Strategy for purifying maltose binding protein fusion proteins by affinity precipitation

The maltose binding protein (MBP) affinity tag has been extensively used for protein purification. A commercial grade cationic starch could precipitate MBP or an MBP-tagged protein quantitatively by simultaneous addition of 10% (w/v) polyethylene glycol (PEG) and 50 mM calcium chloride. The precipitated MBP or MBP-tagged protein could be selectively dissociated by suspending the precipitate in 1 M NaCl. In the case of a soluble MBP fusion with a fragment of human immunodeficiency virus protein gp120, 38% of the contaminating proteins could be removed by precipitation with PEG/CaCl(2) and 100% of the fusion protein was recovered. In all cases, the purified proteins showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the expected changes in fluorescence emission spectra upon binding to maltose.     

Boronate functionalised polymer monoliths for microscale affinity chromatography

Novel macroporous monolithic stationary phase materials suitable for microscale boronate affinity chromatography were developed.     

"Smart" mobile affinity matrix for microfluidic immunoassays

There is a current need for simple methods for immobilizing biomolecules within microfluidic channels. Here, a technique is reported for reversibly immobilizing immunoassay components in a channel zone that can be simply controlled by integrated heating elements. Latex beads were modified with the temperature-responsive polymer poly(N-isopropylacrylamide)(PNIPAAm) and co-modified with biotinylated poly(ethylene glycol)(PEG). PNIPAAm undergoes a hydrophilic-to-hydrophobic transition when the temperature is raised above the lower critical solution temperature (LCST)( approximately 28 degrees C in the solutions used here). This reversible transition drives the aggregation and dis-aggregation of the modified beads in heated zones within poly(ethylene terephthalate)(PET) microchannels. Biotinylated monoclonal antibodies for the drug digoxin were bound via streptavidin to the biotin-PEG-coated beads. These antibody-functionalized beads were then reversibly immobilized by aggregation and hydrophobic adhesion to the surface of PET microfluidic channels in response to a thermal stimulus. The antibodies on the beads immobilized in the channel were shown to bind digoxin and a competitor fluorescent ligand from a flow stream in a quantitative competitive assay format that reported the digoxin concentration. The antibodies could be replenished for each immunoassay trial, using the reversible, temperature-controlled immobilization process. This technique allows reagent immobilization immediately prior to an analytical procedure, following the removal of previously utilized beads, guaranteeing fresh and active immobilized biomolecules. Furthermore, it provides a simple approach to multiplexing through the simultaneous or sequential injection of different antibody-coated bead species, potentially at multiple sites in the integrated device channels.     

Bioskin as an affinity matrix for the separation of glycoproteins

Bioskin is a natural product produced by a mixed culture of Acetobacter xylinum, Saccharomyces cerevisiae and S. pombe cultured on media containing sucrose. It is of fibrillar nature able to retain some proteins, such as cytochrome c, by adsorption, and mainly composed of glucosamine and N-acetyl-D-glucosamine. This makes it possible that, at an adequate pH value, proteins charged as polyanionic molecules, such as catalase, can be retained by ionic adsorption using the positively charged amino groups of the matrix. In addition, bioskin can also be used as an affinity matrix to retain glycoproteins able to perform specific affinity reactions with the amino sugars of the matrix, such as invertase, fetuin or ovalbumin. Its possible use as a chromatographic support is discussed.     

Model for reversible nanoparticle assembly in a polymer matrix

The clustering of nanoparticles (NPs) in solutions and polymer melts depends sensitively on the strength and directionality of the NP interactions involved, as well as the molecular geometry and interactions of the dispersing fluids. Since clustering can strongly influence the properties of polymer-NP materials, we aim to better elucidate the mechanism of reversible self-assembly of highly symmetric NPs into clusters under equilibrium conditions. Our results are based on molecular dynamics simulations of icosahedral NP with a long-ranged interaction intended to mimic the polymer-mediated interactions of a polymer-melt matrix. To distinguish effects of polymer-mediated interactions from bare NP interactions, we compare the NP assembly in our coarse-grained model to the case where the NP interactions are purely short ranged. For the "control" case of NPs with short-ranged interactions and no polymer matrix, we find that the particles exhibit ordinary phase separation. By incorporating physically plausible long-ranged interactions, we suppress phase separation and qualitatively reproduce the thermally reversible cluster formation found previously in computations for NPs with short-ranged interactions in an explicit polymer-melt matrix. We further characterize the assembly process by evaluating the cluster properties and the location of the self-assembly transition. Our findings are consistent with a theoretical model for equilibrium clustering when the particle association is subject to a constraint. In particular, the density dependence of the average cluster mass exhibits a linear concentration dependence, in contrast to the square root dependence found in freely associating systems. The coarse-grained model we use to simulate NP in a polymer matrix shares many features of potentials used to model colloidal systems. The model should be practically valuable for exploring factors that control the dispersion of NP in polymer matrices where explicit simulation of the polymer matrix is too time consuming.     

Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis

An optimization strategy for ternary solvent-strength gradient elution RP chromatography is described in which a two-dimensional model of gradient time (2 levels) against ternary proportions of organic modifiers (4 levels) was constructed. From the resolution surface the optimum ratio of organic modifiers could be selected. Excellent retention time and acceptable peak width and resolution simulations were obtained. The separation could be further optimized from the same input data by using a standard one-dimensional model in order to optimize for gradient slope, duration and shape. Excellent retention time and acceptable peak width and resolution simulations were obtained (< 1, 2 and 6% error, respectively).     

Fabrication and characterization of hexahistidine-tagged protein functionalized multi-walled carbon nanotubes for selective solid-phase extraction of Cu and Ni

Hexahistidine-tagged protein functionalized multi-walled carbon nanotubes (MWCNTs/6His-tagged protein) were prepared and characterized by ultraviolet-visible spectrophotometry and atomic force microscopy. Both static and dynamical adsorption experiments showed that the MWCNTs/6His-tagged protein served as good sorbent for the solid-phase extraction of Cu²⁺ and Ni²⁺. Effective on-line sorption of Cu²⁺ and Ni²⁺ on the MWCNTs/6His-tagged protein packed microcolumn was achieved in a pH range of 3.0-4.5 and 4.5-6.0, respectively. The retained Cu²⁺ and Ni²⁺ were efficiently eluted with 0.2 mol L⁻¹ imidazole-HCl solution for on-line flame atomic absorption spectrometric determination. The MWCNTs/6His-tagged protein exhibited fairly fast kinetics for the sorption of Cu²⁺ and Ni²⁺, and offered up to 20,000 and 1800 times improvement in the tolerable concentrations of co-existing ions over the MWCNTs for solid-phase extraction of Cu²⁺ and Ni²⁺, respectively. On-line solid-phase extraction at a flow rate of 5.0 mL min⁻¹ for 60 s gave an enhancement factor of 29 for Cu²⁺ and 28 for Ni²⁺, a sample throughput of 45 h⁻¹, and a detection limit (3s) of 0.31 μg L⁻¹ for Cu²⁺ and 0.63 μg L⁻¹ for Ni²⁺. The precision for 11 replicate measurements was 2.4% for 10 μg L⁻¹ Cu²⁺, and 2.5% for 15 μg L⁻¹ Ni²⁺.     

A polymer scaffold for protein oligomerization

We report the design and synthesis of well-defined polymers for the noncovalent oligomerization of proteins. The reported scaffolds, which were generated by atom-transfer radical polymerization (ATRP), take advantage of the well-characterized interaction between a Ni2+ complex and an oligohistidine sequence (His tag). Thus, our polymers are designed to facilitate the oligomerization of any protein possessing a His tag. We demonstrate that they can oligomerize fibroblast growth factor-8b (FGF-8b) and promote FGF-8b-mediated cell proliferation in the absence of heparin.     

Membrane-immobilized haptoglobin as affinity matrix for a hemoglobin-A1c immunosensor

An amperometric immunosensor for hemoglobin-A1c (HbA1c) determination has been developed utilizing membrane-immobilized haptoglobin as affinity matrix fixed in front of a Pt-working electrode. The HbA1c assay was carried out in a two-step procedure including the selective hemoglobin enrichment on the sensor surface and the specific HbA1c detection by a glucose oxidase (GOx) labeled anti-HbA1c antibody. Hydrogen peroxide generated by the enzyme label was oxidized at +600 mV versus Ag/AgCl. A standard curve for HbA1c was obtained with a linear range between 0 and 25% HbA1c of total hemoglobin which correspond to 7.8–39 nM. ELISA studies confirmed the advantage of a sandwich-type format with haptoglobin as capture molecule for selective hemoglobin binding over the direct adsorption method. Results by the sandwich immunoassay showed a linear correlation within the clinically relevant range 5–20% (CV < 3). For sensor application the immobilization procedure of haptoglobin onto CDI-activated cellulose membranes was optimized.     

Photo-initiated base-formation in a polymer matrix

It was found that amines were formed efficiently by the photolysis of O-acyloximes followed by hydrolysis in polystyrene films and the relationship between structures of O-acyloximes and yields of amines were investigated. O-phenylacetyl acetophenone oxime (PaApO), O-pivaloyl acetophenone oxime (PApO), and O-benzoyl acetophenone oxime (BApO) as monofunctional O-acyloximes and O,O′-succinyl diacetophenone oxime (SDApO) and O,O′-glutaryl diacetophenone oxime (GDApO) as bifunctional O-acyloximes were examined. The yields of amines for PaApO and SDApO under N₂ were ca. 70%, which was the highest among O-acyloximes examined in this experiment. On the other hand, the yields for PApO, BApO, and GDApO were less than 15% and it was verified that the hydrogen abstraction by imino radicals via 6-membered cyclic intermediates resulted in the lowering of yields. Although the effect of oxygen under photolysis on the yields of amine for PaApO was negligible under 50% conversion of PaApO, the yield decreased with further increase in the conversion and was 50% at 90% conversion. © 1994 John Wiley & Sons, Inc.     

Hydroxyapatite-based immobilized metal affinity adsorbents for protein purification

The employment of metal ion-charged hydroxyapatite for the one-step purification of poly(His)-tagged recombinant proteins was investigated. Fe(III) showed the highest selectivity toward the poly(His)-tagged D-hydantoinase and the best operation stability. The optimal selectivity was observed in 20 mM pH 8.0 buffer containing 150 mM NaCl and 50 mM NaF. The adsorbed poly(His)-tagged enzyme could be quantitatively recovered from hydroxyapatite with 150 mM pH 8.0 phosphate buffer. The capacity of Fe(III)-loaded hydroxyapatite for poly(His)-tagged D-hydantoinase was 4.9 mg/g hydroxyapatite, comparable to commercial agarose-based Ni-NTA adsorbents. Under optimal conditions, a D-hydantoinase preparation with a purity above 95% from crude cellular lysate could be obtained with the one-step purification process employing Fe(III)-loaded hydroxyapatite. The application of Fe(III)-loaded hydroxyapatite for the purification of poly(His)-tagged N-acetyl-D-glucosamine 2-epimerase under denaturing conditions was also demonstrated. These results demonstrate that hydroxyapatite is a promising adsorbent for immobilized metal affinity chromatography.     

Computer-aided selection of a polymer matrix for polyaniline conductive blends

The selection of a polymer matrix for a conductive blend with polyaniline and para-toluene sulfonic acid (PANI-pTSA) was performed using molecular simulation techniques, both a fast quantitative structure–properties relationship method as a first screening phase followed by atomistic simulation. Using the atomistic simulation method, the solubility parameters and the heat of mixing of each blend were calculated to enable the determination of compatible matrices in blends with PANI-pTSA, which was validated by experimental scanning electron microscopy fractographs. Based on such calculations, polycaprolactone (PCL)/PANI-pTSA phase diagrams were estimated, showing slight miscibility of polydispersed PANI in PCL, particularly the short chains fraction, at the elevated melt processing temperature. It was suggested that this partial miscibility at the elevated temperature might lead to a conductive network morphology of PANI in PCL at room temperature, because of phase separation and precipitation of soluble PANI molecules, upon cooling and solidification of the melt. © 1998 John Wiley & Sons, Ltd.     

Scale-up development of high-performance polymer matrix for DNA sequencing analysis

Linear polyacrylamide (LPA) has been widely used as a replaceable separation matrix in CE. An increase in the molecular weight of the separation medium favors the separation of larger DNA fragments. In order to obtain ultrahigh-molecular-weight (UHMW) LPA, a "frozen" method was developed to synthesize the LPA homopolymer. This approach has three major advantages when compared with other existing routes of LPA synthesis: (i) long LPA chains could be obtained easily, with their average molecular weight (MW) being in the high 10 MDa range; (ii) the desired MW could be adjusted over a broad range by controlling the temperature and the concentration of initiators during synthesis; (iii) the product solution contains only a tiny amount of impurity besides the solvent and LPA. Both static and dynamic laser light scattering measurements were carried out to characterize the synthesized LPA in the buffer solution. The DNA sequencing matrix prepared from LPA using this method was studied and the results were compared with the newly developed commercial product POP7 from Applied Biosystems. It should be noted that this approach can be applied to synthesize other water-soluble polymers, resulting in UHMW products because the chain transfer constant is smaller at lower temperatures.     

Biodegradable polymer matrix nanocomposites for tissue engineering: A review

Nanocomposites have emerged in the last two decades as an efficient strategy to upgrade the structural and functional properties of synthetic polymers. Aliphatic polyesters as polylactide (PLA), poly(glycolides) (PGA), poly(?-caprolactone) (PCL) have attracted wide attention for their biodegradability and biocompatibility in the human body. A logic consequence has been the introduction of organic and inorganic nanofillers into biodegradable polymers to produce nanocomposites based on hydroxyapatite, metal nanoparticles or carbon nanotructures, in order to prepare new biomaterials with enhanced properties. Consequently, the improvement of interfacial adhesion between the polymer and the nanostructures has become the key technique in the nanocomposite process. In this review, different results on the fabrication of nanocomposites based on biodegradable polymers for specific field of tissue engineering are presented. The combination of bioresorbable polymers and nanostructures open new perspectives in the self-assembly of nanomaterials for biomedical applications with tuneable mechanical, thermal and electrical properties.     

Functional polymer colloids with ordered interior

Polymer colloids with internal ordering were synthesized using hydrolytic condensation of octadecyl-dimethyl(3-trimethoxysilylpropyl)ammonium chloride (ODMACl) and a mixture of ODMACl and the trisodium salt of the triacetic acid N-(trimethoxysilylpropyl)ethylenediamine (TANED). The structure and morphology of these colloids were studied with small-angle X-ray scattering, transmission electron microscopy, nuclear magnetic resonance, sedimentation in ultracentrifuge; and other methods. When polymer colloids are obtained from a single precursor (ODMACl), their local structure, molecular weight characteristics, and morphology strongly depend on the reaction conditions, while lamellar ordering remains nearly unaffected. Use of a mixture of cationic and anionic silanes (ODMACl and TANED) as precursors in hydrolytic condensation results in novel zwitterionic copolymer colloids with two-dimensional hexagonal packing. Interaction of the ODMACl quaternary ammonium groups with the three carboxy groups of TANED leads to replacement of sodium and chloride ions and formation of gegenions, resulting in a molar ratio ODMACl:TANED = 3:1 (each TANED molecule contains three carboxy groups). Due to their ordered interior, polyODMACl (PODMACl) and PODMACl-TANED colloids can be used as templates for controlled positioning of nanoparticles within these colloids. For example, lamellar ordering controls Pt nanoparticle formation within PODMACl colloids providing Pt nanoparticle alignment within the lamellar structure. Loading of PODMACl-TANED colloids with iron salts followed by pH increase results in the formation of iron oxide nanoparticles located within PODMACl-TANED cylinders.     

Functional polymer thin films designed for antifouling materials and biosensors

Polymer thin films offer a versatile and ubiquitous platform for a wide variety of real-world applications in biomedicine, nanotechnology, catalysis, photovoltaic devices, and energy conversion and storage. Depending on the chemical composition of the polymers and the associated microenvironment, the physicochemical properties (biocompatibility, stability, wettability, adhesion, morphology, surface free energy, and others) of polymer films can be tuned for a specific application through precisely controlled surface synthesis and the incorporation of desirable and responsive functional groups. In this short review, we first summarise the methods most commonly used for the fabrication of polymer thin films. Then we discuss how these polymer thin films can be used in a selection of biomedical applications in antifouling materials and biosensors. Some directions for the rational design of polymer thin films to achieve a specific function or application are also provided.     

Oxidative polymerization of pyrrole in polymer matrix

The oxidative matrix polymerization of pyrrole (Py) by Ce(IV) in the presence of Polyacrylic acid (PAA) has been studied to obtain water-soluble and insoluble products. The role of the PAA, Pyrrole, and Ce(IV) concentration, order of component addition, the structure of polymer matrix (PAA, Hydroxy Ethyl Cellulose (HES), Poly-N-vinylpyrrolidone (PVP)], and model unit of PAA (propionic acid), on the polymerization system were investigated. Interaction of PAA with insoluble polypyrrole (PPy) and the interpolymer complex formation were investigated along with the aggregation of PPy onto the matrix polymer followed by spectral shifts. FTIR results of insoluble products obtained from the PAA–Py–Ce(IV) system and solubility of the system is explained in light of the mechanism of the polymerization of pyrrole on the polymer matrix. © 1995 John Wiley & Sons, Inc.     

Polygonal gold nanoplates in a polymer matrix

Polygonal gold nanoplates are generated in situ in poly(vinyl alcohol) film through thermal treatment, the polymer serving as the reducing agent and stabilizer for the nanoparticle formation and enforcing preferential orientation of the plates. The rare pentagonal as well as the more commonly observed hexagonal, triangular and square/rectangle shapes are obtained by fine-tuning the Au/PVA ratio and the time and temperature of fabrication.     

Photogeneration of heptacene in a polymer matrix

Heptacene (1) was generated by the photodecarbonylation of 7,16-dihydro-7,16-ethanoheptacene-19,20-dione (2) in a polymer matrix using a UV-LED lamp (395 +/- 25 nm). Compound 1 showed a long wavelength absorption band extending from 600 to 825 nm (lambdamax approximately 760 nm) and was found to be stable up to 4 h in the polymer matrix. However, irradiation of a solution of 2 in toluene produced only oxygen adducts.