increase in the concentration of the salt may weaken the action of protein as a compaction agent and cause the reverse transition of a DNA macromolecule to the coiled state.     

阴离子型双功能含环氧基团酶固定化载体的制备及其固定辣根过氧化酶

利用接枝聚合在微米级硅胶微粒表面的甲基丙烯酸缩水甘油酯(GMA)和对羟基苯磺酸钠(SHBS)发生环氧基团的部分开环反应,制得阴离子型双功能复合载体SHBS-PGMA/SiO2,研究了辣根过氧化酶(HRP)与载体SHBS-PGMA/SiO2间的静电相互作用在共价固定HRP过程中的作用及其机理. 结果表明,在较大的pH值范围内,复合载体SHBS-PGMA/SiO2表面携带高密度负电荷,pH=6.0时HRP分子荷正电,酶蛋白分子与载体间会产生强静电相互作用,显著促进HRP的固定化;当载体表面SHBS的键合率约为18%时,静电相互作用对固酶的促进作用最强,固定化酶的偶联率和比活力最高. 离子强度对酶的固定化也有很大影响,加入NaCl对载体与酶蛋白之间的静电相互作用力产生屏蔽作用,增大NaCl浓度可使固定化酶的偶联率和比活力降低.     

Effect of surface charge of polyethyleneimine-modified multiwalled carbon nanotubes on the improvement of polymerase chain reaction

In molecular biology, polymerase chain reaction (PCR) has played an important role but suffers a general problem with low efficiency and specificity. Development of suitable additives to improve the PCR specificity and efficiency and the understanding of the PCR enhancing mechanism still remain a great challenge. Here we report the use of polyethyleneimine (PEI)-modified multiwalled carbon nanotubes (MWCNTs) with different surface charge polarities as a novel class of enhancers to improve the specificity and efficiency of PCR. The materials used included the positively charged PEI-modified MWCNTs (CNT/PEI), the neutral CNT/PEI modified with acetic anhydride (CNT/PEI.Ac), and the negatively charged CNT/PEI modified with succinic anhydride (CNT/PEI.SAH). We show that the specificity and efficiency of an error-prone two-round PCR are greatly impacted by the surface charge polarity of the PEI-modified MWCNTs. Positively charged CNT/PEI could significantly enhance the specificity and efficiency of PCR with an optimum concentration as low as 0.39 mg L(-1), whereas neutral CNT/PEI.Ac had no such effect. Although negatively charged CNT/PEI.SAH could enhance the PCR, the optimum concentration required (630 mg L(-1)) was more than 3 orders of magnitude higher than that of positively charged CNT/PEI. The present study suggests that the PCR enhancing effect may be primarily based on the electrostatic interaction between the positively charged CNT/PEI and the negatively charged PCR components, rather than only on the thermal conductivity of MWCNTs.     

聚丙烯微孔膜荷负电表面的构建与阻抗蛋白质吸附性能

报道一种有效构建聚丙烯微孔膜(PPMM)荷负电表面的新方法.组合大气压介质阻挡放电等离子体预处理和界面交联技术,制得表面荷正电的PPMM;通过动态静电自组装技术将阴离子聚电解质固定到膜表面,从而获得荷负电的PPMM,ATR-FTIR,XPS和FESEM分析确证了修饰过程中膜表面化学组成与形貌的变化.静态水接触角和纯水通...     

Effect of Polyphosphorylation on Behavior of Protein Disordered Regions

Proteins interact with many charged biological macromolecules (polyelectrolytes), including inorganic polyphosphates. Recently a new protein post-translational modification, polyphosphorylation, or a covalent binding of polyphosphate chain to lysine, was demonstrated in human and yeast. Herein, we performed the first molecular modeling study of a possible effect of polyphosphorylation on behavior of the modified protein using replica exchange molecular dynamics simulations in atomistic force field with explicit water. Human endoplasmin (GRP-94), a member of heat shock protein 90 family, was selected as a model protein. Intrinsically disordered region in N-terminal domain serving as a charged linker between domains and containing a polyacidic serine and lysine-rich motif, was selected as a potent polyphosphorylation site according to literature data. Polyphosphorylation, depending on exact modification site, has been shown to influence on the disordered loop flexibility and induce its further expanding, as well as induce changes in interaction with ordered part of the molecule. As a result, polyphosphorylation in N-terminal domain might affect interaction of HSP90 with client proteins since these chaperones play a key role in protein folding.