Protein Loading Capacity Research Articles

Serial displacement chromatofocusing and its applications in multidimensional chromatography and gel electrophoresis: I. Theory and general considerations

The technique of “serial displacement chromatofocusing” (SDC) is investigated both theoretically and experimentally with model mixtures of proteins and peptides. The method employs a multistep, retained pH gradient formed using adsorbed buffering species to produce a series of discrete effluent fractions. Each of these fractions may contain several displaced protein bands under conditions of sufficient mass overloading, so that several displacement trains of adjoined bands can be produced in a single chromatographic run. Numerical simulations and experimental results showed selective concentration effects for minor components in a fraction when the feed amount was sufficient large. A computer-aided design method was developed to facilitate the use of the method and was applied to both anion- and cation-exchange column packings. Good agreement was achieved between the designed pH gradients and experimental results. The characteristics of SDC were also explored in terms of its loading capacity, scalability, repeatability, recovery, and differentiation of proteins between their true and apparent isoelectric point values.

The effects of salt on the physicochemical properties and immunogenicity of protein based vaccine formulated in cationic liposome

Recently, we have developed a simple and potent therapeutic cancer vaccine consisting of a cationic lipid and a peptide antigen. In this report, we expanded the utility of this formulation to protein based vaccines. First, we formulated the human papillomavirus (HPV) 16 E7 protein (E7) in different doses of DOTAP liposome. The results showed that these formulations failed to regress an established tumor. However, when sodium chloride (30mM) was added to the DOTAP (100nmol)/E7 (20μg) formulation, anti-tumor activity was generated in the immunized mice. Correlatively, 30mM NaCl in the DOTAP/E7 protein formulation increased the particle size from ∼350 to 550nm, decreased the protein loading capacity (from 95 to 90%), and finally increased the zeta potential (from 29 to 38mV). Next, a model protein antigen ovalbumin (OVA) was formulated in different doses of DOTAP liposomes. Similarly, the results showed that 20μg OVA formulated in 200nmol DOTAP with 30mM NaCl had the best OVA-specific antibody response, including both IgG1 and IgG2a, suggesting both Th1 and Th2 immune responses were generated by this formulation. In conclusion, we have expanded the application of cationic DOTAP liposome formulation to protein based vaccines and also identified that small amounts of salt could change the physicochemical properties of the vaccine formulation and enhance the activity of the DOTAP/protein based vaccine. The enhancement of immune responses by salt is possibly due to its interference of the electrostatic interaction between the cationic lipid and the protein antigen to facilitate the antigen release from the carrier and at the same time activate the antigen presenting cells.

A New Integrated Membrane Filtration and Chromatographic Device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.

Derivatized graphitic nanofibres (GNF) as a new support material for mass spectrometric analysis of peptides and proteins

Graphitic nanofibres (GNFs), 100-200 nm in diameter and 5-20 microm in length have been modified in order to yield different affinities (Cu2+ and Fe3+ loaded immobilized metal affinity chromatography (IMAC) as well as cation and anion exchange materials) for the extraction of a range of biomolecules by their inherited hydrophobicity and the hydrophilic chemical functionalities, obtained by derivatization. Modified GNFs have for the first time been employed as carrier materials for protein profiling in material-enhanced laser desorption/ionization (MELDI) for the enrichment and screening of biofluids. For that purpose, the derivatized GNF materials have comprehensively been characterized regarding surface area, structural changes during derivatization, IMAC, as well as ion exchange and protein-loading capacity and recovery. GNF derivatives revealed high protein-binding capacity (2,000 microg ml(-1) for insulin) and ideal sensitivities, resulting in a detection limit of 50 fmol microl(-1) (for insulin), which is crucial for the detection of low abundant species in biological samples. Compared to other MELDI carrier materials, sensitivity was enhanced on GNF derivatives, which might be ascribed to the fact that GNFs support desorption and ionization mechanisms and by absorbing laser energy in addition to matrix.

Biotransformation of tea catechins into theaflavins with immobilized polyphenol oxidase

Theaflavins, an active antioxidant, a natural pigment and pharmacologically active molecule obtained from black tea were bioprocessed on an immobilized tea polyphenol oxidase (PPO) system by the conversion of tea catechins extracted from green tea leaves with an overall conversion efficiency of 85% about 14-fold increase over maximum achievable in normal black teas. The immobilized enzyme (IE) system consists of activated cellulose matrix on to which the freshly extracted tea leaf polyphenol oxidase was covalently linked. Cellulose as a matrix of choice was considered primarily for its non-toxic nature, natural origin, low cost and easy availability. The kinetic parameters of the IE system were; protein loading capacity 11.8mg/g; pH optimum 5.9; temperature optimum 37.5°C; Km 4.76±0.08mM; Vmax 20±1.80nmol/min; enzyme activity retention (83.58%) and number of batches per turnover without loss of activity was 14. The product from IE system was identified by HPLC and ESI-QTOF-MS spectrometry.

Novel tetrazole-functionalized ion exchanger for weak cation-exchange chromatography of proteins

A new type of weak cation exchanger, tetrazole-functionalized silica, was developed for bioseparation. It was prepared conveniently by modifying silica gel initially with γ-glycidoxypropyltrimethoxysilane, then with 3-hydroxypropionitrile and finally with ammonium-catalyzed (3 + 2) azide–nitrile cycloaddition, which is an element of click chemistry. The prepared stationary phase was characterized and evaluated for its separation performance, protein retention behavior, loading capacity, protein recovery and chemical stability. The results show that the stationary phase developed has excellent performance for protein separations with high mass recoveries, and has long-term stability. Some remarkable differences were observed between this new exchanger and a carboxylic methyl-functionalized ion exchanger, which is typically used in weak cation-exchange chromatography of proteins. The obtained column was also used for the purification of lysozyme from chicken egg white, and the purity and specific bioactivity of the obtained lysozyme were about 90% and 67 IU/mg, respectively.

Charged nanoparticles as protein delivery systems: A feasibility study using lysozyme as model protein

The aim of this study was to investigate the feasibility of negatively charged nano-carriers (nanoparticles), consisting of polymer blends of poly(lactide-co-glycolide) (PLGA) and poly(styrene-co-4-styrene-sulfonate) (PSS), to improve the loading capacity and release properties of a positively charged model protein, lysozyme, through an adsorption process. Nanoparticles were prepared by a solvent displacement method and characterized in terms of size, ζ-potential, morphology, as well as loading capacity of model protein lysozyme. Morphology of these particles was investigated using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The loading capacity of lysozyme was evaluated as a function of polymer blend ratio, protein concentration, pH, and ionic strength; in vitro release profiles were also studied. The results show that negatively charged nanoparticles were obtained using polymer blends of PLGA and PSS, characterized by increased net negative surface charge with increasing ratios of PSS. Moreover, protein loading capacity increased as function of PSS/PLGA ratio. Increased pH facilitated the adsorption process and improved the loading capacity. Maximum loading efficiency was achieved at salt concentrations of 50 mM. In vitro release of lysozyme from the polymer blend nanoparticles was dependent on drug loading and full bioactivity of lysozyme was preserved throughout the process. These findings suggest that this is a feasible method to prepare nanoparticles with high surface charge density to efficiently adsorb oppositely charged protein through electrostatic interactions.

Polysiloxane–polyvinyl alcohol discs as support for antibody immobilization: Ultra-structural and physical–chemical characterization

A semi-interpenetrated polymer network of posiloxane and polyvinyl alcohol (POS–PVA) was synthesized through sol–gel process in the form of discs and used as solid support for antibody immobilization. POS–PVA discs presented a protein loading capacity of approximately 210 μg/disc and can covalently bind an anti-S100 protein antibody via glutaraldehyde. The POS–PVA matrix network resulted in hard and spherical discs of uniform size, diameter of 4.0 mm 2. Scanning electron microscopy (SEM) analysis revealed a uniformly formed porous network. The SEM and infrared absorption spectra confirmed that the activation with glutaraldehyde do not alter the POS–PVA discs structure is similar to that discs before antibody immobilization. The elemental analysis of discs (POS–PVA + Glutaraldehyde + Antibody) was evaluated and the following constitution was obtained (%): carbon (6.57), nitrogen (0.67), hydrogen (4.25) and sulphur (0.32), suggesting that the antibody completely fixed after adsorption. The shelf-life of discs was performed at six months and showed better protein loading capacity by POS–PVA discs incubated at 4 °C and hydrated with phosphate buffer solution when compared with same discs exposed to different environment store (room temperature and dry). In conclusion, microarrays of antibody immobilized on POS–PVA support demonstrated sensitivity, its antibody covalent immobilization and discs synthesis involves simple steps and the reagents employed are inexpensive. Hence the present semi-interpenetrating polymer network can be used as an alternative of solid support for sera assays.

Preparation and In Vitro Evaluation of bFGF-Loaded Chitosan Nanoparticles

The objective of our study was to prepare and characterize basic fibroblast growth factor (bFGF)-loaded nanoparticles. Protein-loaded chitosan nanoparticles were obtained by ionotropic gelation process based on the interaction between chitosan and tripolyphosphate (TPP). The protein-loading capacity and encapsulation efficiency were 0.021% and 27.388%, respectively. The bFGF-loaded nanoparticles have a mean diameter of 424 nm, a narrow size distribution, spherical shape and positive surface charges. In vitro release showed that the extent of release was 68% at 24 hr. The protein integrity was investigated by SDS-PAGE analysis that confirmed protein integrity was not affected by the encapsulation procedure and release conditions.

Preparation of Medium Cation Exchange Stationary Phase of Polymeric Matrix and Their Chromatographic Properties

AbstractBased on the monodisperse poly(glycidyl methacrylate‐co‐ethylenedimethacrylate) beads (PGMA/EDMA) with macropore as a medium, a new hydrophilic medium cation exchange (MCX) stationary phase for HPLC was synthesized by a new chemically modified method. The stationary phase was evaluated with the property of ion exchange, separability, reproducibility, hydrophilicity, effect of salt concentration, salt types, column loading and pH on the separation and retention of proteins in detail. It was found that it follows ion exchange chromatographic (IEC) retention mechanism. The measured bioactivity recovery for lysozyme was (96±5)%. The dynamic protein loading capacity of the synthesized MCX packings was 21.8 mg/g. Five proteins were almost completely separated within 6.0 min at a flow rate of 4 mL/min using the synthesized MCX resin. The MCX resin was also used for the rapid separation and purification of lysozyme from egg white with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70345 U/mg, respectively.

A sensitive impedance immunosensor based on functionalized gold nanoparticle–protein composite films for probing apolipoprotein A-I

A sensitive and label-free electrochemical impedance immunosensor via covalent coupling the antibody with functionalized gold nanoparticles (FAuNP) for probing apolipoprotein A-I was presented. The hybrid gold nanoparticles were prepared with a two-in-one strategy, i.e. via the stepwise employment of self-assembled monolayer (SAM) and sol–gel techniques, to improve the performance of such a label-free immunosensor, which was investigated by electrochemical impedance spectroscopy. It was found that this novel FAuNP immunosensor showed higher protein-loading capacity and better response properties (6–17 times) than that fabricated by normal SAM technique did. The remarkably improved properties of the immunosensor were ascribed to FAuNP with the larger surface-to-volume ratio, more free amino linkage groups, and the lower nonspecific protein adsorption. As a result, the thus-prepared antibody-modified immunosensor showed reproducible (R.S.D. = ±3.2%, n = 10) linear response to apolipoprotein A-I (Apo A-I) antigens in the range of 0.1–10 ng mL −1. The detection limit of this immunosensor was 50 pg mL −1 (corresponding to 1.8 pmol L −1), which was two orders of magnitude lower than that of the traditional methods. These results exhibited the novel immunosensor had a high sensitivity, stability and selectivity for the determination of Apo A-I, especially in clinic microanalysis.

Preparation of Weak Anion Exchange Chromatographic Packings Based on Monodisperse Polymer Beads and their Application in the Separation of Bioploymers

Based on the 8.0 µm monodisperse, macroporous poly‐(glycidylmethacrylate‐co‐ethylene‐dimethacrylate) beads (PGMA/EDMA), a new hydrophilic weak anion‐exchange (WAX) stationary phase for HPLC was synthesized by a new chemically modified method. The stationary phase was evaluated in detail to determine its ion‐exchange properties, separability, reproducibility, hydrophilicity, and the effect of column loading and pH on the separation and retention of proteins. It was found to have an ion‐exchange chromatographic (IEC) retention mechanism. The dynamic protein loading capacity of the synthesized SAX packings for BSA was 27.4 mg · g−1. Four proteins were separated within 6.0 minutes using the synthesized WAX resin. The WAX resin was also used for the rapid separation and purification of recombinant human granulocyte colony stimulating factor (rhG‐CFS) from the coarse extract solution with only one step. The purity of the purified rhG‐CFS was more than 95%.

Involvement of endoplasmic reticulum (ER) stress in podocyte injury induced by excessive protein accumulation

An imbalance between protein load and folding capacity is referred to as endoplasmic reticulum (ER) stress. As a defense mechanism, cells express ER stress inducible chaperons, such as oxygen-regulated proteins 150 (ORP150) and glucose-regulated proteins (GRPs). While ER stress is important in various diseases, a pathophysiologic role for ER stress in kidney disease remains elusive. Here we investigate expression of ER stress proteins in cultured rat podocytes as well as in our recently developed animal model of abnormal protein retention within the ER of podocytes (i.e., megsin transgenic rat). The expression of ER stress inducible proteins (ORP150, GRP78, or GRP94) in cultured podocytes treated with tunicamycin, A23187, SNAP, hypoxia, or hyperglycemia, and the renal tissues or isolated glomeruli from megsin transgenic rats was analyzed by Western blotting analysis, immunohistochemistry, or confocal microscopy. Cultured podocytes demonstrated that treatment with tunicamycin, A23187, and SNAP, but not hypoxia or hyperglycemia, up-regulate expression of ER stress proteins. Extracts of isolated glomeruli from megsin transgenic rats reveal marked up-regulation of ER stress chaperones in podocytes, which was supported by immunohistochemical analysis. Confocal microscopy revealed that ER stress in podocytes was associated with cellular injury. Podocytes of transgenic rats overexpressing a mutant megsin, without the capacity for polymerization within the ER, do not exhibit ER stress or podocyte damage, suggesting a pathogenic role of ER retention of polymerized megsin. This paper implicates a crucial role for the accumulation of excessive proteins in the podocyte ER in the induction of ER stress and associated podocyte injury.

Isoelectric Point-Based Prefractionation of Proteins from Crude Biological Samples Prior to Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in the presence or absence of epigallocatechin-3-gallate was achieved in fewer than 30 min using an anion-exchange resin and two expressly designed buffers. The procedure was carried out in a centrifuge tube and standard instrumentation was used. The cell lysates were prefractionated into two fractions: proteins with pI values above 7 and between 4 and 7, respectively. The fractions were then analyzed by 2-DE, selecting appropriate pH ranges for the IPG strips, and the gels were compared with those of unprefractionated cell lysates. Protein loading capacity was optimized and resolution and visualization of the less abundant and differentially expressed proteins were greatly improved.

Synthesis of monodisperse poly(chloromethyl-styrene-co-divinylbenzene) beads and their application in separation of biopolymers

The monodisperse, porous poly(chloromethylstyrene-co-divinylbenzene) beads of 7.9 microm were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. Based on this media, a weak cation-exchange (WCX) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery, and good resolution for proteins. The dynamic protein-loading capacity of the synthesized WCX packings was 18.2 mg/g. Five proteins were separated in 3.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 5.0 min with only one step. The purity and specific bioactivity of the purified lysozyme were found to be more than 93% and 70 245 U/mg, respectively.

Preparation of strong anion-exchange chromatographic packings based on monodisperse polymeric beads and their application in the separation of biopolymers

A new hydrophilic strong anion-exchange (SAX) stationary phase for HPLC has been synthesized by chemical modification of macroporous 8.0-microm monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads (P(GMA/EDMA)). The stationary phase was evaluated in detail to determine its ion-exchange properties, separability, reproducibility, hydrophilicity, and the effect of column loading and pH on the separation and retention of proteins. It was found to have an ion-exchange chromatographic (IEC) retention mechanism. The highest dynamic protein loading capacity of the synthesized SAX packing for BSA was 22.6 mg g(-1). Five proteins were separated within 6.0 min using the synthesized SAX resin. The SAX resin was also used for rapid separation and purification of recombinant human stem cell factor (rhSCF) from a crude extract solution in only one step. The purity of the purified of rhSCF was >92.4%.

Synthesis of non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application in separation of biopolymers

The monodisperse, 5.0 μm non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) (P GMA/EDMA) beads were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. Based on this media, a weak cation exchange (WCX) stationary phase for high performance liquid chromatography (HPLC) was synthesized by a new chemical modification method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The measured bioactivity recovery for lysozyme was 97 ± 5%. The dynamic protein loading capacity of the synthesized WCX packings was 20.5 mg/g. Four proteins were completely separated in 3.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 3.0 min with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70.264 IU/mg, respectively.

Microencapsulated chitosan nanoparticles for lung protein delivery

It has already been demonstrated that spray drying is a very valuable technique for producing dry powders adequate for pulmonary delivery of drugs. We have developed chitosan/tripolyphosphate nanoparticles that promote peptide absorption across mucosal surfaces. The aim of this work was to microencapsulate protein-loaded chitosan nanoparticles using typical aerosol excipients, such as mannitol and lactose, producing microspheres as carriers of protein-loaded nanoparticles to the lung. The results showed that the obtained microspheres are mostly spherical and possess appropriate aerodynamic properties for pulmonary delivery (aerodynamic diameters between 2 and 3 μm, apparent density lower than 0.45 g/cm 3). Moreover, microspheres morphology was strongly affected by the content of chitosan nanoparticles. These nanoparticles show a good protein loading capacity (65–80%), providing the release of 75–80% insulin within 15 min, and can be easily recovered from microspheres after contact with an aqueous medium with no significant changes in their size and zeta potential values. Therefore, this work demonstrated that protein-loaded nanoparticles could be successfully incorporated into microspheres with adequate characteristics to reach the deep lung, which after contact with its aqueous environment are expected to be able to release the nanoparticles, and thus, the therapeutic macromolecule.

Synthesis of Monodisperse Poly(glycidylmethacrylate‐co‐ethylene dimethacrylate) Beads and Their Application in Separation of Biopolymers

AbstractThe monodisperse poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) beads with macroporous in the range of 8.0–12.0 μn were prepared by a single‐step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instruction method. By using this media, a weak cation exchange (WCX) stationary phase for HPLC was synthesized by a new chemical modification method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The measured bioactivity recovery for lysozyme was (96 5)%. The dynamic protein loading capacity of the synthesized WCX packing was 21.3 mg/g. Five proteins were completely separated in 8.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 8 min with only one step . The purity and specific bioactivity of the purified lysozyme was found more than 92.0% and 70184 U/mg, respectively.

A New Isoelectric Focusing System for Fast Two-Dimensional Gel Electrophoresis Using a Low-Concentration Polyacrylamide Gel Supported by a Loose Multifilament String

A new isoelectric focusing (IEF) system for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been proposed. In this system, a super-soft and tough IEF gel was achieved by casting polyacrylamide gel down to 2.0% T using a loose multifilament string (LMS) of nylon as a gel support. The IEF apparatus for the LMS-gel, fabricated from acrylic boards, had a cooling water chamber, and eliminated the need of electrode solutions by directly connecting the two ends of individual gels to platinum electrodes. The carrier ampholyte-generated pH gradients using the new IEF system was stable over a long duration of time and a wide range of voltages, and the IEF time became shorter using a 2.0% T gel than using a 4.0% T gel. Also, the LMS-gels prepared in different runs exhibited excellent reproducibility. The new IEF system was applied to 2-D PAGE of a chicken skeletal muscle extract, and it was found that the protein loading capacity, protein entry into the LMS-gels, and protein transfer efficiency from the first-dimensional to the second-dimensional gels were significantly improved by using a low-concentration (2.5% T) gel. Also, proteins of high molecular weight of more than 200 kDa were observed in the 2-D maps, and therefore the new IEF system has a very good potential to be applied for fast 2-D PAGE of high molecular-weight proteins.