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Sample records for protein compatible polymer

  1. Direct observation of interaction between proteins and blood-compatible polymer surfaces.

    PubMed

    Hayashi, Tomohiro; Tanaka, Masaru; Yamamoto, Sadaaki; Shimomura, Masatsugu; Hara, Masahiko

    2007-12-01

    The adhesion force between blood-compatible polymer (poly(2-methoxyethyl acrylate: PMEA) and proteins (fibrinogen and bovine serum albumin (BSA)) were measured by atomic force microscopy. The PMEA surface showed almost no adhesion to native protein molecules, whereas non-blood-compatible poly(n-butyl acrylate): PBA strongly adhered to proteins. Interestingly, adhesion did appear between PMEA and proteins when the proteins were denatured. In all cases, these trends were not affected by the conditions of the solution. Combining the results with previous reports, the authors conclude that interfacial water molecules play a critical role in the protein resistance of PMEA. PMID:20408647

  2. Compatibility of hydrosoluble polymers with corrodible materials

    SciTech Connect

    Audibert, A.; Lecourtier, J. )

    1992-05-01

    This paper reports that application of water-soluble polymers in the oil industry (e.g., fluid-loss reducer, polymer flooding, and water-based drilling muds) requires hydrosoluble polymers to be compatible with corrodible materials. The behavior of polyacrylamides and xanthans in the presence of various materials used for oil production (steel, stainless steel, carbon steel, and Inconel) has been studied vs. different water salinities, oxygen contents, and temperatures. The influence of such commonly used additives as oxygen scavengers and sequestrants on corrosion and polymer stability has also been investigated. For both types of polymers, as corrosion occurs under anaerobic conditions, strong interactions between polymer chains and divalent cations (Fe{sup 2+} to Fe{sup 2+}) are observed. Such interactions also depend on polymer quality. In the presence of oxygen, corrosion induces a molecular-weight degradation of the polymer followed by a gelation process for xanthan. Some additives may accelerate the transformation of Fe{sup 2+} to Fe{sup 3+}, thus inducing polymer degradation, but this reaction depends on the nature of the chelating agent. These results provide guidelines for the implementation of polymers in oil production, including the selection of materials, water treatment, or mud formulation.

  3. A triple-function zwitterion for preparing water compatible diclofenac imprinted polymers.

    PubMed

    Shen, Feng; Zhang, Qingxi; Ren, Xueqin

    2015-01-01

    A novel zwitterion acting as both a functional monomer and a crosslinker with the protein-resistant ability concomitantly was synthesized for preparing water compatible diclofenac imprinted polymers. This new imprinted polymer showed high imprinting efficiency for template and strong anti-protein adsorption in aqueous medium. PMID:25387988

  4. Polymer Matrix Composite Material Oxygen Compatibility

    NASA Technical Reports Server (NTRS)

    Owens, Tom

    2001-01-01

    Carbon fiber/polymer matrix composite materials look promising as a material to construct liquid oxygen (LOX) tanks. Based on mechanical impact tests the risk will be greater than aluminum, however, the risk can probably be managed to an acceptable level. Proper tank design and operation can minimize risk. A risk assessment (hazard analysis) will be used to determine the overall acceptability for using polymer matrix composite materials.

  5. Compatibility of polymers and chemical oxidants for enhanced groundwater remediation.

    PubMed

    Smith, Megan M; Silva, Jeff A K; Munakata-Marr, Junko; McCray, John E

    2008-12-15

    Polymer floods provide a promising method to more effectively deliver conventional groundwater treatment agents to organic contaminants distributed within heterogeneous aquifer systems. Combinations of nontoxic polymers (xanthan and hydrolyzed polyacrylamide) and common chemical oxidants (potassium permanganate and sodium persulfate) were investigated to determine the suitability of these mixtures for polymer-enhanced in situ chemical oxidation applications. Oxidant demand and solution viscosity were utilized as initial measures of chemical compatibility. After 72 h of reaction with both test oxidants, solution viscosities in mixtures containing hydrolyzed polyacrylamide were decreased by more than 90% (final viscosities approximately 2 cP), similar to the 95% viscosity loss (final viscosities approximately 1 cP, near that of water) observed in xanthan/persulfate experiments. In contrast, xanthan solutions exposed to potassium permanganate preserved 60-95% of initial viscosity after 72 h. Permanganate depletion in xanthan-containing experiments ranged from 2% to 24% over the same test period. Although oxidant consumption in xanthan/permanganate solutions appeared to be correlated with increasing xanthan concentrations, solutions of up to 2000 mg/L xanthan did not inhibit permanganate from oxidizing a dissolved-phase test contaminant (tetrachloroethene, PCE) in xanthan solution. These advantageous characteristics (high viscosity retention, moderate oxidant demand, and lack of competitive effects on PCE oxidation rate) render xanthan/permanganate the most compatible polymer/oxidant combination of those tested for remediation by polymer-enhanced chemical oxidation. PMID:19174907

  6. Facile surface modification of silicone rubber with zwitterionic polymers for improving blood compatibility.

    PubMed

    Liu, Pingsheng; Chen, Qiang; Yuan, Bo; Chen, Mengzhou; Wu, Shishan; Lin, Sicong; Shen, Jian

    2013-10-01

    A facile approach to modify silicone rubber (SR) membrane for improving the blood compatibility was investigated. The hydrophobic SR surface was firstly activated by air plasma, after which an initiator was immobilized on the activated surface for atom transfer radical polymerization (ATRP). Three zwitterionic polymers were then grafted from SR membrane via surface-initiated atom transfer radical polymerization (SI-ATRP). The surface composition, wettability, and morphology of the membranes before and after modification were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (WCA) measurement, and atomic force microscopy (AFM). Results showed that zwitterionic polymers were successfully grafted from SR surfaces, which remarkably improved the wettability of the SR surface. The blood compatibility of the membranes was evaluated by protein adsorption and platelet adhesion tests in vitro. As observed, all the zwitterionic polymer modified surfaces have improved resistance to nonspecific protein adsorption and have excellent resistance to platelet adhesion, showing significantly improved blood compatibility. This work should inspire many creative uses of SR based materials for biomedical applications such as vessel, catheter, and microfluidics. PMID:23910289

  7. Effects of compatability on the conductivity of conducting polymer blends

    SciTech Connect

    Liu, Mingjun; Nowak, C.K.; Gregory, R.V.

    1995-12-01

    The electrical conductivity of chemically synthesized polyaniline (PANI) blends with nylon 6,6 and polystyrene was measured. The conductivities of the top and bottom of the films cast from blend solutions were found to differ. This effect was most pronounced at low percent loadings of PANI. The maximum difference in conductivity between two sides of the same film was found to be five orders of magnitude in the case of a 5% PANI blend with polystyrene. In this case the conductive polymer appears to be rich on one side of the film rather than more homogeneously dispersed on both sides. SEM provides evidence for the formation of a percolation cluster on one side of the film which is most notable in polystyrene blends. X-ray and FTIR indicated that greater interaction between PANI and nylon 6,6 than PANI and polystyrene. It is proposed that the magnitude of the variation in conductivity between the two sides of the film depends on the compatibility of the conducting and insulating host polymers.

  8. Zwitterionic polymer functionalization of polysulfone membrane with improved antifouling property and blood compatibility by combination of ATRP and click chemistry.

    PubMed

    Xiang, Tao; Lu, Ting; Xie, Yi; Zhao, Wei-Feng; Sun, Shu-Dong; Zhao, Chang-Sheng

    2016-08-01

    The chemical compositions are very important for designing blood-contacting membranes with good antifouling property and blood compatibility. In this study, we propose a method combining ATRP and click chemistry to introduce zwitterionic polymer of poly(sulfobetaine methacrylate) (PSBMA), negatively charged polymers of poly(sodium methacrylate) (PNaMAA) and/or poly(sodium p-styrene sulfonate) (PNaSS), to improve the antifouling property and blood compatibility of polysulfone (PSf) membranes. Attenuated total reflectance-Fourier transform infrared spectra, X-ray photoelectron spectroscopy and water contact angle results confirmed the successful grafting of the functional polymers. The antifouling property and blood compatibility of the modified membranes were systematically investigated. The zwitterionic polymer (PSBMA) grafted membranes showed good resistance to protein adsorption and bacterial adhesion; the negatively charged polymer (PNaSS or PNaMAA) grafted membranes showed improved blood compatibility, especially the anticoagulant property. Moreover, the PSBMA/PNaMAA modified membrane showed both antifouling property and anticoagulant property, and exhibited a synergistic effect in inhibiting blood coagulation. The functionalization of membrane surfaces by a combination of ATRP and click chemistry is demonstrated as an effective route to improve the antifouling property and blood compatibility of membranes in blood-contact. PMID:27039977

  9. Protein crystallization facilitated by molecularly imprinted polymers

    PubMed Central

    Saridakis, Emmanuel; Khurshid, Sahir; Govada, Lata; Phan, Quan; Hawkins, Daniel; Crichlow, Gregg V.; Lolis, Elias; Reddy, Subrayal M.; Chayen, Naomi E.

    2011-01-01

    We present a previously undescribed initiative and its application, namely the design of molecularly imprinted polymers (MIPs) for producing protein crystals that are essential for determining high-resolution 3D structures of proteins. MIPs, also referred to as “smart materials,” are made to contain cavities capable of rebinding protein; thus the fingerprint of the protein created on the polymer allows it to serve as an ideal template for crystal formation. We have shown that six different MIPs induced crystallization of nine proteins, yielding crystals in conditions that do not give crystals otherwise. The incorporation of MIPs in screening experiments gave rise to crystalline hits in 8–10% of the trials for three target proteins. These hits would have been missed using other known nucleants. MIPs also facilitated the formation of large single crystals at metastable conditions for seven proteins. Moreover, the presence of MIPs has led to faster formation of crystals in all cases where crystals would appear eventually and to major improvement in diffraction in some cases. The MIPs were effective for their cognate proteins and also for other proteins, with size compatibility being a likely criterion for efficacy. Atomic force microscopy (AFM) measurements demonstrated specific affinity between the MIP cavities and a protein-functionalized AFM tip, corroborating our hypothesis that due to the recognition of proteins by the cavities, MIPs can act as nucleation-inducing substrates (nucleants) by harnessing the proteins themselves as templates. PMID:21690356

  10. Characterization of the attachment mechanisms of tissue-derived cell lines to blood-compatible polymers.

    PubMed

    Hoshiba, Takashi; Nikaido, Mayo; Tanaka, Masaru

    2014-05-01

    Recent advances in biomedical engineering require the development of new types of blood-compatible polymers that also allow non-blood cell attachment for the isolation of stem cells and circulating tumor cells (CTCs) from blood and for the development of artificial organs for use under blood-contact conditions. Poly(2-methoxyethyl acrylate) (PMEA) and poly(tetrafurfuryl acrylate) (PTHFA) were previously identified as blood-compatible polymers. Here, it is demonstrated that cancer cells can attach to the PMEA and PTHFA substrates, and the differences in the attachment mechanisms to the PMEA and PTHFA substrates between cancer cells and platelets are investigated. It is also found that the adsorption-induced deformation of fibrinogen, which is required for the attachment and activation of platelets, does not occur on the PMEA and PTHFA substrates. In contrast, fibronectin is deformed on the PMEA and PTHFA substrates. Therefore, it is concluded that cancer cells and not platelets can attach to the PMEA and PTHFA substrates based on this protein-deformation difference between these substrates. Moreover, it is observed that cancer cells attach to the PMEA substrate via both integrin-dependent and -independent mechanisms and attach to the PTHFA substrate only through an integrin-dependent mechanism. It is expected that PMEA and PTHFA will prove useful for blood-contact biomedical applications. PMID:24105989

  11. Compatibility of Medical-Grade Polymers with Dense CO2

    PubMed Central

    Jiménez, A; Thompson, G L; Matthews, M A; Davis, T A; Crocker, K; Lyons, J S; Trapotsis, A

    2009-01-01

    This study reports the effect of exposure to liquid carbon dioxide on the mechanical properties of selected medical polymers. The tensile strengths and moduli of fourteen polymers are reported. Materials were exposed to liquid CO2, or CO2 + trace amounts of aqueous H2O2, at 6.5 MPa and ambient temperature. Carbon dioxide uptake, swelling, and distortion were observed for the more amorphous polymers while polymers with higher crystallinity showed little effect from CO2 exposure. Changes in tensile strength were not statistically significant for most plastics, and most indicated good tolerance to liquid CO2. These results are relevant to evaluating the potential of liquid CO2-based sterilization technology. PMID:19756235

  12. Quick setting water-compatible furfuryl alcohol polymer concretes

    DOEpatents

    Sugama, Toshifumi; Kukacka, Lawrence E.; Horn, William H.

    1982-11-30

    A novel quick setting polymer concrete composite comprising a furfuryl alcohol monomer, an aggregate containing a maximum of 8% by weight water, and about 1-10% trichlorotoluene initiator and about 20-80% powdered metal salt promoter, such as zinc chloride, based on the weight of said monomer, to initiate and promote polymerization of said monomer in the presence of said aggregate, within 1 hour after mixing at a temperature of -20.degree. C. to 40.degree. C., to produce a polymer concrete having a 1 hour compressive strength greater than 2000 psi.

  13. Cell compatibility of a maghemite/polymer biomedical nanoplatform.

    PubMed

    Ali, Lamiaa M A; Piñol, Rafael; Villa-Bellosta, Ricardo; Gabilondo, Lierni; Millán, Angel; Palacio, Fernando; Sorribas, Victor

    2015-08-01

    We are reporting the cytocompatibility and cellular fate of an iron oxide/polymer nanoplatform (IONP) in its most basic formulation, using both mesenchymal (vascular smooth muscle cells, VSMC), and epithelial (opossum kidney, OK) cells. The cytotoxicity and cell internalization of the nanoplatform has been evaluated in relation to time of exposure and concentration of different components. A series of samples with different iron oxide nanoparticle, sizes, hydrodynamic sizes and iron/polymer ratio have been examined. In all cases cytotoxicity is low, and it is mostly determined by the internalization rate, being higher in VSMC than in OK cells. The mean lethal dose has a very narrow threshold, and necrosis is the only cell death type. IONP uptake shows little incidence on oxidative stress, and inflammasome activation is only observed with the smaller IONP at high concentration. The internalization rate in VSMC is determined by the polymer concentration exclusively. In OK cells, internalization rate seems to increase with decreasing hydrodynamic size. Internalization occurs through clathrin-dependent endocytosis, as it is prevented by potassium depletion and chlorpromazine. IONP are directed and accumulated in lysosomes. Under IONP overload, lysosomal dysfunction would cause cell death using concentrations that are hardly achieved in vivo. PMID:25891827

  14. Polymer principles and protein folding.

    PubMed Central

    Dill, K. A.

    1999-01-01

    This paper surveys the emerging role of statistical mechanics and polymer theory in protein folding. In the polymer perspective, the folding code is more a solvation code than a code of local phipsi propensities. The polymer perspective resolves two classic puzzles: (1) the Blind Watchmaker's Paradox that biological proteins could not have originated from random sequences, and (2) Levinthal's Paradox that the folded state of a protein cannot be found by random search. Both paradoxes are traditionally framed in terms of random unguided searches through vast spaces, and vastness is equated with impossibility. But both processes are partly guided. The searches are more akin to balls rolling down funnels than balls rolling aimlessly on flat surfaces. In both cases, the vastness of the search is largely irrelevant to the search time and success. These ideas are captured by energy and fitness landscapes. Energy landscapes give a language for bridging between microscopics and macroscopics, for relating folding kinetics to equilibrium fluctuations, and for developing new and faster computational search strategies. PMID:10386867

  15. RAFT polymers for protein recognition

    PubMed Central

    Tominey, Alan F; Liese, Julia; Wei, Sun; Kowski, Klaus

    2010-01-01

    Summary A new family of linear polymers with pronounced affinity for arginine- and lysine-rich proteins has been created. To this end, N-isopropylacrylamide (NIPAM) was copolymerized in water with a binding monomer and a hydrophobic comonomer using a living radical polymerization (RAFT). The resulting copolymers were water-soluble and displayed narrow polydispersities. They formed tight complexes with basic proteins depending on the nature and amount of the binding monomer as well as on the choice of the added hydrophobic comonomer. PMID:20703378

  16. Oxygen Compatibility of Brass-Filled PTFE Compared to Commonly Used Fluorinated Polymers for Oxygen Systems

    NASA Technical Reports Server (NTRS)

    Herald, Stephen D.; Frisby, Paul M.; Davis, Samuel Eddie

    2009-01-01

    Safe and reliable seal materials for high-pressure oxygen systems sometimes appear to be extinct species when sought out by oxygen systems designers. Materials that seal well are easy to find, but these materials are typically incompatible with oxygen, especially in cryogenic liquid form. This incompatibility can result in seals that leak, or much worse, seals that easily ignite and burn during use. Materials that are compatible with oxygen are easy to find, such as the long list of compatible metals, but these metallic materials are limiting as seal materials. A material that seals well and is oxygen compatible has been the big game in the designer's safari. Scientists at the Materials Combustion Research Facility (MCRF), part of NASA/Marshall Space Flight Center (MSFC), are constantly searching for better materials and processes to improve the safety of oxygen systems. One focus of this effort is improving the characteristics of polymers used in the presence of an oxygen enriched environment. Very few systems can be built which contain no polymeric materials; therefore, materials which have good impact resistance, low heat of combustion, high auto-ignition temperature and that maintain good mechanical properties are essential. The scientists and engineers at the Materials Combustion Research Facility, in cooperation with seal suppliers, are currently testing a new formulation of polytetrafluoroethylene (PTFE) with Brass filler. This Brass-filled PTFE is showing great promise as a seal and seat material for high pressure oxygen systems. Early research has demonstrated very encouraging results, which could rank this material as one of the best fluorinated polymers ever tested. This paper will compare the data obtained for Brass-filled PTFE with other fluorinated polymers, such as TFE-Teflon (PTFE) , Kel-F 81, Viton A, Viton A-500, Fluorel , and Algoflon . A similar metal filled fluorinated polymer, Salox-M , was tested in comparison to Brass-filled PTFE to

  17. Determination of solute-polymer interaction properties and their application to parenteral product container compatibility evaluations.

    PubMed

    Kenley, R A; Jenke, D R

    1990-09-01

    Kinetic and thermodynamic interaction properties between dialkyl phthalate test compounds and a polyolefin polymer were examined via a permeation-cell experimental design. Disappearance and appearance rates of solute in the receptor and donor solutions, as well as the equilibrium composition of the test system, are used to determine sorption and diffusion coefficients and the solute/polymer equilibrium binding constant. Sorption rate constants and diffusion coefficients exhibit Arrenhius-type behavior. The binding constants obtained correlate well with the solute's octanol-water partition coefficient. The kinetic and thermodynamic data generated combine with proposed interaction models to identify solute/polymer interactions (binding and leaching) pertinent to evaluating container/solution compatibility for parenteral products. PMID:2235889

  18. NEXT GENERATION SOLVENT MATERIALS COMPATIBILITY WITH POLYMER COMPONENTS WITHIN MODULAR CAUSTIC-SIDE SOLVENT EXTRACTION UNIT

    SciTech Connect

    Fondeur, F.; Peters, T.; Fink, S.

    2011-09-29

    The Office of Waste Processing, within the Office of Technology Innovation and Development, is funding the development of an enhanced solvent for deployment at the Savannah River Site for removal of cesium from High Level Waste. The technical effort is collaboration between Oak Ridge National Laboratory (ORNL), Savannah River National Laboratory (SRNL), and Argonne National Laboratory. The first deployment target for the technology is within the Modular Caustic-Side Solvent Extraction Unit (MCU). Deployment of a new chemical within an existing facility requires verification that the chemical components are compatible with the installed equipment. In the instance of a new organic solvent, the primary focus is on compatibility of the solvent with organic polymers used in the facility. This report provides the data from exposing these polymers to the Next Generation Solvent (NGS). The test was conducted over six months. An assessment of the dimensional stability of polymers present in MCU (i.e., PEEK, Grafoil{reg_sign}, Tefzel{reg_sign} and Isolast{reg_sign}) in the modified NGS (where the concentration of the guanidine suppressor and MaxCalix was varied systematically) showed that guanidine (LIX{reg_sign}79) selectively affected Tefzel{reg_sign} (by an increase in size and lowering its density). The copolymer structure of Tefzel{reg_sign} and possibly its porosity allows for the easier diffusion of guanidine. Tefzel{reg_sign} is used as the seat material in some of the valves at MCU. Long term exposure to guanidine, may make the valves hard to operate over time due to the seat material (Tefzel{reg_sign}) increasing in size. However, since the physical changes of Tefzel{reg_sign} in the improved solvent are comparable to the changes in the CSSX baseline solvent, no design changes are needed with respect to the Tefzel{reg_sign} seating material. PEEK, Grafoil{reg_sign} and Isolast{reg_sign} were not affected by guanidine and MaxCalix within six months of exposure. The

  19. CMOS compatible IR sensors by cytochrome c protein

    NASA Astrophysics Data System (ADS)

    Liao, Chien-Jen; Su, Guo-Dung

    2013-09-01

    In recent years, due to the progression of the semiconductor industrial, the uncooled Infrared sensor - microbolometer has opened the opportunity for achieving low cost infrared imaging systems for both military and commercial applications. Therefore, various fabrication processes and different materials based microbolometer have been developed sequentially. The cytochrome c (protein) thin film has be reported high temperature coefficient of resistance (TCR), which is related to the performance of microbolometer directly. Hence the superior TCR value will increase the performance of microbolometer. In this paper, we introduced a novel fabrication process using aluminum which is compatible with the Taiwan Semiconductor Manufacture Company (TSMC) D35 2P4M process as the main structure material, which benefits the device to integrate with readout integrated circuit (ROIC).The aluminum split structure is suspended by sacrificial layer utilizing the standard photolithography technology and chemical etching. The height and thickness of the structure are already considered. Besides, cytochrome c solutions were ink-jetted onto the aluminum structure by using the inkjet printer, applying precise control of the Infrared absorbing layer. In measurement, incident Infrared radiation can be detected and later the heat can be transmitted to adjacent pads to readout the signal. This approach applies an inexpensive and simple fabrication process and makes the device suitable for integration. In addition, the performance can be further improved with low noise readout circuits.

  20. Polymer displacement/shielding in protein chromatography.

    PubMed

    Kumar, A; Galaev, I Y; Mattiasson, B

    2000-05-12

    An overview of different applications of polymer interactions with ion-exchange and dye-affinity chromatographic matrices is presented here. The strength of interaction between the ligand and the polymer plays a crucial role in deciding the mode of chromatographic application. Charged, non-ionic and thermosensitive polymers such as poly(ethylene imine), poly(N-vinyl pyrrolidone) and poly(vinyl caprolactam) respectively, show different degrees of interaction with the dye molecules in dye ligand chromatography. Polymers, with their ability of multipoint and hence strong attachment to the chromatographic matrices, were used as efficient displacers in displacement chromatography. The polymer displacement resulted in better recoveries and sharper elution profiles than traditional salt elutions. The globule-coil transition of the thermosensitive reversible soluble-insoluble polymer, poly(vinyl caprolactam), can be exploited in dye-affinity columns for the temperature induced displacement of the bound protein. In another situation, prior to the column chromatography of crude protein extract, polymers formed complexes with the dye matrix and "shielded" the column. The polymer shielding decreased the nonspecific interactions without affecting the specific interactions of the target protein to the dye matrix. PMID:10872581

  1. Creating Patterned Conjugated Polymer Images Using Water-Compatible Reactive Inkjet Printing.

    PubMed

    Jeon, Seongho; Park, Sumin; Nam, Jihye; Kang, Youngjong; Kim, Jong-Man

    2016-01-27

    The fabrication of patterned conjugated polymer images on solid substrates has gained significant attention recently. Office inkjet printers can be used to generate flexible designs of functional materials on substrates on a large scale and in an inexpensive manner. Although creating patterns of conjugated polymers on paper using common office inkjet printers has been reported, only a few examples exist, such as polyaniline (PANI) and poly(3,4-ethylenedioxythiophene) (PEDOT), because only water-compatible inks can be utilized. Herein, we describe the production of poly(phenylenevinylene) (PPV) patterns on paper by employing a reactive inkjet printing (RIJ) method. In this process, printing of a hydrophilic terephthaldehyde, bis(triphenylphosphonium salt) and potassium t-butoxide using a common office inkjet printer leads to formation PPV patterns as a consequence of an in situ Wittig reaction. In addition, microarrayed PPV patterns are also readily generated on solid substrates, such as glass and PDMS, when a piezoelectric dispenser system is employed. The in situ prepared PPV was found to be insoluble in water and chloroform. As a result, unreacted excess reagents and byproducts can be efficiently removed by washing with these solvents. PMID:26731170

  2. Blood compatibility assessment of polymers used in drug eluting stent coatings.

    PubMed

    Szott, Luisa Mayorga; Irvin, Colleen A; Trollsas, Mikael; Hossainy, Syed; Ratner, Buddy D

    2016-06-01

    Differences in thrombosis rates have been observed clinically between different drug eluting stents. Such differences have been attributed to numerous factors, including stent design, injury created by the catheter delivery system, coating application technologies, and the degree of thrombogenicity of the polymer. The relative contributions of these factors are generally unknown. This work focuses on understanding the thrombogenicity of the polymer by examining mechanistic interactions with proteins, human platelets, and human monocytes of a number of polymers used in drug eluting stent coatings, in vitro. The importance for blood interactions of adsorbed albumin and the retention of albumin was suggested by the data. Microscopic imaging and immunostaining enhanced the interpretation of results from the lactate dehydrogenase cell counting assay and provided insight into platelet interactions, total quantification, and morphometry. In particular, highly spread platelets may be surface-passivating, possibly inhibiting ongoing thrombotic events. In many of the assays used here, poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) showed a differentiated protein deposition pattern that may contribute to the explanation of the consistently thromboresistant blood-materials interaction for fluororpolymers cited in literature. These results are supportive of one of several possible factors contributing to the good thromboresistant clinical safety performance of PVDF-HFP coated drug eluting stents. PMID:27083991

  3. Water-compatible halloysite-imprinted polymer by Pickering emulsion polymerization for the selective recognition of herbicides.

    PubMed

    Zhou, Chengyun; Li, Huan; Zhou, Hui; Wang, Hui; Yang, Pengjie; Zhong, Shian

    2015-05-01

    A water-compatible molecularly imprinted polymer was prepared by Pickering emulsion polymerization using halloysite nanotubes as stabilized solid particles. During polymerization, we used 4-vinylpyridine as monomer, divinylbenzene as cross-linking agent, toluene as porogen, 2,2-azobisisobutyronitrile as initiator, 2,4-dichlorophenoxyacetic acid as template to form the oil phase, and Triton X-100 aqueous solution to form the water phase. The halloysite nanotubes molecularly imprinted polymer was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Kinetic and equilibrium bindings were also employed to evaluate the adsorption properties of the imprinted polymer. The imprinted polymer showed better selectivity, more rapid kinetic binding (60 min) for 2,4-dichlorophenoxyacetic acid in pure water compared with rebinding in toluene. The imprinted polymer was used as a sorbent to enrich and separate 2,4-dichlorophenoxyacetic acid from water, and was detected by high-performance liquid chromatography with UV detection. PMID:25650303

  4. Chemical virology: Packing polymers in protein cages

    NASA Astrophysics Data System (ADS)

    Cornelissen, Jeroen J. L. M.

    2012-10-01

    The combination of addressable synthetic macromolecules with proteins of precise structure and function often leads to materials with unique properties, as is now shown by the efficient multi-site initiation of polymer growth inside the cavity of a virus capsid.

  5. A New Hidden Markov Model for Protein Quality Assessment Using Compatibility Between Protein Sequence and Structure

    PubMed Central

    He, Zhiquan; Ma, Wenji; Zhang, Jingfen; Xu, Dong

    2015-01-01

    Protein structure Quality Assessment (QA) is an essential component in protein structure prediction and analysis. The relationship between protein sequence and structure often serves as a basis for protein structure QA. In this work, we developed a new Hidden Markov Model (HMM) to assess the compatibility of protein sequence and structure for capturing their complex relationship. More specifically, the emission of the HMM consists of protein local structures in angular space, secondary structures, and sequence profiles. This model has two capabilities: (1) encoding local structure of each position by jointly considering sequence and structure information, and (2) assigning a global score to estimate the overall quality of a predicted structure, as well as local scores to assess the quality of specific regions of a structure, which provides useful guidance for targeted structure refinement. We compared the HMM model to state-of-art single structure quality assessment methods OPUSCA, DFIRE, GOAP, and RW in protein structure selection. Computational results showed our new score HMM.Z can achieve better overall selection performance on the benchmark datasets. PMID:26221066

  6. Polymer-Oxygen Compatibility Testing: Effect of Oxygen Aging on Ignition and Combustion Properties

    NASA Technical Reports Server (NTRS)

    Waller, Jess M.; Haas, Jon P.; Wilson, D. Bruce; Fries, Joseph (Technical Monitor)

    2000-01-01

    The oxygen compatibility of six polymers used in oxygen service was evaluated after exposure for 48 hours to oxygen pressures ranging from 350 to 6200 kPa (50 to 900 psia), and temperatures ranging from 50 to 250 C (122 to 302 F). Three elastomers were tested: CR rubber (C873-70), FKM fluorocarbon rubber (Viton A), and MPQ silicone rubber (MIL-ZZ-765, Class 2); and three thermoplastics were tested: polyhexamethylene adipamide (Zytel 42), polytetrafluoroethylene (Teflon TFE), and polychlorotrifluoroethylene (Neoflon CTFE M400H). Post-aging changes in mass, dimensions, tensile strength, elongation at break, and durometer hardness were determined. Also, the compression set was determined for the three elastomers. Results show that the properties under investigation were more sensitive to oxygen pressure at low to moderate temperatures, and more sensitive to temperature at low to moderate oxygen pressures. Inspection of the results also suggested that both chain scissioning and cross-linking processes were operative, consistent with heterogeneous oxidation. Attempts are underway to verify conclusively the occurrence of heterogeneous oxidation using a simple modulus profiling technique. Finally, the effect of aging at 620 kpa (90 psia) and 121 C (250 F) on ignition and combustion resistance was determined. As expected, aged polymers were less ignitable and combustible (had higher AlTs and lower heats of combustion). Special attention was given to Neoflon CTFE. More specifically, the effect of process history (compression versus extrusion molding) and percent crystallinity (quick- versus slow-quenched) on the AIT, heat of combustion, and impact sensitivity of Neoflon CTFE was investigated. Results show the AIT, heat of combustion, and impact sensitivity to be essentially independent of Neoflon CTFE process history and structure.

  7. Approach for achieving flame retardancy while retaining physical properties in a compatible polymer matrix

    NASA Technical Reports Server (NTRS)

    Williams, Martha K. (Inventor); Smith, Trent M. (Inventor)

    2011-01-01

    The invention provides polymer blends containing polyhydroxyamide and one or more flammable polymers. The polymer blends are flame retardant and have improved durability and heat stability compared to the flammable polymer portion of the blends. Articles containing the polymer blends are also provided.

  8. Approach for achieving flame retardancy while retaining physical properties in a compatible polymer matrix

    NASA Technical Reports Server (NTRS)

    Williams, Martha K. (Inventor); Smith, Trent M. (Inventor)

    2007-01-01

    The invention provides polymer blends containing polyhydroxyamide and one or more flammable polymers. The polymer blends are flame retardant and have improved durability and heat stability compared to the flammable polymer portion of the blends. Articles containing the polymer blends are also provided.

  9. Magnetic Resonance Imaging Compatibility of the Polymer-based Cochlear Implant

    PubMed Central

    Kim, Jin Ho; Min, Kyou Sik; An, Soon Kwan; Jeong, Joon Soo; Jun, Sang Beom; Cho, Min Hyoung; Son, Young-Don; Cho, Zang-Hee

    2012-01-01

    Objectives In this study, we compared the magnetic resonance (MR) image artifacts caused by a conventional metal-based cochlear implant and a newly developed liquid crystal polymer (LCP)-based device. Methods The metal-based cochlear implant system (Nurobiosys Co.) was attached to side of the head of a subject and the LCP-based device was attached to opposite side. In both devices, alignment magnets were removed for safety. Magnetic resonance imaging (MRI) was performed on a widely used 3.0 T and an ultra-high 7.0 T MRI machine. 3.0 and 7.0 T MR images were acquired using T1- and T2*-weighted gradient echo sequences, respectively. Results In the 3.0 T images, the metal-based device on the left side generated the significant amount of artifacts. The MR images in the proximity of the metal package were obscured by the artifacts in both axial and sagittal views. On the other hand, the MR images near the LCP-based device were relatively free from the artifacts and clearly showed the brain structures. 7.0 T MR images showed the more severe distortion in the both sides but the metal-based cochlear implant system caused a much larger obscure area than the LCP-based system. Conclusion The novel LCP-based cochlear implant provides a good MRI compatibility beyond present-day cochlear implants. Thus, MR images can be obtained from the subjects even with the implanted LCP-based neural prosthetic systems providing useful diagnostic information. Furthermore, it will be also useful for functional MRI studies of the auditory perception mechanism after cochlear implantations as well as for positron emission tomography-MRI hybrid imaging. PMID:22701769

  10. Effect of polymer surface modification on polymer-protein interaction via hydrophilic polymer grafting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Surface modification of flat sheet ultrafiltration membranes, polyethersulfone (PES) was investigated to improve the hydrophilicity of the membrane surface thereby reducing adsorption of the proteins onto the membrane. Grafting of hydrophilic polymers onto UV/ozone treated PES was used to improve t...

  11. A review of the fundamentals of polymer-modified asphalts: Asphalt/polymer interactions and principles of compatibility.

    PubMed

    Polacco, Giovanni; Filippi, Sara; Merusi, Filippo; Stastna, George

    2015-10-01

    During the last decades, the number of vehicles per citizen as well as the traffic speed and load has dramatically increased. This sudden and somehow unplanned overloading has strongly shortened the life of pavements and increased its cost of maintenance and risks to users. In order to limit the deterioration of road networks, it is necessary to improve the quality and performance of pavements, which was achieved through the addition of a polymer to the bituminous binder. Since their introduction, polymer-modified asphalts have gained in importance during the second half of the twentieth century, and they now play a fundamental role in the field of road paving. With high-temperature and high-shear mixing with asphalt, the polymer incorporates asphalt molecules, thereby forming a swallowed network that involves the entire binder and results in a significant improvement of the viscoelastic properties in comparison with those of the unmodified binder. Such a process encounters the well-known difficulties related to the poor solubility of polymers, which limits the number of macromolecules able to not only form such a structure but also maintain it during high-temperature storage in static conditions, which may be necessary before laying the binder. Therefore, polymer-modified asphalts have been the subject of numerous studies aimed to understand and optimize their structure and storage stability, which gradually attracted polymer scientists into this field that was initially explored by civil engineers. The analytical techniques of polymer science have been applied to polymer-modified asphalts, which resulted in a good understanding of their internal structure. Nevertheless, the complexity and variability of asphalt composition rendered it nearly impossible to generalize the results and univocally predict the properties of a given polymer/asphalt pair. The aim of this paper is to review these aspects of polymer-modified asphalts. Together with a brief description of

  12. Fluorinated amino acids: compatibility with native protein structures and effects on protein-protein interactions.

    PubMed

    Salwiczek, Mario; Nyakatura, Elisabeth K; Gerling, Ulla I M; Ye, Shijie; Koksch, Beate

    2012-03-21

    Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids--aliphatic, aromatic, charged and polar as well as proline--is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references). PMID:22130572

  13. Molecular cloning of protein-based polymers.

    PubMed

    Mi, Lixin

    2006-07-01

    Protein-based biopolymers have become a promising class of materials for both biomedical and pharmaceutical applications, as they have well-defined molecular weights, monomer compositions, as well as tunable chemical, biological, and mechanical properties. Using standard molecular biology tools, it is possible to design and construct genes encoding artificial proteins or protein-based polymers containing multiple repeats of amino acid sequences. This article reviews some of the traditional methods used for constructing DNA duplexes encoding these repeat-containing genes, including monomer generation, concatemerization, iterative oligomerization, and seamless cloning. A facile and versatile method, called modules of degenerate codons (MDC), which uses PCR and codon degeneracy to overcome some of the disadvantages of traditional methods, is introduced. Re-engineering of the random coil spacer domain of a bioactive protein, WPT2-3R, is used to demonstrate the utility of the MDC method. MDC re-constructed coding sequences facilitate further manipulations, such as insertion, deletion, and swapping of various sequence modules. A summary of some promising emerging techniques for synthesizing repetitive sequence-containing artificial proteins is also provided. PMID:16827576

  14. Water-compatible molecularly imprinted polymers for selective solid phase extraction of dencichine from the aqueous extract of Panax notoginseng.

    PubMed

    Ji, Wenhua; Xie, Hongkai; Zhou, Jie; Wang, Xiao; Ma, Xiuli; Huang, Luqi

    2016-01-01

    Specific molecularly imprinted polymers for dencichine were developed for the first time in this study by the bulk polymerization using phenylpyruvic acid and dl-tyrosine as multi-templates. The photographs confirmed that molecularly imprinted polymers prepared using N,N'-methylene diacrylamide as cross-linker and glycol dimethyl ether as porogen displayed excellent hydrophilicity. Selectivity, adsorption isotherm and adsorption kinetics were investigated. The sample loading-washing-eluting solvent was optimized to evaluate the property of molecularly imprinted solid phase extract. Compared with LC/WCX-SPE, water-compatible molecularly imprinted solid phase extraction displayed more excellent specific adsorption performance. The extracted dencichine from Panax notoginseng with the purity of 98.5% and the average recovery of 85.6% (n=3) was obtained. PMID:26680322

  15. Protein Chips Compatible with MALDI Mass Spectrometry Prepared by Ambient Ion Landing.

    PubMed

    Pompach, Petr; Benada, Oldřich; Rosůlek, Michal; Darebná, Petra; Hausner, Jiří; Růžička, Viktor; Volný, Michael; Novák, Petr

    2016-09-01

    We present a technology that allows the preparation of matrix-assisted laser desorption/ionization (MALDI)-compatible protein chips by ambient ion landing of proteins and successive utilization of the resulting protein chips for the development of bioanalytical assays. These assays are based on the interaction between the immobilized protein and the sampled analyte directly on the protein chip and subsequent in situ analysis by MALDI mass spectrometry. The electrosprayed proteins are immobilized on dry metal and metal oxide surfaces, which are nonreactive under normal conditions. The ion landing of electrosprayed protein molecules is performed under atmospheric pressure by an automated ion landing apparatus that can manufacture protein chips with a predefined array of sample positions or any other geometry of choice. The protein chips prepared by this technique are fully compatible with MALDI ionization because the metal-based substrates are conductive and durable enough to be used directly as MALDI plates. Compared to other materials, the nonreactive surfaces show minimal nonspecific interactions with chemical species in the investigated sample and are thus an ideal substrate for selective protein chips. Three types of protein chips were used in this report to demonstrate the bioanalytical applications of ambient ion landing. The protein chips with immobilized proteolytic enzymes showed the usefulness for fast in situ peptide MALDI sequencing; the lectin-based protein chips showed the ability to enrich glycopeptides from complex mixtures with subsequent MALDI analysis, and the protein chips with immobilized antibodies were used for a novel immunoMALDI workflow that allowed the enrichment of antigens from the serum followed by highly specific MALDI detection. PMID:27478994

  16. NEXT GENERATION SOLVENT-MATERIALS COMPATIBILITY WITH POLYMER COMPONENTS WITHIN MODULAR CAUSTIC-SIDE SOLVENT EXTRACTION UNIT (FINAL REPORT)

    SciTech Connect

    Fondeur, F.; Peters, T.; Fink, S.

    2012-01-17

    The Office of Waste Processing, within the Office of Technology Innovation and Development, is funding the development of an enhanced solvent for deployment at the Savannah River Site for removal of cesium from High Level Waste. The technical effort is collaboration between Oak Ridge National Laboratory (ORNL), Savannah River National Laboratory (SRNL), and Argonne National Laboratory. The first deployment target for the technology is within the Modular Caustic-Side Solvent Extraction Unit (MCU). Deployment of a new chemical within an existing facility requires verification that the chemical components are compatible with the installed equipment. In the instance of a new organic solvent, the primary focus is on compatibility of the solvent with organic polymers used in the facility. This report provides the data from exposing these polymers to the Next Generation Solvent (NGS). The test was conducted over six months. An assessment of the dimensional stability of polymers present in MCU (i.e., PEEK, Grafoil, Tefzel and Isolast) in the modified NGS (where the concentration of LIX{reg_sign}79 and MaxCalix was varied systematically) showed that LIX{reg_sign}79 selectively affected Tefzel and its different grades (by an increase in size and lowering its density). The copolymer structure of Tefzel and possibly its porosity allows for the easier diffusion of LIX{reg_sign}79. Tefzel is used as the seat material in some of the valves at MCU. Long term exposure to LIX{reg_sign}79, may make the valves hard to operate over time due to the seat material (Tefzel) increasing in size. However, since the physical changes of Tefzel in the improved solvent are comparable to the changes in the CSSX baseline solvent, no design changes are needed with respect to the Tefzel seating material. PEEK, Grafoil and Isolast were not affected by LIX{reg_sign}79 and MaxCalix within six months of exposure. The initial rapid weight gain observed in every polymer is assigned to the finite and

  17. Compatibility of Astragalus and Salvia extract inhibits myocardial fibrosis and ventricular remodeling by regulation of protein kinase D1 protein

    PubMed Central

    Mao, Bingyu; Nuan, Liu; Yang, Lei; Zeng, Xiaotao

    2015-01-01

    Aims: This study is to determine the effect of astragalus and salvia extract on the alteration of myocardium in a rat model of myocardial infarction. Methods: A total of 40 male Sprague-Dawley rats were randomly divided into the sham-operated group, the control group, the Astragalus group, the Salvia group, and the compatibility of Astragalus and Salvia and group. The cardiac functions were determined at 8 weeks after treatment. Hematoxylin-eosin staining was performed to observe the morphology and arrangement of cardiomyocytes. Masson’s trichrome staining was performed to investigate the distribution of myocardial interstitial collagen. Immunohistochemical staining was performed to determine the expression ofprotein kinase D1 in myocardial tissues. Results: In the sham-operated group, the Astragalus group, the Salvia group, and the compatibility of Astragalus and Salvia group, the left ventricular systolic pressure and the maximum rate of left ventricular pressure were significantly increased while the left ventricular end diastolic pressure were significantly decreased when compared with those in the control group (P < 0.05). Normal morphology and arrangement of cardiomyocytes were maintained in the compatibility of Astragalus and Salvia group. Contents of collagen fibers in myocardial tissues were decreased in the compatibility of Astragalus and Salvia group (P < 0.05). Expression levels of protein kinase D1 were significantly decreased in cardiomyocytes of the compatibility of Astragalus and Salvia group. Conclusions: Compatibility of Astragalus and Salvia extract may inhibit myocardial fibrosis and ventricular remodeling by regulation of protein kinase D1 protein in a rat model of myocardial infarction. PMID:26064267

  18. Emerging Synthetic Techniques for Protein-Polymer Conjugations

    PubMed Central

    Broyer, Rebecca M.; Grover, Gregory N.; Maynard, Heather D.

    2011-01-01

    Protein-polymer conjugates are important in diverse fields including drug delivery, biotechnology, and nanotechnology. This feature article highlights recent advances in the synthesis and application of protein-polymer conjugates by controlled radical polymerization techniques. Special emphasis on new applications of the materials, particularly in biomedicine, are highlighted. PMID:21229146

  19. Advances in Polymer and Polymeric Nanostructures for Protein Conjugation

    PubMed Central

    González-Toro, Daniella C.; Thayumanavan, S.

    2013-01-01

    Linear polymers have been considered the best molecular structures for the formation of efficient protein conjugates due to their biological advantages, synthetic convenience and ease of functionalization. In recent years, much attention has been dedicated to develop synthetic strategies that produce the most control over protein conjugation utilizing linear polymers as scaffolds. As a result, different conjugate models, such as semitelechelic, homotelechelic, heterotelechelic and branched or star polymer conjugates, have been obtained that take advantage of these well-controlled synthetic strategies. Development of protein conjugates using nanostructures and the formation of said nanostructures from protein-polymer bioconjugates are other areas in the protein bioconjugation field. Although several polymer-protein technologies have been developed from these discoveries, few review articles have focused on the design and function of these polymers and nanostructures. This review will highlight some recent advances in protein-linear polymer technologies that employ protein covalent conjugation and successful protein-nanostructure bioconjugates (covalent conjugation as well) that have shown great potential for biological applications. PMID:24058205

  20. Physical properties and blood compatibility of surface-modified segmented polyurethane by semi-interpenetrating polymer networks with a phospholipid polymer.

    PubMed

    Morimoto, Nobuyuki; Iwasaki, Yasuhiko; Nakabayashi, Nobuo; Ishihara, Kazuhiko

    2002-12-01

    Segmented polyurethanes, (SPU)s, are widely used in the biomedical fields because of their excellent mechanical property. However, when blood is in contact with the SPU, non-specific biofouling on the SPU occurs which reduces its mechanical property. To obtain novel blood compatible elastomers, the surface of the SPU was modified with 2-methacryloyloxyethyl phosphorylcholine (MPC) by forming a semi-interpenetrating polymer network (semi-IPN). The SPU film modified by MPC polymer with the semi-IPN (MS-IPN film) was prepared by visible light irradiation of the SPU film in which the monomers were diffused. X-ray photoelectron spectroscopy confirmed that the MPC units were exposed on the MS-IPN film surface. The mechanical properties of the MS-IPN film characterized by tensile testing were similar to those of the SPU film. Platelet adhesion on MS-IPN films was also investigated before and after stress loading to determine the effects of the surface modification on the blood compatibility. Many platelets did adhere on the SPU film before and after stress loading. On the other hand, the MS-IPN film prevented platelet adhesion even after repeated stress loading. PMID:12361629

  1. Chemical compatibility of PU/PAN interpenetrating polymer network membrane with substituted aromatic solvents.

    PubMed

    Kumar, H; Siddaramaiah

    2007-09-01

    Polyethylene glycol (PEG)-based polyurethane/polyacrylonitrile (PU/PAN, 50/50) semi-interpenetrating polymer network (SIPN) membrane has been studied from sorption/desorption cycles and diffusion behaviour with substituted aromatic probe molecules at 20, 40 and 60 degrees C. Sorption/desorption cycles have been repeated to evaluate polymer-solvent interaction. Organic solvents taken up or given out by IPN are measured periodically till equilibrium. Using these data, sorption (S), diffusion (D) and permeation (P) coefficients have been calculated from Fick's equation. Sorption data is correlated with solubility parameter of solvents and polymer. It was found that solvents of comparable solubility parameter with IPN interact more and thus there is an increase in sorption. Molecular mass between cross-link has been calculated using Flory Rehner equation. The cross-link density and degree of cross-linking of the membrane is calculated. From the temperature dependence of sorption and diffusion coefficients, the Arrhenius activation parameters like activation energy for diffusion (E(D)) and permeation (E(P)) processes have been calculated. Furthermore, the sorption results have been interpreted in terms of thermodynamic parameters such as change in enthalpy (DeltaH) and entropy (DeltaS). Concentration profiles of penetrants at different penetration depths in the polymer sample at different time intervals have also been calculated theoretically from a solution of Fick's equation under appropriate initial boundary conditions. PMID:17418943

  2. Compatibility and Impact Resistance of Biodegradable Polymer Blends Using Clays and Natural Nanotubes

    NASA Astrophysics Data System (ADS)

    Guo, Yichen; Yuan, Xue; Zuo, Xianghao; Rafailovich, Miriam

    Montmorillonite clays and Halloysite nanotubes (HNTs) were modified by surface adsorption of resorcinol di (phenyl phosphate) (RDP) oligomers. Biodegradable poly (lactic acid) (PLA) and poly (butylene adipate-co-butylene terephthalate) (PBAT) polymers were blended together with RDP coated clays and tubes. TEM images of thin sections indicated that even though both RDP coated clay nanotubes and platelets located on the interfacial region between two immiscible polymers, only the platelets, having the larger aspect ratio, were able to reduce the PBAT domain sizes. The ability of clay platelets to partially compatibilize the blend was further confirmed by the dynamic mechanical analysis (DMA) which showed that the glass transition temperatures of two polymers tend to shift closer. Izod impact testing demonstrated that the rubbery PBAT phase greatly increased the impact strength of the unfilled blend, but addition of only 5% of clay filler decrease the impact strength by nearly 50% while a small increase was observed with nanotubes at that concentration. A simple model is proposed. The clay platelets are observed to cover the interfacial area. Although they are effective at reducing the interfacial tension, they block the entanglements between two polymer phase and increase the overall brittleness. On the other hand, the HNTs are observed to lie perpendicular to the interface, which makes them less effective in reducing interfacial tension, but far more effective at retarding micro-crack propagation.

  3. Improved thermal cycling durability and PCR compatibility of polymer coated quantum dot

    NASA Astrophysics Data System (ADS)

    Xun, Zhe; Zhao, Xiaoyun; Guan, Yifu

    2013-09-01

    Quantum dots have experienced rapid development in imaging, labeling and sensing in medicine and life science. To be suitable for polymerase chain reaction (PCR) assay, we have tested QD thermal cycling durability and compatibility, which have not been addressed in previous reports. In this study, we synthesized CdSe/ZnS QDs with a surface modification with high-MW amphiphilic copolymers and observed that Mg2+ ions in the PCR reaction could induce the QDs to precipitate and reduce their fluorescence signal significantly after thermal cycling. To overcome this problem, we used mPEG2000 to conjugate the QD surface for further protection, and found that this modification enables QDs to endure 40 thermal cycles in the presence of other components essential for PCR reactions. We have also identified that QDs have different effects on rTaq and Ex Taq polymerization systems. A high QD concentration could apparently reduce the PCR efficiency, but this inhibition was relieved significantly in the Ex PCR system as the concentration of Ex Taq polymerase was increased. Real-time PCR amplification results showed that QDs could provide a sufficiently measurable fluorescence signal without excessively inhibiting the DNA amplification. Based on this improved thermal cycling durability and compatibility with the PCR system, QDs have the potential to be developed as stable fluorescent sensors in PCR and real-time PCR amplification.

  4. Improved thermal cycling durability and PCR compatibility of polymer coated quantum dot.

    PubMed

    Xun, Zhe; Zhao, Xiaoyun; Guan, Yifu

    2013-09-01

    Quantum dots have experienced rapid development in imaging, labeling and sensing in medicine and life science. To be suitable for polymerase chain reaction (PCR) assay, we have tested QD thermal cycling durability and compatibility, which have not been addressed in previous reports. In this study, we synthesized CdSe/ZnS QDs with a surface modification with high-MW amphiphilic copolymers and observed that Mg²⁺ ions in the PCR reaction could induce the QDs to precipitate and reduce their fluorescence signal significantly after thermal cycling. To overcome this problem, we used mPEG2000 to conjugate the QD surface for further protection, and found that this modification enables QDs to endure 40 thermal cycles in the presence of other components essential for PCR reactions. We have also identified that QDs have different effects on rTaq and Ex Taq polymerization systems. A high QD concentration could apparently reduce the PCR efficiency, but this inhibition was relieved significantly in the Ex PCR system as the concentration of Ex Taq polymerase was increased. Real-time PCR amplification results showed that QDs could provide a sufficiently measurable fluorescence signal without excessively inhibiting the DNA amplification. Based on this improved thermal cycling durability and compatibility with the PCR system, QDs have the potential to be developed as stable fluorescent sensors in PCR and real-time PCR amplification. PMID:23924819

  5. Water-Stable Metal-Organic Framework/Polymer Composites Compatible with Human Hepatocytes.

    PubMed

    Neufeld, Megan J; Ware, Brenton R; Lutzke, Alec; Khetani, Salman R; Reynolds, Melissa M

    2016-08-01

    Metal-organic frameworks (MOFs) have demonstrated promise in biomedical applications as vehicles for drug delivery, as well as for the ability of copper-based MOFs to generate nitric oxide (NO) from endogenous S-nitrosothiols (RSNOs). Because NO is a participant in biological processes where it exhibits anti-inflammatory, antibacterial, and antiplatelet activation properties, it has received significant attention for therapeutic purposes. Previous work has shown that the water-stable MOF H3[(Cu4Cl)3-(BTTri)8] (H3BTTri = 1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene), or CuBTTri, produces NO from RSNOs and can be included within a polymeric matrix to form NO-generating materials. While such materials demonstrate potential, the possibility of MOF degradation leading to copper-related toxicity is a concern that must be addressed prior to adapting these materials for biomedical applications. Herein, we present the first cytotoxicity evaluation of an NO-generating CuBTTri/polymer composite material using 3T3-J2 murine embryonic fibroblasts and primary human hepatocytes (PHHs). CuBTTri/polymer films were prepared from plasticized poly(vinyl chloride) (PVC) and characterized via PXRD, ATR-FTIR, and SEM-EDX. Additionally, the ability of the CuBTTri/polymer films to enhance NO generation from S-nitroso-N-acetylpenicillamine (SNAP) was evaluated. Enhanced NO generation in the presence of the CuBTTri/polymer films was observed, with an average NO flux (0.90 ± 0.13 nmol cm(-2) min(-1)) within the range associated with antithrombogenic surfaces. The CuBTTri/polymer films were analyzed for stability in phosphate buffered saline (PBS) and cell culture media under physiological conditions for a 4 week duration. Cumulative copper release in both cell media (0.84 ± 0.21%) and PBS (0.18 ± 0.01%) accounted for less than 1% of theoretical copper present in the films. In vitro cell studies performed with 3T3-J2 fibroblasts and PHHs did not indicate significant toxicity, providing further

  6. Blood-Compatible Polymer for Hepatocyte Culture with High Hepatocyte-Specific Functions toward Bioartificial Liver Development.

    PubMed

    Hoshiba, Takashi; Otaki, Takayuki; Nemoto, Eri; Maruyama, Hiroka; Tanaka, Masaru

    2015-08-19

    The development of bioartificial liver (BAL) is expected because of the shortage of donor liver for transplantation. The substrates for BAL require the following criteria: (a) blood compatibility, (b) hepatocyte adhesiveness, and (c) the ability to maintain hepatocyte-specific functions. Here, we examined blood-compatible poly(2-methoxyethyl acrylate) (PMEA) and poly(tetrahydrofurfuryl acrylate) (PTHFA) (PTHFA) as the substrates for BAL. HepG2, a human hepatocyte model, could adhere on PMEA and PTHFA substrates. The spreading of HepG2 cells was suppressed on PMEA substrates because integrin contribution to cell adhesion on PMEA substrate was low and integrin signaling was not sufficiently activated. Hepatocyte-specific gene expression in HepG2 cells increased on PMEA substrate, whereas the expression decreased on PTHFA substrates due to the nuclear localization of Yes-associated protein (YAP). These results indicate that blood-compatible PMEA is suitable for BAL substrate. Also, PMEA is expected to be used to regulate cell functions for blood-contacting tissue engineering. PMID:26258689

  7. Investigation of Polymer Resin/Fiber Compatibility in Natural Fiber Reinforced Composite Automotive Materials

    SciTech Connect

    Fifield, Leonard S.; Huang, Cheng; Simmons, Kevin L.

    2010-01-01

    Natural fibers represent a lower density and potentially lower cost alternative to glass fibers for reinforcement of polymers in automotive composites. The high specific modulus and strength of bast fibers make them an attractive option to replace glass not only in non-structural automotive components, but also in semi-structural and structural components. Significant barriers to insertion of bast fibers in the fiber reinforced automotive composite market include the high moisture uptake of this lignocellulosic material relative to glass and the weak inherent interface between natural fibers and automotive resins. This work seeks to improve the moisture uptake and resin interfacing properties of natural fibers through improved fundamental understanding of fiber physiochemical architecture and development of tailored fiber surface modification strategies.

  8. Synthesis of Maleimide-End Functionalized Star Polymers and Multimeric Protein-Polymer Conjugates

    PubMed Central

    Tao, Lei; Kaddis, Catherine S.; Loo, Rachel R. Ogorzalek; Grover, Gregory N.; Loo, Joseph A.; Maynard, Heather D.

    2011-01-01

    Protein-polymer conjugates exhibit superior properties to unmodified proteins, generating a high demand for these materials in the fields of medicine, biotechnology, and nanotechnology. Multimeric conjugates are predicted to surpass the activity of monomeric conjugates. Herein, we report a straightforward method to synthesize multimeric polymer-conjugates. Four armed poly(N-isopropylacrylamide) (pNIPAAm) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization in the presence of a tetra-functionalized trithiocarbonate chain transfer agent (CTA). The polymer molecular weight, architecture and polydispersity index (PDI) were verified by gel permeation chromatography (GPC), dynamic light scattering gel permeation chromatography (DLS-GPC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This approach afforded well-defined polymers (PDI's < 1.06) and the ability to target various molecular weights. Maleimide functional groups were introduced at the chain ends by heating the polymers in the presence of a furan-protected azo-initiator. This allowed for site-specific conjugation of V131C T4 lysozyme to the polymers to generate multimeric protein-polymer conjugates. MALDI-TOF mass spectrometry, electrospray ionization gas-phase electrophoretic-mobility macromolecule analysis (ESI-GEMMA), gel electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS) of the trypsin digests demonstrated that multimeric protein-polymer conjugates had formed. This simple strategy provides ready access to star protein-polymer conjugates for application in the fields of drug discovery, drug delivery, and nanotechnology. PMID:21544227

  9. Pretreatment of amphiphilic comb polymer surfaces dramatically affects protein adsorption.

    PubMed

    Zhang, Zhanping; Ma, Hongwei; Hausner, Douglas B; Chilkoti, Ashutosh; Beebe, Thomas P

    2005-01-01

    New applications in regenerative biotechnology require the ability to understand and control protein-surface interactions on micrometer and submicrometer length scales. Evidence presented here shows that micropatterned amphiphilic comb polymer films exhibit a pretreatment-dependent behavior with respect to protein adsorption for the proteins fibronectin, laminin, and for serum. A micropatterned surface, consisting of protein-reactive regions, separated by comb polymer, was created and tested for protein adsorption using the surface-sensitive imaging tool TOF-SIMS. Immersion of micropatterned surfaces in solutions of fibronectin or laminin resulted in uniform protein coverage on both the comb polymer and protein-reactive regions. However, preimmersion of similarly patterned surfaces in water for 2 h prior to protein incubation was found to dramatically improve the protein-resistant properties of the comb polymer regions. These results are consistent with poly(ethylene glycol) (PEG) side chain reorientation and/or hydration and poly(methyl methacrylate) (PMMA) backbone segregation away from the interface region. PMID:16283770

  10. Construction of monomer-free, highly crosslinked, water-compatible polymers.

    PubMed

    Dailing, E A; Lewis, S H; Barros, M D; Stansbury, J W

    2014-12-01

    Polymeric dental adhesives require the formation of densely crosslinked network structures to best ensure mechanical strength and durability in clinical service. Monomeric precursors to these materials typically consist of mixtures of hydrophilic and hydrophobic components that potentially undergo phase separation in the presence of low concentrations of water, which is detrimental to material performance and has motivated significant investigation into formulations that reduce this effect. We have investigated an approach to network formation based on nanogels that are dispersed in inert solvent and directly polymerized into crosslinked polymers. Monomers of various hydrophilic or hydrophobic characteristics were copolymerized into particulate nanogels bearing internal and external polymerizable functionality. Nanogel dispersions were stable at high concentrations in acetone or, with some exceptions, in water and produced networks with a wide range of mechanical properties. Networks formed rapidly upon light activation and reached high conversion with extremely low volumetric shrinkage. Prepolymerizing monomers into reactive nanostructures significantly changes how hydrophobic materials respond to water compared with networks obtained from polymerizations involving free monomer. The modulus of fully hydrated networks formed solely from nanogels was shown to equal or exceed the modulus in the dry state for networks based on nanogels containing a hydrophobic dimethacrylate and hydrophilic monomethacrylate, a result that was not observed in a hydroxyethyl methacrylate (HEMA) homopolymer or in networks formed from nanogels copolymerized with HEMA. These results highlight the unique approach to network development from nanoscale precursors and properties that have direct implications in functional dental materials. PMID:25248612

  11. Construction of Monomer-free, Highly Crosslinked, Water-compatible Polymers

    PubMed Central

    Dailing, E.A.; Lewis, S.H.; Barros, M.D.; Stansbury, J.W.

    2014-01-01

    Polymeric dental adhesives require the formation of densely crosslinked network structures to best ensure mechanical strength and durability in clinical service. Monomeric precursors to these materials typically consist of mixtures of hydrophilic and hydrophobic components that potentially undergo phase separation in the presence of low concentrations of water, which is detrimental to material performance and has motivated significant investigation into formulations that reduce this effect. We have investigated an approach to network formation based on nanogels that are dispersed in inert solvent and directly polymerized into crosslinked polymers. Monomers of various hydrophilic or hydrophobic characteristics were copolymerized into particulate nanogels bearing internal and external polymerizable functionality. Nanogel dispersions were stable at high concentrations in acetone or, with some exceptions, in water and produced networks with a wide range of mechanical properties. Networks formed rapidly upon light activation and reached high conversion with extremely low volumetric shrinkage. Prepolymerizing monomers into reactive nanostructures significantly changes how hydrophobic materials respond to water compared with networks obtained from polymerizations involving free monomer. The modulus of fully hydrated networks formed solely from nanogels was shown to equal or exceed the modulus in the dry state for networks based on nanogels containing a hydrophobic dimethacrylate and hydrophilic monomethacrylate, a result that was not observed in a hydroxyethyl methacrylate (HEMA) homopolymer or in networks formed from nanogels copolymerized with HEMA. These results highlight the unique approach to network development from nanoscale precursors and properties that have direct implications in functional dental materials. PMID:25248612

  12. Microscopic Model of the Metal-Organic Framework/Polymer Interface: A First Step toward Understanding the Compatibility in Mixed Matrix Membranes.

    PubMed

    Semino, Rocio; Ramsahye, Naseem A; Ghoufi, Aziz; Maurin, Guillaume

    2016-01-13

    An innovative computational methodology integrating density functional theory calculations and force field-based molecular dynamics simulations was developed to provide a first microscopic model of the interactions at the metal-organic framework (MOF) surface/polymer interface. This was applied to the case of the composite formed by the polymer of intrinsic microporosity, PIM-1, and the zeolitic imidazolate framework, ZIF-8, as a model system. We found that the structure of the composite at the interface is the result of both the chemical affinity between PIM-1 and ZIF-8 and the rigidity of the polymer. Specifically, there is a preferential interaction between the -CN groups of PIM-1 and the NH terminal functions of the organic linker at the ZIF-8 surface. Additionally, the resulting conformation of the polymer gives rise to interfacial microvoids at the vicinity of the MOF surface. The porosity, rigidity, and density of the interfacial polymer were analyzed and compared to those for the bulk polymer. It was shown that the polymer still feels the impact of the MOF surface even at long distances above 15-20 Å. Further, both the polydispersity of the polymer and the flexibility of the MOF surface were revealed to only slightly affect the properties of the MOF/interface. This work, which delivers a microscopic picture of the MOF surface/polymer interactions at the interface, would lead, in turn, to the understanding of the compatibility in MOF-based mixed-matrix membranes. PMID:26653765

  13. Water-compatible temperature and magnetic dual-responsive molecularly imprinted polymers for recognition and extraction of bisphenol A.

    PubMed

    Wu, Xiaqing; Wang, Xiaoyan; Lu, Wenhui; Wang, Xinran; Li, Jinhua; You, Huiyan; Xiong, Hua; Chen, Lingxin

    2016-02-26

    Versatile molecularly imprinted polymers (MIPs) have been widely applied to various sample matrices, however, molecular recognition in aqueous media is still difficult. Stimuli-responsive MIPs have received increasing attentions due to their unique feature that the molecular recognition is regulated by specific external stimuli. Herein, water-compatible temperature and magnetic dual-responsive MIPs (WC-TMMIPs) with hydrophilic brushes were prepared via reversible addition-fragmentation chain transfer precipitation polymerization for reversible and selective recognition and extraction of bisphenol A (BPA). Transmission electron microscopy (TEM), Fourier transform infrared spectrometer (FT-IR) and vibrating sample magnetometry (VSM) as characterization methods were used to examine the successful synthesis of polymers, and the resultant WC-TMMIPs showed excellent thermosensitivity and simple rapid magnetic separation. Controlled adsorption and release of BPA by temperature regulation were investigated systematically, and the maximum adsorption and removal efficiency toward BPA in aqueous solutions were attained at 35 °C and 45 °C, respectively, as well as a good recoverability was exhibited with the precision less than 5% through five adsorption-desorption cycles. Phenolic structural analogs were tested and good recognition specificity for BPA was displayed. Accordingly, the WC-TMMIPs were employed as adsorbents for magnetic solid-phase extraction (MSPE) and packed SPE of BPA from seawater samples. Using the two modes followed by HPLC-UV determination, excellent linearity was attained in the range of 0.1-14.5 μM and 1.3-125 nM, with low detection limits of 0.02 μM and 0.18 nM, respectively. Satisfactory recoveries for spiked seawater samples were achieved ranging from 86.3-103.5% and 96.2-104.3% with RSD within 2.12-4.33%. The intelligent WC-TMMIPs combining water-compatibility, molecular recognition, magnetic separation, and temperature regulation proved

  14. Applications of Polymer Brushes in Protein Analysis and Purification

    NASA Astrophysics Data System (ADS)

    Jain, Parul; Baker, Gregory L.; Bruening, Merlin L.

    2009-07-01

    This review examines the application of polymer brush-modified flat surfaces, membranes, and beads for protein immobilization and isolation. Modification of porous substrates with brushes yields membranes that selectively bind tagged proteins to give 99% pure protein at capacities as high as 100 mg of protein per cubic centimeter of membrane. Moreover, enrichment of phosphopeptides on brush-modified matrix-assisted laser desorption/ionization (MALDI) plates allows detection and characterization of femtomole levels of phosphopeptides by MALDI mass spectrometry. Because swollen hydrophilic brushes can resist nonspecific protein adsorption while immobilizing a high density of proteins, they are attractive as substrates for protein microarrays. This review highlights the advantages of polymer brush-modified surfaces over self-assembled monolayers and identifies some research needs in this area.

  15. Reinforcement effect of soy protein and carbohydrates in polymer composites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The modulus of soft polymer material can be increased by filler reinforcement. A review of using soy protein and carbohydrates as alternative renewable reinforcement material is presented here. Dry soy protein and carbohydrates are rigid and can form strong filler networks through hydrogen-bonding...

  16. Effect of film compatibility on electro-optic properties of dye doped polymer DR1/SU-8

    NASA Astrophysics Data System (ADS)

    Sun, Xiaoqiang; Xie, Ying; Zhao, Xuliang; Li, Dehui; Zhao, Shimin; Yue, Yuanbin; Wang, Xibin; Sun, Jian; Liang, Lei; Chen, Changming; Zhang, Daming; Wang, Fei; Xie, Zhiyuan

    2013-11-01

    The physic-chemical compatibility of passive cladding and poled Dispersed Red 1 (DR1) doped ultraviolet (UV) curable polymer SU-8 was investigated. The multilayer films consisting of DR1/SU-8 core and Norland Optical Adhensive 73 (NOA73), SU-8, polydimethylsiloxane (PDMS), or polymethylmethacrylate (PMMA) upper-cladding were fabricated on the silicon substrate, respectively. The interface morphologies were characterized through scan electronic microscope. Parallel plate electric field poling was carried out to align the polarity of chromophores in SU-8. The core-cladding interface with no chemical erosion or delamination was obtained by adopting an excess UV exposure and higher temperature dealing when NOA73 was used as the upper-cladding. The root mean square roughness of the upper-cladding surface was measured by atomic force microscope to verify the poling process. The electro-optic (EO) signal response amplitude of these multilayer films was used to characterize the polarizability alignment of DR1 chromophores by means of Teng-Man method after poling. Resistivity of claddings was measured at the glass transition temperature of DR1/SU-8 to explain the EO response difference. The configuration of NOA73/(DR1/SU-8) exhibited the best EO performance and time relaxation in amplitude within 550 h by prolonging the cooling time in poling process. A channel waveguide was fabricated to study the poling-induced optical loss. The results show that the selection of passive cladding with favorable electrical and chemical property is essential to establish optical nonlinearity in the dye-polymer system.

  17. Protein-based supramolecular polymers: progress and prospect.

    PubMed

    Luo, Quan; Dong, Zeyuan; Hou, Chunxi; Liu, Junqiu

    2014-09-11

    Proteins are naturally evolved macromolecules with highly sophisticated structures and diverse properties. The design and controlled self-assembly of proteins into polymeric architectures via supramolecular interactions offers unique advantages in understanding the spontaneously self-organisational process and fabrication of various bioactive materials. This feature article highlights recent advances and future trends in supramolecular polymers that are directly assembled from the building blocks of proteins. Non-covalent interactions capable of inducing polymerization include aromatic π-π stacking, host-guest interactions, metal coordination, and interprotein interactions combined with site-selective protein modification to explore the dynamic and specific unidirectional aggregation behaviours among protein units. We also discuss some extended supramolecular protein polymers achieved by rational design and fine-tuning the protein-protein interactions, which may help to inspire future design of more complicated polymeric protein assemblies. The protein-based supramolecular polymer system provides a versatile platform for functionalization and thereby shows great potential in the development of novel biomaterials with controlled structures and properties. PMID:25005829

  18. Elastic repulsion from polymer brush layers exhibiting high protein repellency.

    PubMed

    Inoue, Yuuki; Nakanishi, Tomoaki; Ishihara, Kazuhiko

    2013-08-27

    Hydrophilic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly(2-hydroxyethyl methacrylate) (PHEMA) brush layers with different thicknesses and graft densities were prepared to construct a model surface to elucidate protein-surface interactions. In particular, we focused on the steric repulsion of hydrophilic polymer layers as one of the surface properties that strongly influence protein adsorption and employed force-versus-distance (f-d) curve measurements obtained via atomic force microscopy to quantitatively evaluate the steric repulsion force, which is also referred to as the "elastic repulsion energy." We also analyzed direct interactions between the surface and proteins via the f-d curve, because these interactions trigger the protein-adsorption phenomenon. Protein-surface interactions were extremely suppressed at surfaces with high elastic repulsion energies and highly dense polymer brush structures, which is in contrast to those at surfaces with low elastic repulsion energies and low density of the grafted polymer layers. These results indicate that the elastic repulsion from the grafted polymer layer at the surface is an important parameter for controlling protein-surface interactions and protein adsorption phenomenon. PMID:23898820

  19. Translational Dynamics of Lipidated Ras Proteins in the Presence of Crowding Agents and Compatible Osmolytes.

    PubMed

    Patra, Satyajit; Erwin, Nelli; Winter, Roland

    2016-07-18

    Ras proteins are small GTPases and are involved in transmitting signals that control cell growth, differentiation, and proliferation. Since the cell cytoplasm is crowded with different macromolecules, understanding the translational dynamics of Ras proteins in crowded environments is crucial to yielding deeper insight into their reactivity and function. Herein, the translational dynamics of lipidated N-Ras and K-Ras4B is studied in the bulk and in the presence of a macromolecular crowder (Ficoll) and the compatible osmolyte and microcrowder sucrose by fluorescence correlation spectroscopy. The results reveal that N-Ras forms dimers due to the presence of its lipid moiety in the hypervariable region, whereas K-Ras4B remains in its monomeric form in the bulk. Addition of a macromolecular crowding agent gradually favors clustering of the Ras proteins. In 20 wt % Ficoll N-Ras forms trimers and K-Ras4B dimers. Concentrations of sucrose up to 10 wt % foster formation of N-Ras trimers and K-Ras dimers as well. The results can be rationalized in terms of the excluded-volume effect, which enhances the association of the proteins, and, for the higher concentrations, by limited-hydration conditions. The results of this study shed new light on the association state of these proteins in a crowded environment. This is of particular interest for the Ras proteins, because their solution state-monomeric or clustered-influences their membrane-partitioning behavior and their interplay with cytosolic interaction partners. PMID:27028423

  20. Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility.

    PubMed

    Jin, Li-Tai; Li, Xiao-Kun; Cong, Wei-Tao; Hwang, Sun-Young; Choi, Jung-Kap

    2008-12-15

    A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1h with a detection limit of 0.2 ng/band. PMID:18804088

  1. Encapsulation of Semiconducting Polymers in Vault Protein Cages

    SciTech Connect

    Ng, B.C.; Yu, M.; Gopal, A.; Rome, L.H.; Monbouquette, H.G.; Tolbert, S.H.

    2009-05-22

    We demonstrate that a semiconducting polymer [poly(2-methoxy-5-propyloxy sulfonate phenylene vinylene), MPS-PPV] can be encapsulated inside recombinant, self-assembling protein nanocapsules called 'vaults'. Polymer incorporation into these nanosized protein cages, found naturally at {approx}10,000 copies per human cell, was confirmed by fluorescence spectroscopy and small-angle X-ray scattering. Although vault cellular functions and gating mechanisms remain unknown, their large internal volume and natural prevalence within the human body suggests they could be used as carriers for therapeutics and medical imaging reagents. This study provides the groundwork for the use of vaults in encapsulation and delivery applications.

  2. Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences

    PubMed Central

    2016-01-01

    Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate–polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity

  3. Hydrophobic blocks facilitate lipid compatibility and translocon recognition of transmembrane protein sequences.

    PubMed

    Stone, Tracy A; Schiller, Nina; von Heijne, Gunnar; Deber, Charles M

    2015-02-24

    Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity in

  4. Cell mimetic lateral stabilization of outer cell mimetic bilayer on polymer surfaces by peptide bonding and their blood compatibility.

    PubMed

    Kaladhar, K; Sharma, Chandra P

    2006-10-01

    The biological lipid bilayer membranes are stabilized laterally with the help of integral proteins. We have simulated this with an optimized ternary phospholipid/glycolipid/cholesterol system, and stabilized laterally on functionalized poly methyl methacrylate (PMMA) surfaces, using albumin, heparin, and polyethylene glycol as anchors. We have earlier demonstrated the differences due to orientation and packing of the ternary phospholipid monolayers in relation to blood compatibility (Kaladhar and Sharma, Langmuir 2004;20:11115-11122). The structure of albumin is changed here to expose its interior hydrophobic core by treating with organic solvent. The interaction between the hydrophobic core of the albumin molecule and the hydrophobic core of the lipid molecules is confirmed by incorporating the molecule into bilayer membranes. The secondary structure of the membrane incorporated albumin is studied by CD spectral analysis. The structure of the altered albumin molecule contains more beta-sheet as compared to the native albumin. This conformation is also retained in membranes. The partitioning of the different anchors based on its polarity and ionic interactions in the monolayer is studied from the pressure-area (pi-A) isotherm of the lipid monolayers at the air/water interface using Langmuir-Blodgett (LB) trough facility. Such two monolayers are deposited onto the functionalized PMMA surface using LB trough and crosslinked by carbodiimide chemistry. The structure of the deposited bilayer is studied by depth analysis using contact mode AFM in dry conditions. The stabilized bilayer shows stability up to 1 month by contact angle studies. Preliminary blood compatibility studies reveal that the calcification, protein adsorption, as well as blood-cell adhesion is significantly reduced after the surface modification. The reduced adsorption of ions, proteins, and cells to the modified surfaces may be due to the fluidity of the microenvironment along with the contribution of

  5. Polymer and protein interfacial competition in a shell production process

    NASA Astrophysics Data System (ADS)

    Willard, Emma; Randall, Greg

    2015-11-01

    We are exploring oil-in-aqueous polymer compound droplet formulations to UV polymerize into shells while in a strong AC electric field (kV/cm, 20 MHz). The electric field drives the drops to adopt a concentric configuration so that a ``perfect'' spherical shell can be polymerized with a uniform wall thickness. In our previous study of oil-in-water droplet centering, we determined that droplet stretching in the electric field was a problem, which we overcame by using protein additives to strengthen the oil/water interface. However, adding polymer to the shell fluid has been shown to weaken the droplet interface and further complicates T junction droplet generation. In this work, we study the adsorption competition between bovine serum albumin and polyethylene glycol diacrylate with the pendant drop method to generate a polymer/protein shell formulation that will resist stretching in the centering electric field. Furthermore, we explore droplet generation of polymer/protein shell formulations in a double T junction and stretching in an electric field. Work supported by General Atomics IR&D funds.

  6. Chromatographic removal of endotoxin from protein solutions by polymer particles.

    PubMed

    Hirayama, Chuichi; Sakata, Masayo

    2002-12-01

    Endotoxins, constituents of cell walls of gram-negative bacteria, are potential contaminants of the protein solutions originating from biological products. Such contaminants have to be removed from solutions used for intravenous administration, because of their potent biological activities causing pyrogenic reactions. Separation methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. To remove endotoxin from a solution of high-molecular-mass compounds, such as proteins, the adsorption method has proven to be most effective. In this review, we first introduce endotoxin-specific properties in an aqueous solution, and then provide various methods of chromatographic separation of endotoxins from cellular products using polymer adsorbents. We also provide the design of novel endotoxin-specific polymer adsorbents. PMID:12450672

  7. Oriented immobilization of proteins on grafted porous polymers

    NASA Astrophysics Data System (ADS)

    Carbajal, M. Laura; Smolko, Eduardo E.; Grasselli, Mariano

    2003-08-01

    The modification of polymers by radiation grafting has been utilized for several decades. The penetrability of gamma rays allows to modify the internal surfaces of porous materials retaining its mechanical properties. In recent years applications of these materials to obtain chromatographic supports and biocatalysts have been reported. In this work, we described the grafting of glycidyl methacrylate (GMA) onto a macroporous polysulfone polymer. Reproducible amount of grafting, from 10% to 60% was obtained by choosing favourable monomer concentration and gamma radiation doses from 6 kGy up. Afterwards, iminodiacetic acid (IDA) and amino phenyl arsine oxide (PAO) were covalently attached to the grafted polyGMA, in correspondence with the grafting degree. Later on, a recombinant histidin-patch thioredoxin protein (HP-rTrx) was immobilized onto this surface by two different ways, involving specific protein orientations. The first one involves an IDA-Ni 2+ complex and three HP-rTrx's histidines and the other one involves a co-ordination site between PAO and two proximal HP-rTrx's cysteines, which corresponds to the active site of the enzyme. Specific polyclonal antibodies recognize HP-rTrx on the polymer. Proper orientation of the protein was confirmed by HP-rTrx activity measurements. The described procedure allows the successful oriented immobilization of a protein onto a macroporous polysulfone material.

  8. DMA Modulus as a Screening Parameter for Compatibility of Polymeric Containment Materials with Various Solutions for use in Space Shuttle Microgravity Protein Crystal Growth (PCG) Experiments

    NASA Technical Reports Server (NTRS)

    Wingard, Charles Doug; Munafo, Paul M. (Technical Monitor)

    2002-01-01

    Protein crystals are grown in microgravity experiments inside the Space Shuttle during orbit. Such crystals are basically grown in a five-component system containing a salt, buffer, polymer, organic and water. During these experiments, a number of different polymeric containment materials must be compatible with up to hundreds of different PCG solutions in various concentrations for durations up to 180 days. When such compatibility experiments are performed at NASA/MSFC (Marshall Space Flight Center) simultaneously on containment material samples immersed in various solutions in vials, the samples are rather small out of necessity. DMA4 modulus was often used as the primary screening parameter for such small samples as a pass/fail criterion for incompatibility issues. In particular, the TA Instruments DMA 2980 film tension clamp was used to test rubber O-rings as small in I.D. as 0.091 in. by cutting through the cross-section at one place, then clamping the stretched linear cord stock at each end. The film tension clamp was also used to successfully test short length samples of medical/surgical grade tubing with an O.D. of 0.125 in.

  9. Magnetic Surfactants and Polymers with Gadolinium Counterions for Protein Separations.

    PubMed

    Brown, Paul; Bromberg, Lev; Rial-Hermida, M Isabel; Wasbrough, Matthew; Hatton, T Alan; Alvarez-Lorenzo, Carmen

    2016-01-26

    New magnetic surfactants, (cationic hexadecyltrimethlyammonium bromotrichlorogadolinate (CTAG), decyltrimethylammonium bromotrichlorogadolinate (DTAG), and a magnetic polymer (poly(3-acrylamidopropyl)trimethylammonium tetrachlorogadolinate (APTAG)) have been synthesized by the simple mixing of the corresponding surfactants and polymer with gadolinium metal ions. A magnetic anionic surfactant, gadolinium tri(1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate) (Gd(AOT)3), was synthesized via metathesis. Both routes enable facile preparation of magnetically responsive magnetic polymers and surfactants without the need to rely on nanocomposites or organic frameworks with polyradicals. Electrical conductivity, surface tensiometry, SQUID magnetometry, and small-angle neutron scattering (SANS) demonstrate surface activity and self-aggregation behavior of the magnetic surfactants similar to their magnetically inert parent analogues but with added magnetic properties. The binding of the magnetic surfactants to proteins enables efficient separations under low-strength (0.33 T) magnetic fields in a new, nanoparticle-free approach to magnetophoretic protein separations and extractions. Importantly, the toxicity of the magnetic surfactants and polymers is, in some cases, lower than that of their halide analogues. PMID:26725503

  10. Self-Assembly of Globular Protein-Polymer Diblock Copolymers

    NASA Astrophysics Data System (ADS)

    Thomas, C. S.; Olsen, B. D.

    2011-03-01

    The self-assembly of globular protein-polymer diblock copolymers into nanostructured phases is demonstrated as an elegant and simple method for structural control in biocatalysis or bioelectronics. In order to fundamentally investigate self-assembly in these complex block copolymer systems, a red fluorescent protein was expressed in E. coli and site-specifically conjugated to a low polydispersity poly(N-isopropyl acrylamide) (PNIPAM) block using thiol-maleimide coupling to form a well-defined model globular protein-polymer diblock. Functional protein materials are obtained by solvent evaporation and solvent annealing above and below the lower critical solution temperature of PNIPAM in order to access different pathways toward self-assembly. Small angle x-ray scattering and microscopy are used to show that the diblock forms lamellar nanostructures and to explore dependence of nanostructure formation on processing conditions. Circular dichroism and UV-vis show that a large fraction of the protein remains in its folded state after conjugation, and wide angle x-ray scattering demonstrates that diblock copolymer self-assembly changes the protein packing symmetry.

  11. [Molecularly imprinted polymers in electro analysis of proteins].

    PubMed

    Shumyantseva, V V; Bulko, T V; Baychorov, I Kh; Archakov, A I

    2015-01-01

    In the review the main approaches to creation of recognition materials capable of competing with biological specific receptors, (polymeric analogs of antibodies or molecularly imprinted polymers, MIP) for the electro analysis of functionally significant proteins such as a myoglobin, troponin T, albumin, human ferritin, calmodulin are considered. The main types of monomers for MIP fabrication, and methods for MIP/protein interactions, such as a surface plasmon resonance (SPR), nanogravimetry with use of the quartz crystal resonator (QCM), spectral and electrochemical methods are discussed. Experimental data on electrochemical registration of a myoglobin using MIP/electrode are presented. For a development of electrochemical sensor systems based on MIPs, o-phenylenediamine (1,2-diaminobenzene was used as a monomer. It was shown that the imprinting factor Imax(MIP)/Imax(NIP), calculated as a myoglobin signal ratio when embedding in MIP to a myoglobin signal when embedding in the polymer received without molecular template (NIP) corresponds 2-4. PMID:26215409

  12. Protein markers of Bursaphelenchus xylophilus Steiner & Buhrer, 1934 (Nickle, 1970) populations using quantitative proteomics and character compatibility.

    PubMed

    Ciordia, Sergio; Robertson, Lee; Arcos, Susana C; González, María Rosa; Mena, María Del Carmen; Zamora, Paula; Vieira, Paulo; Abrantes, Isabel; Mota, Manuel; Castagnone-Sereno, Philippe; Navas, Alfonso

    2016-03-01

    The Pine Wood Nematode (PWN) Bursaphelenchus xylophilus is a severe forest pathogen in countries where it has been introduced and is considered a worldwide quarantine organism. In this study, protein markers for differentiating populations of this nematode were identified by studying differences among four selected Iberian and one American population. These populations were compared by quantitative proteomics (iTRAQ). From a total of 2860 proteins identified using the public database from the B. xylophilus genome project, 216 were unambiguous and significantly differentially regulated in the studied populations. Comparisons of their pairwise ratio were statistically treated and supported in order to convert them into discrete character states, suggesting that 141 proteins were not informative as population specific markers. Application of the Character Compatibility methodology on the remaining 75 proteins (belonging to families with different biological functions) excludes 27 which are incompatible among them. Considering only the compatible proteins, the method selects a subset of 30 specific unique protein markers which allowed the compared classification of the Iberian isolates. This approach makes it easier search for diagnostic tools and phylogenetic inference within species and populations of a pathogen exhibiting a high level of genetic diversity. PMID:26718462

  13. Synthesis of Heterotelechelic Polymers for Conjugation of Two Different Proteins

    PubMed Central

    Heredia, Karina L.; Grover, Gregory N.; Tao, Lei

    2011-01-01

    In this report we describe a straightforward approach to synthesize polymers with end-groups that bind site-specifically to two different proteins. Telechelic biotin, maleimide poly(N-isopropylacrylamide) (pNIPAAm) was synthesized for the formation of streptavidin (SAv)-bovine serum albumin (BSA) polymer conjugates. Reversible addition-fragmentation chain transfer (RAFT) polymerization of NIPAAm was conducted in the presence of biotinylated chain transfer agents (CTAs) with either ester or amide linkages, and the resultant α-biotinylated pNIPAAm were formed with low polydispersity indices (PDI ≤ 1.09). UV-Vis analysis of the trithiocarbonate chain-ends indicated 88% or greater retention of the group. A maleimide was introduced to the ω chain-end via a radical cross-coupling reaction with a functionalized azo-initiator. The polymer structures were characterized by 1H NMR spectroscopy and gel permeation chromatography (GPC). The resultant biotin-maleimide heterotelechelic polymer was used to form a SAv-BSA heterodimer conjugate. Bioconjugate formation was confirmed by gel electrophoresis. PMID:25378715

  14. Function of Amphiphilic Biomolecular Machines: Elastic Protein-based Polymers

    NASA Astrophysics Data System (ADS)

    Urry, Dan W.

    2000-03-01

    Elastic protein-based polymers function as biomolecular machines due to inverse temperature transitions of hydrophobic folding and assembly. The transitions occur either on raising the temperature from below to above the transition temperature, Tt, or on isothermally lowering Tt from above to below an operating temperature. The inverse temperature transition involves a decrease in entropy of the polymer component of the system on raising the temperature and a larger increase in solvent entropy on hydrophobic association. Tt depends on the quantity of hydrophobic hydration that undergoes transition to bulk water. Designed amphiphilic polymers perform free energy transductions involving the intensive variables of mechanical force, pressure, temperature, chemical potential, electrochemical potential and electromagnetic radiation and define a set of five axioms for their function as machines. The physical basis for these diverse energy conversions is competition for hydration between apolar (hydrophobic) and polar (e.g., charged) moieties. The effectiveness of these Tt-type entropic elastic protein-based machines is due to repeating peptide sequences that form regular, dynamic repeating structures and exhibit damping of backbone torsional oscillations on extension.

  15. Synthesis of protein-containing polymers in organic solvents.

    PubMed

    Yang, Z; Williams, D; Russell, A J

    1995-01-01

    Subtilisin has been modified with polyethylene glycol (PEG) monomethacrylate (MW 8000) by reductive alkylation, and incorporated into polymethyl methacrylate durring free-radical initiated polymerization. The activity and stability of the PEG-modified enzymes have been determined in aqueous buffer and organic solvents. The K(m) and V(max) values for unmodified, singly and doubly modified subtilisin were compared in these environments, and the half-lives of both modified enzymes were remarkably high (up to 2 months). The protein-containing polymer was analyzed for activity and polymer properties, and our results indicate that active subtilisin can be incorporated into polymethyl methacrylate during polymerization in organic solvents while retaining its activity and stability. (c) 1995 John Wiley & Sons, Inc. PMID:18623046

  16. Photocatalytic formulations for protein fibers: experimental analysis of the effect of preparation on compatibility and photocatalytic activities.

    PubMed

    Tung, Wing Sze; Daoud, Walid A

    2008-10-01

    In this article, we report an optimization study of a photocatalytic self-cleaning sol-gel formulation. In particular we studied the effect of formulation preparation time on the formation of anatase titanium dioxide sol and its compatibility to protein keratin-type wool fibers. The sols were formed by a low temperature sol-gel process. The nucleated anatase was characterized by UV-vis transmission, particle size distribution, X-ray diffraction, and transmission electron microscopy. The compatibility between the formulations and wool fibers is evaluated by field-emission scanning electron microscopy, UV transmission, and mechanical properties. The photocatalytic self-cleaning activity of coated fibers and its reproducibility are also discussed. PMID:18691723

  17. Characterization of Hyaluronan-Protein Microstructures and Polymer Solutions

    NASA Astrophysics Data System (ADS)

    Curtis, J. E.; McLane, L.; Bedoya, M.; Beatty, R.; Kramer, A.; Boehm, H.; Scrimgeour, J.

    2010-03-01

    Evidence is mounting that mechanical and topographical features of biomaterials can be as critical for cellular behavior as chemical properties. A case in point is hyaluronan (HA), a large polysaccharide with unique mechanical and hydrodynamic properties, found in many tissues and bodily fluids. Thanks to a large variety of accessible conformations and aggregation states, this remarkable polymer can impart on its biological environment a diverse range of structural and viscoelastic properties with far-reaching consequences for cell physiology (migration, inflammation, cancer). Supramolecular assembly of HA is typically mediated by HA-binding proteins. These specialized molecules are known to assist the formation of organized structures, such as cross-linked bundles, gels, or the all-important pericellular coat, a polymer network anchored to many cell surfaces. Precisely how the material properties of HA-rich matrices and aggregates are modified by the associated proteins, however, is largely a matter of speculation. We will present new insights concerning the cell coat and HA-protein solutions characterized using passive microrheology, fluorescence recovery after photobleaching (FRAP), and optical force probe microscopy.

  18. Localized entrapment of green fluorescent protein within nanostructured polymer films

    NASA Astrophysics Data System (ADS)

    Ankner, John; Kozlovskaya, Veronika; O'Neill, Hugh; Zhang, Qiu; Kharlampieva, Eugenia

    2012-02-01

    Protein entrapment within ultrathin polymer films is of interest for applications in biosensing, drug delivery, and bioconversion, but controlling protein distribution within the films is difficult. We report on nanostructured protein/polyelectrolyte (PE) materials obtained through incorporation of green fluorescent protein (GFP) within poly(styrene sulfonate)/poly(allylamine hydrochloride) multilayer films assembled via the spin-assisted layer-by-layer method. By using deuterated GFP as a marker for neutron scattering contrast we have inferred the architecture of the films in both normal and lateral directions. We find that films assembled with a single GFP layer exhibit a strong localization of the GFP without intermixing into the PE matrix. The GFP volume fraction approaches the monolayer density of close-packed randomly oriented GFP molecules. However, intermixing of the GFP with the PE matrix occurs in multiple-GFP layer films. Our results yield new insight into the organization of immobilized proteins within polyelectrolyte matrices and open opportunities for fabrication of protein-containing films with well-organized structure and controllable function, a crucial requirement for advanced sensing applications.

  19. Semi-permeable coatings fabricated from comb-polymers efficiently protect proteins in vivo.

    PubMed

    Liu, Mi; Johansen, Pål; Zabel, Franziska; Leroux, Jean-Christophe; Gauthier, Marc A

    2014-01-01

    In comparison to neutral linear polymers, functional and architecturally complex (that is, non-linear) polymers offer distinct opportunities for enhancing the properties and performance of therapeutic proteins. However, understanding how to harness these parameters is challenging, and studies that capitalize on them in vivo are scarce. Here we present an in vivo demonstration that modification of a protein with a polymer of appropriate architecture can impart low immunogenicity, with a commensurably low loss of therapeutic activity. These combined properties are inaccessible by conventional strategies using linear polymers. For the model protein L-asparaginase, a comb-polymer bio-conjugate significantly outperformed the linear polymer control in terms of lower immune response and more sustained bioactivity. The semi-permeability characteristics of the coatings are consistent with the phase diagram of the polymer, which will facilitate the application of this strategy to other proteins and with other therapeutic models. PMID:25407758

  20. Synthesis of water-compatible surface-imprinted polymer via click chemistry and RAFT precipitation polymerization for highly selective and sensitive electrochemical assay of fenitrothion.

    PubMed

    Zhao, Lijuan; Zhao, Faqiong; Zeng, Baizhao

    2014-12-15

    A novel water-compatible fenitrothion imprinted polymer was prepared on Au nanoparticles (AuNPs) by click chemistry and reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization (RAFTPP). The RAFT chain-transfer agent was synthesized on the surface of AuNPs using click chemistry, then an imprinted polymer with hydrophilic polymer brushes was prepared on the RAFT chain-transfer agent modified AuNPs by RAFTPP, mediated by hydrophilic polyethylene glycol macromolecular cochain-transfer agent. The obtained molecularly imprinted material showed improved accessibility to fenitrothion and recognition property in water medium. When the material was immobilized on an ionic liquid functionalized graphene coated glassy carbon electrode for the electrochemical determination of fenitrothion, the resulting electrochemical sensor presented linear response in the range of 0.01-5 μM, with a sensitivity of 6.1 μA/μM mm(2). The low limit of detection was 8 nM (S/N=3). The sensor was successfully applied to the determination of real samples and the recovery for standard added was 95-108%. PMID:24973538

  1. Hierarchically Ordered Supramolecular Protein-Polymer Composites with Thermoresponsive Properties

    PubMed Central

    Välimäki, Salla; Mikkilä, Joona; Liljeström, Ville; Rosilo, Henna; Ora, Ari; Kostiainen, Mauri A.

    2015-01-01

    Synthetic macromolecules that can bind and co-assemble with proteins are important for the future development of biohybrid materials. Active systems are further required to create materials that can respond and change their behavior in response to external stimuli. Here we report that stimuli-responsive linear-branched diblock copolymers consisting of a cationic multivalent dendron with a linear thermoresponsive polymer tail at the focal point, can bind and complex Pyrococcus furiosus ferritin protein cages into crystalline arrays. The multivalent dendron structure utilizes cationic spermine units to bind electrostatically on the surface of the negatively charged ferritin cage and the in situ polymerized poly(di(ethylene glycol) methyl ether methacrylate) linear block enables control with temperature. Cloud point of the final product was determined with dynamic light scattering (DLS), and it was shown to be approximately 31 °C at a concentration of 150 mg/L. Complexation of the polymer binder and apoferritin was studied with DLS, small-angle X-ray scattering, and transmission electron microscopy, which showed the presence of crystalline arrays of ferritin cages with a face-centered cubic (fcc Fm3¯m) Bravais lattice where lattice parameter a = 18.6 nm. The complexation process was not temperature dependent but the final complexes had thermoresponsive characteristics with negative thermal expansion. PMID:25950765

  2. Hierarchically ordered supramolecular protein-polymer composites with thermoresponsive properties.

    PubMed

    Välimäki, Salla; Mikkilä, Joona; Liljeström, Ville; Rosilo, Henna; Ora, Ari; Kostiainen, Mauri A

    2015-01-01

    Synthetic macromolecules that can bind and co-assemble with proteins are important for the future development of biohybrid materials. Active systems are further required to create materials that can respond and change their behavior in response to external stimuli. Here we report that stimuli-responsive linear-branched diblock copolymers consisting of a cationic multivalent dendron with a linear thermoresponsive polymer tail at the focal point, can bind and complex Pyrococcus furiosus ferritin protein cages into crystalline arrays. The multivalent dendron structure utilizes cationic spermine units to bind electrostatically on the surface of the negatively charged ferritin cage and the in situ polymerized poly(di(ethylene glycol) methyl ether methacrylate) linear block enables control with temperature. Cloud point of the final product was determined with dynamic light scattering (DLS), and it was shown to be approximately 31 °C at a concentration of 150 mg/L. Complexation of the polymer binder and apoferritin was studied with DLS, small-angle X-ray scattering, and transmission electron microscopy, which showed the presence of crystalline arrays of ferritin cages with a face-centered cubic (fcc, Fm3m)) Bravais lattice where lattice parameter a=18.6 nm. The complexation process was not temperature dependent but the final complexes had thermoresponsive characteristics with negative thermal expansion. PMID:25950765

  3. Toward a Universal Method for Preparing Molecularly Imprinted Polymer Nanoparticles with Antibody-like Affinity for Proteins.

    PubMed

    Xu, Jingjing; Ambrosini, Serena; Tamahkar, Emel; Rossi, Claire; Haupt, Karsten; Tse Sum Bui, Bernadette

    2016-01-11

    We describe a potentially universal, simple and cheap method to prepare water-compatible molecularly imprinted polymer nanoparticles (MIP-NPs) as synthetic antibodies against proteins. The strategy is based on a solid phase synthesis approach where glass beads (GBs) are functionalized with a metal chelate, acting as a general affinity ligand to attract surface-bound histidines present on proteins. This configuration enables an oriented immobilization of the proteins, upon which thermoresponsive MIP-NPs are synthesized. The GBs play the role of both a reactor and a separation column since, after synthesis, the MIP-NPs are released from the support by a simple temperature change, resulting in protein-free polymers. The resulting MIP-NPs are endowed with improved binding site homogeneity, since the binding sites have the same orientation. Moreover, they are stable (no aggregation) in a buffer solution for prolonged storage time and exhibit apparent dissociation constants in the nanomolar range, with little or no cross-reactivity toward other proteins. PMID:26644006

  4. Tyrosine Coupling Creates a Hyperbranched Multivalent Protein Polymer Using Horseradish Peroxidase via Bipolar Conjugation Points.

    PubMed

    Minamihata, Kosuke; Yamaguchi, Sou; Nakajima, Kei; Nagamune, Teruyuki

    2016-05-18

    Protein polymers of covalently cross-linked protein monomers are highly attractive biomaterials because each monomer unit possesses distinct protein functions. Protein polymers often show enhancement effects on the function by integrating a large number of molecules into one macromolecule. The cross-linking site of component proteins should be precisely controlled to avoid diminishing the protein function. However, preparing protein polymers that are cross-linked site-specifically with a high cross-linking degree is a challenge. Here, we demonstrate the preparation of a site-specifically cross-linked protein polymer that has a hyperbranched polymer-like structure with a high cross-linking degree. A horseradish peroxidase (HRP) reaction was used to achieve the protein polymerization through a peptide tag containing a tyrosine residue (Y-tag). Y-tag sequences were introduced to both N- and C-termini of a model protein, protein G. The dual Y-tagged protein G (Y-pG-Y) was treated with HRP to form a Y-pG-Y polymer possessing average and maximum cross-linking degree of approximately 70-mer and 150-mer, respectively. The Y-pG-Y polymer shows the highest cross-linking degree among the protein polymers reported, which are completely soluble in water and cross-linked via covalent bonding. The Y-pG-Y was cross-linked site-specifically at the Tyr residue in the Y-tag, retaining its function, and the Y-pG-Y polymer showed extremely strong avidity against immunoglobulin G. The reactivities of N- and C-terminal Y-tags were evaluated, and we revealed that the difference in the radical formation rate by HRP was the key for yielding highly cross-linked protein polymers. PMID:27093089

  5. An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies.

    PubMed

    Schulenberg, Birte; Arnold, Brad; Patton, Wayne F

    2003-07-01

    Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively. PMID:12872220

  6. Role of Polymer Architecture on the Activity of Polymer-Protein Conjugates for the Treatment of Accelerated Bone Loss Disorders.

    PubMed

    Tucker, Bryan S; Stewart, Jon D; Aguirre, J Ignacio; Holliday, L Shannon; Figg, C Adrian; Messer, Jonathan G; Sumerlin, Brent S

    2015-08-10

    Polymers of similar molecular weights and chemical constitution but varying in their macromolecular architectures were conjugated to osteoprotegerin (OPG) to determine the effect of polymer topology on protein activity in vitro and in vivo. OPG is a protein that inhibits bone resorption by preventing the formation of mature osteoclasts from the osteoclast precursor cell. Accelerated bone loss disorders, such as osteoporosis, rheumatoid arthritis, and metastatic bone disease, occur as a result of increased osteoclastogenesis, leading to the severe weakening of the bone. OPG has shown promise as a treatment in bone disorders; however, it is rapidly cleared from circulation through rapid liver uptake, and frequent, high doses of the protein are necessary to achieve a therapeutic benefit. We aimed to improve the effectiveness of OPG by creating OPG-polymer bioconjugates, employing reversible addition-fragmentation chain transfer polymerization to create well-defined polymers with branching densities varying from linear, loosely branched to densely branched. Polymers with each of these architectures were conjugated to OPG using a "grafting-to" approach, and the bioconjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The OPG-polymer bioconjugates showed retention of activity in vitro against osteoclasts, and each bioconjugate was shown to be nontoxic. Preliminary in vivo studies further supported the nontoxic characteristics of the bioconjugates, and measurement of the bone mineral density in rats 7 days post-treatment via peripheral quantitative computed tomography suggested a slight increase in bone mineral density after administration of the loosely branched OPG-polymer bioconjugate. PMID:26151628

  7. Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

    PubMed

    Monroe, Nicole; Hill, Christopher P

    2016-05-01

    Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. PMID:26555750

  8. Plasma protein adsorbed biomedical polymers: activation of human monocytes and induction of interleukin 1.

    PubMed

    Bonfield, T L; Colton, E; Anderson, J M

    1989-06-01

    These studies involved the evaluation of human monocyte/macrophage activation by biomedical polymers coated with human blood proteins. The biomedical polymers were polyethylene, polydimethylsiloxane, woven Dacron fabric, expanded polytetrafluoroethylene, Biomer, and tissue culture treated polystyrene as the control. They were adsorbed with human blood proteins: albumin, fibrinogen, fibronectin, hemoglobin, and gamma globulin. The protein adsorbed polymers were evaluated for their potential to activate the monocyte/macrophage cellular population in vitro as assessed by the induction of the monocyte/macrophage inflammatory mediator, Interleukin 1 (IL1). Suppression of IL1 was observed when protein adsorbed polymers were compared to the appropriate protein adsorbed control. Protein adsorbed polymers, when compared to polymers without protein adsorption, stimulated IL1 production. The data presented in this manuscript show the level of induction and secretion of IL1 was dependent on the biomedical polymer and the protein adsorbed, as well as the requirement of lipopolysaccharide. These results show differential interactions occur between the proteins, monocytes/macrophages, and biomedical polymers which alter activation and induction of IL1. PMID:2786877

  9. Water-compatible graphene oxide/molecularly imprinted polymer coated stir bar sorptive extraction of propranolol from urine samples followed by high performance liquid chromatography-ultraviolet detection.

    PubMed

    Fan, Wenying; He, Man; You, Linna; Zhu, Xuewei; Chen, Beibei; Hu, Bin

    2016-04-22

    Due to the high selectivity and stability, molecularly imprinted polymers (MIPs) have been successfully applied in stir bar sorptive extraction (SBSE) as a special coating to improve the selective extraction capability for target analytes. However, traditional MIPs usually suffer from incompatibility in aqueous media and low adsorption capacity, which limit the application of MIP coated stir bar in aqueous samples. To solve these problems, a water-compatible graphene oxides (GO)/MIP composite coated stir bar was prepared in this work by in situ polymerization. The prepared water-compatible GO/MIP coated stir bar presented good mechanical strength and chemical stability, and its recognition ability in aqueous samples was improved due to the polymerization of MIP in water environment, the adsorption capacity for target analytes was also increased by the addition of GO in MIP pre-polymer solution. Based on it, a method of water-compatible GO/MIP coated stir bar sorptive extraction combined with high performance liquid chromatography-ultraviolet detector (HPLV-UV) was proposed for the analysis of propranolol (PRO) in aqueous solution. The influencing factors of SBSE, such as sample pH, salt effect, stirring rate, extraction time, desorption solvent and desorption time, were optimized, and the analytical performance of the developed SBSE-HPLC-UV method was evaluated under the optimized conditions. The limit of detection (LOD) of the proposed method for PRO was about 0.37 μg L(-1), and the enrichment factor (EF) was 59.7-fold (theoretical EF was 100-fold). The reproducibility was also investigated at concentrations of 5 μg L(-1) and the relative standard deviation (RSD) was found to be 7.3% (n=7). The proposed method of GO/MIP coating-SBSE-HPLC-UV was successfully applied for the assay of the interested PRO drug in urine samples, and further extended to the investigation of the excretion of the drugs by monitoring the variation of the concentration of PRO in urine

  10. Water-compatible silica sol-gel molecularly imprinted polymer as a potential delivery system for the controlled release of salicylic acid.

    PubMed

    Li, Bin; Xu, Jingjing; Hall, Andrew J; Haupt, Karsten; Tse Sum Bui, Bernadette

    2014-09-01

    Molecularly imprinted polymers (MIPs) for salicylic acid were synthesized and evaluated in aqueous environments in the aim to apply them as drug delivery carriers. One organic MIP and one inorganic MIP based on the sol-gel process were synthesized. The organic MIP was prepared by radical polymerization using the stoichiometric functional monomer, 1-(4-vinylphenyl)-3-(3,5-bis(trifluoromethyl)phenyl)urea, which can establish strong electrostatic interactions with the -COOH of salicylic acid. The sol-gel MIP was prepared with 3-(aminopropyl)triethoxysilane and trimethoxyphenylsilane, as functional monomers and tetraethyl orthosilicate as the crosslinker. While the organic MIPs bound the target specifically in acetonitrile, they exhibited lower binding in the presence of water, although the imprinting factor increased under these conditions, due to reduced non-specific binding. The sol-gel MIP has a high specificity and capacity for the drug in ethanol, a solvent compatible with drug formulation and biomedical applications. In vitro release profiles of the polymers in water were evaluated, and the results were modelled by Fick's law of diffusion and the power law. Analysis shows that the release mechanism was predominantly diffusion-controlled. PMID:25042710

  11. Preparation of surface-imprinted polymer grafted with water-compatible external layer via RAFT precipitation polymerization for highly selective and sensitive electrochemical determination of brucine.

    PubMed

    Zhao, Lijuan; Zhao, Faqiong; Zeng, Baizhao

    2014-10-15

    A novel brucine imprinted polymer was prepared on multi-walled carbon nanotubes by reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization. The polymer was further grafted with hydrophilic poly(glycerol monomethacrylate) brushes to improve its water-compatibility. The obtained molecularly imprinted material showed enhanced accessibility to brucine and improved selective recognition property in water medium. When the material was supported on an ionic liquid functionalized graphene coated glassy carbon electrode for the electrochemical determination of brucine, the resulting electrochemical sensor presented good analytical performance. Under the optimized conditions, the peak current was linear to brucine concentration in the ranges of 0.006-0.6 μM and 0.6-5.0 μM with sensitivities of 15.3 μA/μMmm(2) and 5.4 μA/μM mm(2), respectively; the detection limit was 2 nM (S/N=3). The sensor was successfully applied to the determination of brucine in practical samples and the recovery for the standards added was 94-104%. PMID:24769450

  12. Molecular interaction forces generated during protein adsorption to well-defined polymer brush surfaces.

    PubMed

    Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

    2015-03-17

    The molecular interaction forces generated during the adsorption of proteins to surfaces were examined by the force-versus-distance (f-d) curve measurements of atomic force microscopy using probes modified with appropriate molecules. Various substrates with polymer brush layers bearing zwitterionic, cationic, anionic, and hydrophobic groups were systematically prepared by surface-initiated atom transfer radical polymerization. Surface interaction forces on these substrates were analyzed by the f-d curve measurements using probes with the same polymer brush layer as the substrate. Repulsive forces, which decreased depending on the ionic strength, were generated between cationic or anionic polyelectrolyte brush layers; these were considered to be electrostatic interaction forces. A strong adhesive force was detected between hydrophobic polymer brush layers during retraction; this corresponded to the hydrophobic interaction between two hydrophobic polymer layers. In contrast, no significant interaction forces were detected between zwitterionic polymer brush layers. Direct interaction forces between proteins and polymer brush layers were then quantitatively evaluated by the f-d curve measurements using protein-immobilized probes consisting of negatively charged albumin and positively charged lysozyme under physiological conditions. In addition, the amount of protein adsorbed on the polymer brush layer was quantified by surface plasmon resonance measurements. Relatively large amounts of protein adsorbed to the polyelectrolyte brush layers with opposite charges. It was considered that the detachment of the protein after contact with the polymer brush layer hardly occurred due to salt formation at the interface. Both proteins adsorbed significantly on the hydrophobic polymer brush layer, which was due to hydrophobic interactions at the interface. In contrast, the zwitterionic polymer brush layer exhibited no significant interaction force with proteins and suppressed

  13. Effect of hydrophilicity of end-grafted polymers on protein adsorption behavior: A Monte Carlo study.

    PubMed

    Han, Yuanyuan; Jin, Jing; Cui, Jie; Jiang, Wei

    2016-06-01

    Monte Carlo simulation is employed to investigate protein adsorption behavior on end-grafted polymers. The effect of hydrophilicity of end-grafted polymers on protein adsorption behavior is investigated in detail. The simulation results indicate that the hydrophilicity of the end-grafted polymers can affect both the amount and speed of protein adsorption. An increase in the hydrophilicity of the end-grafted polymers can significantly decrease the amount and speed of protein adsorption first. However, a further increase in the hydrophilicity of the end-grafted polymers results in the increase in the amount and speed of protein adsorption. This phenomenon is easier to be observed in the end-grafted polymer systems with lower grafting density and longer chain length. In addition, the investigation of the chain conformation of the end-grafted polymers reveals that the end-grafted polymers with mediate hydrophilicity have relatively small size difference along the parallel and perpendicular directions to the substrate, and these end-grafted polymers have relatively wide height distribution. Such characteristics favor covering the space above the hydrophobic substrate and thus can effectively resist protein adsorption. PMID:26925724

  14. Gold Nanoparticle-Polymer/Biopolymer Complexes for Protein Sensing

    PubMed Central

    Moyano, Daniel F.; Rana, Subinoy; Bunz, Uwe H. F.; Rotello, Vincent M.

    2014-01-01

    Nanoparticle-based sensor arrays have been used to distinguish a wide range of biomolecular targets through pattern recognition. Such biosensors require selective receptors that generate a unique response pattern for each analyte. The tunable surface properties of gold nanoparticles make these systems excellent candidates for the recognition process. Likewise, the metallic core makes these particles fluorescence superquenchers, facilitating transduction of the binding event. In this report we analyze the role of gold nanoparticles as receptors in differentiating a diversity of important human proteins different, and the role of the polymer/biopolymer fluorescent probes for transducing the binding event. A structure-activity relationship analysis of both the probes and the nanoparticles is presented, providing direction for the engineering of future sensor systems. PMID:22455037

  15. Protein ProQ Influences Osmotic Activation of Compatible Solute Transporter ProP in Escherichia coli K-12

    PubMed Central

    Kunte, H. Jörg; Crane, Rebecca A.; Culham, Doreen E.; Richmond, Deborah; Wood, Janet M.

    1999-01-01

    ProP is an osmoregulatory compatible solute transporter in Escherichia coli K-12. Mutation proQ220::Tn5 decreased the rate constant for and the extent of ProP activation by an osmotic upshift but did not alter proP transcription or the ProP protein level. Allele proQ220::Tn5 was isolated, and the proQ sequence was determined. Locus proQ is upstream from prc (tsp) at 41.2 centisomes on the genetic map. The proQ220::Tn5 and prc phenotypes were different, however. Gene proQ is predicted to encode a 232-amino-acid, basic, hydrophilic protein (molecular mass, 25,876 Da; calculated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar amino acids). The insertion of PCR-amplified proQ into vector pBAD24 produced a plasmid containing the wild-type proQ open reading frame, the expression of which yielded a soluble protein with an apparent molecular mass of 30 kDa. Antibodies raised against the overexpressed ProQ protein detected cross-reactive material in proQ+ bacteria but not in proQ220::Tn5 bacteria. ProQ may be a structural element that influences the osmotic activation of ProP at a posttranslational level. PMID:10049386

  16. Coffee bean arabinogalactans: acidic polymers covalently linked to protein.

    PubMed

    Redgwell, Robert J; Curti, Delphine; Fischer, Monica; Nicolas, Pierre; Fay, Laurent B

    2002-02-11

    The arabinogalactan content of green coffee beans (Coffea arabica var. Yellow Caturra) was released by a combination of chemical extraction and enzymatic hydrolysis of the mannan-cellulose component of the wall. Several arabinogalactan fractions were isolated, purified by gel-permeation and ion-exchange chromatography and characterised by compositional and linkage analysis. The AG fractions contained between 6 and 8% glucuronic acid, and gave a positive test for the beta-glucosyl-Yariv reagent, a stain specific for arabinogalactan-proteins. The protein component accounted for between 0.5 and 2.0% of the AGPs and contained between 7 and 12% hydroxyproline. The AG moieties displayed considerable heterogeneity with regard to their degree of arabinosylation and the extent and composition of their side-chains. They possessed a MW average of 650 kDa which ranged between 150 and 2000 kDa. An investigation of the structural features of the major AG fraction, released following enzymatic hydrolysis of the mannan-cellulose polymers, allowed a partial structure of coffee arabinogalactan to be proposed. PMID:11844494

  17. A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus.

    PubMed Central

    Olesnicky, N S; Brown, A J; Dowell, S J; Casselton, L A

    1999-01-01

    In the mushroom Coprinus cinereus, the multiallelic B mating type genes are predicted to encode a large family of seven-transmembrane domain receptors and CaaX-modified pheromones. We have shown that a single amino acid change Q229P in transmembrane domain VI of one receptor confers a self-compatible mating phenotype. Using a heterologous yeast assay, we have demonstrated that this C.cinereus pheromone receptor is a G-protein-coupled receptor and that the Q229P mutation is constitutively activating. A C.cinereus pheromone precursor was processed to an active species specifically in yeast MATa cells and activated the co-expressed wild-type receptor. Yeast cells expressing the wild-type receptor were used to test the activity of synthetic peptides, enabling us to predict the structure of the mature C.cinereus pheromone and to show that the Q229P mutation does not compromise normal receptor function. PMID:10329622

  18. Selection and demographic history shape the molecular evolution of the gamete compatibility protein bindin in Pisaster sea stars.

    PubMed

    Popovic, Iva; Marko, Peter B; Wares, John P; Hart, Michael W

    2014-05-01

    Reproductive compatibility proteins have been shown to evolve rapidly under positive selection leading to reproductive isolation, despite the potential homogenizing effects of gene flow. This process has been implicated in both primary divergence among conspecific populations and reinforcement during secondary contact; however, these two selective regimes can be difficult to discriminate from each other. Here, we describe the gene that encodes the gamete compatibility protein bindin for three sea star species in the genus Pisaster. First, we compare the full-length bindin-coding sequence among all three species and analyze the evolutionary relationships between the repetitive domains of the variable second bindin exon. The comparison suggests that concerted evolution of repetitive domains has an effect on bindin divergence among species and bindin variation within species. Second, we characterize population variation in the second bindin exon of two species: We show that positive selection acts on bindin variation in Pisaster ochraceus but not in Pisaster brevispinus, which is consistent with higher polyspermy risk in P. ochraceus. Third, we show that there is no significant genetic differentiation among populations and no apparent effect of sympatry with congeners that would suggest selection based on reinforcement. Fourth, we combine bindin and cytochrome c oxidase 1 data in isolation-with-migration models to estimate gene flow parameter values and explore the historical demographic context of our positive selection results. Our findings suggest that positive selection on bindin divergence among P. ochraceus alleles can be accounted for in part by relatively recent northward population expansions that may be coupled with the potential homogenizing effects of concerted evolution. PMID:24967076

  19. Reducing protein adsorption with polymer-grafted hyaluronic acid coatings.

    PubMed

    Ramadan, Mohamed H; Prata, Joseph E; Karácsony, Orsolya; Dunér, Gunnar; Washburn, Newell R

    2014-07-01

    We report a thermoresponsive chemical modification strategy of hyaluronic acid (HA) for coating onto a broad range of biomaterials without relying on chemical functionalization of the surface. Poly(di(ethylene glycol) methyl ether methacrylate) (PMEO2MA), a polymer with a lower critical solution temperature of 26 °C in water, was grafted onto HA to allow facile formation of biopolymer coatings. While the mechanism for film formation appears to involve a complex combination of homogeneous nucleation followed by heterogeneous film growth, we demonstrate that it resulted in hydrophilic coatings that significantly reduce protein adsorption despite the high fraction of hydrophobic (PMEO2MA). Structural characterization was performed using atomic force microscopy (AFM), which showed the formation of a dense, continuous coating based on 200 nm domains that were stable in protein solutions for at least 15 days. The coatings had a water contact angle of 16°, suggesting the formation of hydrophilic but not fully wetting films. Quartz crystal microbalance with dissipation monitoring (QCM-D) as well as biolayer interferometry (BLI) techniques were used to measure adsorption of bovine serum albumin (BSA), fibrinogen (Fbg), and human immunoglobulin (IgG), with results indicating that HA-PMEO2MA-coated surfaces effectively inhibited adsorption of all three serum proteins. These results are consistent with previous studies demonstrating that this degree of hydrophilicity is sufficient to generate an effectively nonfouling surface and suggest that segregation during the solubility transition resulted in a surface that presented the hydrophilic HA component of the hybrid biopolymer. We conclude that PMEO2MA-grafted HA is a versatile platform for the passivation of hydrophobic biomaterial surfaces without need for substrate functionalization. PMID:24892924

  20. Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus.

    PubMed

    Lamosa, P; Burke, A; Peist, R; Huber, R; Liu, M Y; Silva, G; Rodrigues-Pousada, C; LeGall, J; Maycock, C; Santos, H

    2000-05-01

    Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications. PMID:10788369

  1. Protein microarrays based on polymer brushes prepared via surface-initiated atom transfer radical polymerization.

    PubMed

    Barbey, Raphael; Kauffmann, Ekkehard; Ehrat, Markus; Klok, Harm-Anton

    2010-12-13

    Polymer brushes represent an interesting platform for the development of high-capacity protein binding surfaces. Whereas the protein binding properties of polymer brushes have been investigated before, this manuscript evaluates the feasibility of poly(glycidyl methacrylate) (PGMA) and PGMA-co-poly(2-(diethylamino)ethyl methacrylate) (PGMA-co-PDEAEMA) (co)polymer brushes grown via surface-initiated atom transfer radical polymerization (SI-ATRP) as protein reactive substrates in a commercially available microarray system using tantalum-pentoxide-coated optical waveguide-based chips. The performance of the polymer-brush-based protein microarray chips is assessed using commercially available dodecylphosphate (DDP)-modified chips as the benchmark. In contrast to the 2D planar, DDP-coated chips, the polymer-brush-covered chips represent a 3D sampling volume. This was reflected in the results of protein immobilization studies, which indicated that the polymer-brush-based coatings had a higher protein binding capacity as compared to the reference substrates. The protein binding capacity of the polymer-brush-based coatings was found to increase with increasing brush thickness and could also be enhanced by copolymerization of 2-(diethylamino)ethyl methacrylate (DEAEMA), which catalyzes epoxide ring-opening of the glycidyl methacrylate (GMA) units. The performance of the polymer-brush-based microarray chips was evaluated in two proof-of-concept microarray experiments, which involved the detection of biotin-streptavidin binding as well as a model TNFα reverse assay. These experiments revealed that the use of polymer-brush-modified microarray chips resulted not only in the highest absolute fluorescence readouts, reflecting the 3D nature and enhanced sampling volume provided by the brush coating, but also in significantly enhanced signal-to-noise ratios. These characteristics make the proposed polymer brushes an attractive alternative to commercially available, 2D microarray

  2. Probing the weak interaction of proteins with neutral and zwitterionic antifouling polymers.

    PubMed

    Wu, Jiang; Zhao, Chao; Hu, Rundong; Lin, Weifeng; Wang, Qiuming; Zhao, Jun; Bilinovich, Stephanie M; Leeper, Thomas C; Li, Lingyan; Cheung, Harry M; Chen, Shengfu; Zheng, Jie

    2014-02-01

    Protein-polymer interactions are of great interest in a wide range of scientific and technological applications. Neutral poly(ethylene glycol) (PEG) and zwitterionic poly(sulfobetaine methacrylate) (pSBMA) are two well-known nonfouling materials that exhibit strong surface resistance to proteins. However, it still remains unclear or unexplored how PEG and pSBMA interact with proteins in solution. In this work, we examine the interactions between two model proteins (bovine serum albumin and lysozyme) and two typical antifouling polymers of PEG and pSBMA in aqueous solution using fluorescence spectroscopy, atomic force microscopy and nuclear magnetic resonance. The effect of protein:polymer mass ratios on the interactions is also examined. Collective data clearly demonstrate the existence of weak hydrophobic interactions between PEG and proteins, while there are no detectable interactions between pSBMA and proteins. The elimination of protein interaction with pSBMA could be due to an enhanced surface hydration of zwitterionic groups in pSBMA. New evidence is given to demonstrate the interactions between PEG and proteins, which are often neglected in the literature because the PEG-protein interactions are weak and reversible, as well as the structural change caused by hydrophobic interaction. This work provides a better fundamental understanding of the intrinsic structure-activity relationship of polymers underlying polymer-protein interactions, which are important for designing new biomaterials for biosensor, medical diagnostics and drug delivery applications. PMID:24120846

  3. Uniform polymer-protein conjugate by aqueous AGET ATRP using protein as a macroinitiator.

    PubMed

    Zhu, Binbin; Lu, Diannan; Ge, Jun; Liu, Zheng

    2011-05-01

    In situ aqueous activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP) in air, using an enzyme as a macroinitiator, has been proposed to prepare uniform polymer-protein conjugates with improved stability under adverse conditions. In the first step, an initiator, 2-bromoisobutyryl bromide (BIB), was grafted onto the protein surface by reaction with the amino groups. The second step was in situ AGET ATRP polymerization in air using CuBr(2)/1,1,4,7,7-pentamethyldiethylenetriamine as a catalyst and ascorbic acid as a reducing agent. The effectiveness of this method has been demonstrated using horseradish peroxidase (HRP) as a model protein and acrylamide as the monomer, which yielded HRP-polyacrylamide conjugate with a mean particle size of about 20-30 nm. The grafting of BIB onto HRP and the subsequent polymerization yielding a polyacrylamide chain were confirmed by nuclear magnetic resonance and matrix-assisted laser desorption ionization time-of-flight spectrometry analysis. The size of the conjugate was shown to be a function of monomer loading and reaction time. The HRP conjugates yielded essentially retained the catalytic behavior of HRP in free form, as shown by K(m) and V(max) values, but exhibited significantly enhanced thermal stability against high temperature and trypsin digestion. The use of protein as the macroinitiator prevented the formation of copolymer and thus facilitated purification of the protein conjugate. The uniform size indicates a well-defined composition of protein and polymer, which is essential for applications that request a precise control of the dosage of enzyme activity. PMID:21277397

  4. Effects of compatibility of polymer binders with solvate ionic liquid electrolytes on discharge and charge reactions of lithium-sulfur batteries

    NASA Astrophysics Data System (ADS)

    Nakazawa, Toshitada; Ikoma, Ai; Kido, Ryosuke; Ueno, Kazuhide; Dokko, Kaoru; Watanabe, Masayoshi

    2016-03-01

    Electrochemical reactions in Li-S cells with a solvate ionic liquid (SIL) electrolyte composed of tetraglyme (G4) and Li[TFSA] (TFSA: bis(trifluoromethanesulfonyl)amide) are studied. The sulfur cathode (S cathode) comprises sulfur, carbon powder, and a polymer binder. Poly(ethylene oxide) (PEO) and poly(vinyl alcohol) (PVA-x) with different degrees of saponification (x%) are used as binders to prepare the composite cathodes. For the Li-S cell containing PEO binder, lithium polysulfides (Li2Sm, 2 ≤ m ≤ 8), reaction intermediates of the S cathode, dissolve into the electrolyte, and Li2Sm acts as a redox shuttle in the Li-S cell. In contrast, in the Li-S cell with PVA-x binder, the dissolution of Li2Sm is suppressed, leading to high columbic efficiencies during charge-discharge cycles. The compatibility of the PVA-x binder with the SIL electrolyte changes depending on the degree of saponification. Decreasing the degree of saponification leads to increased electrolyte uptake by the PVA-x binder, increasing the charge and discharge capacities of Li-S cell. The rate capability of Li-S cell is also enhanced by the partial swelling of the PVA-x binder. The enhanced performance of Li-S cell containing PVA-x is attributed to the lowering of resistance of Li+ ion transport in the composite cathode.

  5. Solubilizing and Stabilizing Proteins in Anhydrous Ionic Liquids through Formation of Protein-Polymer Surfactant Nanoconstructs.

    PubMed

    Brogan, Alex P S; Hallett, Jason P

    2016-04-01

    Nonaqueous biocatalysis is rapidly becoming a desirable tool for chemical and fuel synthesis in both the laboratory and industry. Similarly, ionic liquids are increasingly popular anhydrous reaction media for a number of industrial processes. Consequently, the use of enzymes in ionic liquids as efficient, environment-friendly, commercial biocatalysts is highly attractive. However, issues surrounding the poor solubility and low stability of enzymes in truly anhydrous media remain a significant challenge. Here, we demonstrate for the first time that engineering the surface of a protein to yield protein-polymer surfactant nanoconstructs allows for dissolution of dry protein into dry ionic liquids. Using myoglobin as a model protein, we show that this method can deliver protein molecules with near native structure into both hydrophilic and hydrophobic anhydrous ionic liquids. Remarkably, using temperature-dependent synchrotron radiation circular dichroism spectroscopy to measure half-denaturation temperatures, our results show that protein stability increases by 55 °C in the ionic liquid as compared to aqueous solution, pushing the solution thermal denaturation beyond the boiling point of water. Therefore, the work presented herein could provide a platform for the realization of biocatalysis at high temperatures or in anhydrous solvent systems. PMID:26976718

  6. Polymer-based protein engineering can rationally tune enzyme activity, pH-dependence, and stability.

    PubMed

    Murata, Hironobu; Cummings, Chad S; Koepsel, Richard R; Russell, Alan J

    2013-06-10

    The attachment of inert polymers, such as polyethylene glycol, to proteins has driven the emergence of a multibillion dollar biotechnology industry. In all cases, proteins have been stabilized or altered by covalently coupling the pre-existing polymer to the surface of the protein. This approach is inherently limited by a lack of exquisite control of polymer architecture, site and density of attachment. Using a novel water-soluble atom transfer radical polymerization initiator, we have grown temperature- and pH-responsive polymers from the surface of a model protein, the enzyme chymotrypsin. Poly(2-(dimethylamino)ethyl methacrylate) changes in conformation with altered temperature and pH. Growing the polymer from the surface of chymotrypsin we were able to demonstrate that changes in temperature or pH can change predictably the conformation of the polymer surrounding the enzyme, which in turn enabled the rational tailoring of enzyme activity and stability. Using what we now term "Polymer-Based Protein Engineering", we have increased the activity and stability of chymotrypsin by an order of magnitude at pHs where the enzyme is usually inactive or unstable. PMID:23600667

  7. Quantitative ToF-SIMS studies of protein drug release from biodegradable polymer drug delivery membranes

    NASA Astrophysics Data System (ADS)

    Burns, Sarah A.; Gardella, Joseph A.

    2008-12-01

    Biodegradable polymers are of interest in developing strategies to control protein drug delivery. The protein that was used in this study is Keratinocyte Growth Factor (KGF) which is a protein involved in the re-epithelialization process. The protein is stabilized in the biodegradable polymer matrix during formulation and over the course of polymer degradation with the use of an ionic surfactant Aerosol-OT (AOT) which will encapsulate the protein in an aqueous environment. The release kinetics of the protein from the surface of these materials requires precise timing which is a crucial factor in the efficacy of this drug delivery system. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used in the same capacity to identify the molecular ion peak of the surfactant and polymer and use this to determine surface concentration. In the polymer matrix, the surfactant molecular ion peak was observed in the positive and negative mode at m/ z 467 and 421, respectively. These peaks were determined to be [AOT + Na +] and [AOT - Na +]. These methods are used to identify the surfactant and protein from the polymer matrix and are used to measure the rate of surface accumulation. The second step was to compare this accumulation rate with the release rate of the protein into an aqueous solution during the degradation of the biodegradable film. This rate is compared to that from fluorescence spectroscopy measurements using the protein autofluorescence from that released into aqueous solution [C.M. Mahoney, J. Yu, A. Fahey, J.A.J. Gardella, SIMS depth profiling of polymer blends with protein based drugs, Appl. Surf. Sci. 252 (2006), 6609-6614.].

  8. Effects of post-anthesis fertilizer on the protein composition of the gluten polymer in a US bread wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Both genetic and environmental factors influence the types and amounts of wheat proteins that link together to form polymers essential for flour quality. To understand how plant growth conditions might influence gluten polymer formation, protein fractions containing small and large polymers were se...

  9. Quaternary diffusion coefficients in a protein-polymer-salt-water system determined by rayleigh interferometry.

    PubMed

    Annunziata, Onofrio; Vergara, Alessandro; Paduano, Luigi; Sartorio, Roberto; Miller, Donald G; Albright, John G

    2009-10-01

    We have experimentally investigated multicomponent diffusion in a protein-polymer-salt-water quaternary system. Specifically, we have measured the nine multicomponent diffusion coefficients, D(ij), for the lysozyme-poly(ethylene glycol)-NaCl-water system at pH 4.5 and 25 degrees C using precision Rayleigh interferometry. Lysozyme is a model protein for protein-crystallization and enzymology studies. We find that the protein diffusion coefficient, D(11), decreases as polymer concentration increases at a given salt concentration. This behavior can be quantitatively related to the corresponding increase in fluid viscosity only at low polymer concentration. However, at high polymer concentration (250 g/L), protein diffusion is enhanced compared to the corresponding viscosity prediction. We also find that a protein concentration gradient induces salt diffusion from high to low protein concentration. This effect increases in the presence of poly(ethylene glycol). Finally, we have evaluated systematic errors associated with measurements of protein diffusion coefficients by dynamic light scattering. This work overall helps characterize protein diffusion in crowded environments and may provide guidance for further theoretical developments in the field of protein crystallization and protein diffusion in such crowded systems, such as the cytoplasm of living cells. PMID:19746957

  10. Control of protein-ligand recognition using a stimuli-responsive polymer

    NASA Astrophysics Data System (ADS)

    Stayton, Patrick S.; Shimoboji, Tsuyoshi; Long, Cynthia; Chilkoti, Ashutosh; Ghen, Guohua; Harris, J. Milton; Hoffman, Allan S.

    1995-11-01

    STIMULI-responsive polymers exhibit reversible phase changes in response to changes in environmental factors such as pH or temperature1-14. Conjugating such polymers to antibodies and proteins provides molecular systems for applications such as affinity separations, immunoassays and enzyme recovery and recycling15- 25. Here we show that conjugating a temperaturesensitive polymer to a genetically engineered site on a protein allows the protein's ligand binding affinity to be controlled. We synthesized a mutant of the protein streptavidin to enable sitespecific conjugation of the responsive polymer near the protein's binding site. Normal binding of biotin to the modified protein occurs below 32 °C, whereas above this temperature the polymer collapses and blocks binding. The collapse of the polymer and thus the enabling and disabling of binding, is reversible. Such environmentally triggered control of binding may find many applications in biotechnology and biomedicine, such as the control of enzyme reaction rates and of biosensor activity, and the controlled release of drugs.

  11. Effects of Polymer Hydrophobicity on Protein Structure and Aggregation Kinetics in Crowded Milieu.

    PubMed

    Breydo, Leonid; Sales, Amanda E; Frege, Telma; Howell, Mark C; Zaslavsky, Boris Y; Uversky, Vladimir N

    2015-05-19

    We examined the effects of water-soluble polymers of various degrees of hydrophobicity on the folding and aggregation of proteins. The polymers we chose were polyethylene glycol (PEG) and UCON (1:1 copolymer of ethylene glycol and propylene glycol). The presence of additional methyl groups in UCON makes it more hydrophobic than PEG. Our earlier analysis revealed that similarly sized PEG and UCON produced different changes in the solvent properties of water in their solutions and induced morphologically different α-synuclein aggregates [Ferreira, L. A., et al. (2015) Role of solvent properties of aqueous media in macromolecular crowding effects. J. Biomol. Struct. Dyn., in press]. To improve our understanding of molecular mechanisms defining behavior of proteins in a crowded environment, we tested the effects of these polymers on secondary and tertiary structure and aromatic residue solvent accessibility of 10 proteins [five folded proteins, two hybrid proteins; i.e., protein containing ordered and disordered domains, and three intrinsically disordered proteins (IDPs)] and on the aggregation kinetics of insulin and α-synuclein. We found that effects of both polymers on secondary and tertiary structures of folded and hybrid proteins were rather limited with slight unfolding observed in some cases. Solvent accessibility of aromatic residues was significantly increased for the majority of the studied proteins in the presence of UCON but not PEG. PEG also accelerated the aggregation of protein into amyloid fibrils, whereas UCON promoted aggregation to amyloid oligomers instead. These results indicate that even a relatively small change in polymer structure leads to a significant change in the effect of this polymer on protein folding and aggregation. This is an indication that protein folding and especially aggregation are highly sensitive to the presence of other macromolecules, and an excluded volume effect is insufficient to describe their effect. PMID:25919930

  12. Surface modification on microfluidic devices with 2-methacryloyloxyethyl phosphorylcholine polymers for reducing unfavorable protein adsorption.

    PubMed

    Sibarani, James; Takai, Madoka; Ishihara, Kazuhiko

    2007-01-15

    Surface modification of polymer materials for preparing microfluidic devices including poly(dimethyl siloxane) (PDMS) was investigated with phospholipids polymers such as poly(2-methacryloyloxylethyl phosphorylcholine(MPC)-co-n-butyl methacrylate) (PMB) and poly(MPC-co-2-ethylhexyl methacrylate-co-2-(N,N-dimethylamino)ethyl methacrylate) (PMED). The hydrophilicity of every surface on the polymer materials modified with these MPC polymers increased and the value of zeta-potential became close to zero. The protein adsorption on the polymer materials with and without the surface modification was evaluated using a protein mixture of human plasma fibrinogen and serum albumin. Amount of proteins adsorbed on these polymeric materials showed significant reduction by the surface modification with the MPC polymers compared to the uncoated surfaces ranging from 56 to 90%. Furthermore, we successfully prepared PDMS-based microchannel which was modified by simple coating with the PMB and PMED. The modified microchannel also revealed a significant reduction of adsorption of serum albumin. We conclude that the MPC polymers are useful for reducing unfavorable protein adsorption on microfluidic devices. PMID:17112710

  13. Quantitative evaluation of interaction force between functional groups in protein and polymer brush surfaces.

    PubMed

    Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

    2014-03-18

    To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and n-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be

  14. BioC-compatible full-text passage detection for protein-protein interactions using extended dependency graph.

    PubMed

    Peng, Yifan; Arighi, Cecilia; Wu, Cathy H; Vijay-Shanker, K

    2016-01-01

    There has been a large growth in the number of biomedical publications that report experimental results. Many of these results concern detection of protein-protein interactions (PPI). In BioCreative V, we participated in the BioC task and developed a PPI system to detect text passages with PPIs in the full-text articles. By adopting the BioC format, the output of the system can be seamlessly added to the biocuration pipeline with little effort required for the system integration. A distinctive feature of our PPI system is that it utilizes extended dependency graph, an intermediate level of representation that attempts to abstract away syntactic variations in text. As a result, we are able to use only a limited set of rules to extract PPI pairs in the sentences, and additional rules to detect additional passages for PPI pairs. For evaluation, we used the 95 articles that were provided for the BioC annotation task. We retrieved the unique PPIs from the BioGRID database for these articles and show that our system achieves a recall of 83.5%. In order to evaluate the detection of passages with PPIs, we further annotated Abstract and Results sections of 20 documents from the dataset and show that an f-value of 80.5% was obtained. To evaluate the generalizability of the system, we also conducted experiments on AIMed, a well-known PPI corpus. We achieved an f-value of 76.1% for sentence detection and an f-value of 64.7% for unique PPI detection.Database URL: http://proteininformationresource.org/iprolink/corpora. PMID:27170286

  15. Comparison of descriptors for predicting selectivity of protein-imprinted polymers.

    PubMed

    Raim, Vladimir; Zadok, Israel; Srebnik, Simcha

    2016-08-01

    Molecular imprinting is a technique that is used to create artificial receptors by the formation of a polymer network around a template molecule, creating a molecularly imprinted polymer. These artificial receptors may be used in applications that require molecular recognition, such as enantioseparations, biosensors, artificial catalysis, drug delivery and others. Small molecules, such as drugs, have been imprinted with high efficiency and, combined with the low cost of preparation, molecularly imprinted polymers have acquired commercial usage. While attempts at imprinting proteins have been significantly less successful, the great potential of protein-imprinted polymers (PIPs) in medicine and industry attracted much research. Multifunctionality, conformational flexibility, large size of the proteins, and aqueous polymerization environment are some of the obstacles faced by protein imprinting. We explore the relation between PIP selectivity and the properties of the template and competitor proteins. A comprehensive statistical analysis of published studies reveals a statistically significant correlation between four protein descriptors and the corresponding selectivity of PIPs. Namely, a PIP will generally be more selective against large competitor proteins with a smooth surface, whose isoelectric point and aspect ratio are significantly different than those of the template protein. The size of the protein, as measured by its molecular weight, appears to be independent of the template protein characteristics. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26931646

  16. In Situ Infrared Ellipsometry for Protein Adsorption Studies on Ultrathin Smart Polymer Brushes in Aqueous Environment

    SciTech Connect

    Kroning, Annika; Furchner, Andreas; Aulich, Dennis; Bittrich, Eva; Rauch, Sebastian; Uhlmann, Petra; Eichhorn, Klaus-Jochen; Seeber, Michael; Luzinov, Igor; Kilbey, S. Michael; Lokitz, Bradley S.; Minko, Sergiy; Hinrichs, Karsten

    2015-02-10

    The protein-adsorbing and -repelling properties of various smart nanometer-thin polymer brushes with high potential for biosensing and biomedical applications are studied by in-situ infrared-spectroscopic ellipsometry (IRSE). IRSE as a highly sensitive non-destructive technique allows us to investigate protein adsorption on polymer brushes in aqueous environment in dependence of external stimuli like temperature and pH. These stimuli are, for instance, relevant in switchable mixed brushes containing poly(N-isopropyl acrylamide) and poly(acrylic acid), respectively. We use such brushes as model surfaces for controlling protein adsorption of human serum albumin and human fibrinogen. IRSE can distinguish between polymer-specific vibrational bands, which yield insights into the hydration state of the brushes, and changes in the protein-specific amide bands, which are related to changes of the protein secondary structure.

  17. In Situ Infrared Ellipsometry for Protein Adsorption Studies on Ultrathin Smart Polymer Brushes in Aqueous Environment

    DOE PAGESBeta

    Kroning, Annika; Furchner, Andreas; Aulich, Dennis; Bittrich, Eva; Rauch, Sebastian; Uhlmann, Petra; Eichhorn, Klaus-Jochen; Seeber, Michael; Luzinov, Igor; Kilbey, S. Michael; et al

    2015-02-10

    The protein-adsorbing and -repelling properties of various smart nanometer-thin polymer brushes with high potential for biosensing and biomedical applications are studied by in-situ infrared-spectroscopic ellipsometry (IRSE). IRSE as a highly sensitive non-destructive technique allows us to investigate protein adsorption on polymer brushes in aqueous environment in dependence of external stimuli like temperature and pH. These stimuli are, for instance, relevant in switchable mixed brushes containing poly(N-isopropyl acrylamide) and poly(acrylic acid), respectively. We use such brushes as model surfaces for controlling protein adsorption of human serum albumin and human fibrinogen. IRSE can distinguish between polymer-specific vibrational bands, which yield insights intomore » the hydration state of the brushes, and changes in the protein-specific amide bands, which are related to changes of the protein secondary structure.« less

  18. Effects of Crosslinking on the Mechanical Properties Drug Release, and Cytocompatibility of Protein Polymers

    PubMed Central

    Martinez, Adam W.; Caves, Jeffrey M.; Ravi, Swathi; Li, Wehnsheng; Chaikof, Elliot L.

    2013-01-01

    Recombinant elastin-like protein polymers are increasingly being investigated as component materials of a variety of implantable medical devices. This is chiefly a result of their favorable biological properties and the ability to tailor their physical and mechanical properties. In this report, we explore the potential of modulating the water content, mechanical properties, and drug release profiles of protein films through the selection of different crosslinking schemes and processing strategies. We find that the selection of crosslinking scheme and processing strategy has a significant influence on all aspects of protein polymer films. Significantly, utilization of a confined, fixed volume, as well as vapor phase crosslinking strategies decreased protein polymer equilibrium water content. Specifically, as compared to uncrosslinked protein gels, water content was reduced for genipin (15.5%), glutaraldehyde (GTA) (24.5%), GTA vapor crosslinking (31.6%), disulfide (SS) (18.2%), and SS vapor crosslinking (25.5%) (p <0.05). Distinct crosslinking strategies modulated protein polymer stiffness, strain at failure, and ultimate tensile strength (UTS). In all cases, vapor phase crosslinking produced the stiffest films with the highest UTS. Moreover, both confined, fixed volume and vapor phase approaches influenced drug delivery rates; with decreased initial drug burst and release rates as compared to solution phase crosslinking. Tailored crosslinking strategies provide an important option for modulating the physical, mechanical, and drug delivery properties of protein polymers. PMID:23993944

  19. Chemical interactions between protein molecules and polymer membrane materials. Annual progress report, August 1, 1992--July 30, 1993

    SciTech Connect

    Belfort, G.; Koehler, J.; Wood, J.

    1993-07-15

    The Surface Force Apparatus is now operable; data collection is automatic. Hen egg lysozyme was chosen as model protein. Protein-protein, protein-mica, protein-polymer, and protein-surfactant interactions were studied. Circular dichroism was used to study changes in protein structure during adsorption.

  20. In Situ Infrared Ellipsometry for Protein Adsorption Studies on Ultrathin Smart Polymer Brushes in Aqueous Environment.

    PubMed

    Kroning, Annika; Furchner, Andreas; Aulich, Dennis; Bittrich, Eva; Rauch, Sebastian; Uhlmann, Petra; Eichhorn, Klaus-Jochen; Seeber, Michael; Luzinov, Igor; Kilbey, S Michael; Lokitz, Bradley S; Minko, Sergiy; Hinrichs, Karsten

    2015-06-17

    The protein-adsorbing and -repelling properties of various smart nanometer-thin polymer brushes containing poly(N-isopropylacrylamide) and poly(acrylic acid) with high potential for biosensing and biomedical applications are studied by in situ infrared-spectroscopic ellipsometry (IRSE). IRSE is a highly sensitive nondestructive technique that allows protein adsorption on polymer brushes to be investigated in an aqueous environment as external stimuli, such as temperature and pH, are varied. These changes are relevant to conditions for regulation of protein adsorption and desorption for biotechnology, biocatalysis, and bioanalytical applications. Here brushes are used as model surfaces for controlling protein adsorption of human serum albumin and human fibrinogen. The important finding of this work is that IRSE in the in situ experiments in protein solutions can distinguish between contributions of polymer brushes and proteins. The vibrational bands of the polymers provide insights into the hydration state of the brushes, whereas the protein-specific amide bands are related to changes of the protein secondary structure. PMID:25668395

  1. 'All-solid-state' electrochemistry of a protein-confined polymer electrolyte film

    SciTech Connect

    Parthasarathy, Meera; Pillai, Vijayamohanan K. Mulla, Imtiaz S.; Shabab, Mohammed; Khan, M.I.

    2007-12-07

    Interfacial redox behavior of a heme protein (hemoglobin) confined in a solid polymer electrolyte membrane, Nafion (a perfluoro sulfonic acid ionomer) is investigated using a unique 'all-solid-state' electrochemical methodology. The supple phase-separated structure of the polymer electrolyte membrane, with hydrophilic pools containing solvated protons and water molecules, is found to preserve the incorporated protein in its active form even in the solid-state, using UV-visible, Fluorescence (of Tryptophan and Tyrosine residues) and DRIFT (diffuse reflectance infrared Fourier transform) spectroscopy. More specifically, solid-state cyclic voltammetry and electrochemical impedance of the protein-incorporated polymer films reveal that the Fe{sup 2+}-form of the entrapped protein is found to bind molecular oxygen more strongly than the native protein. In the 'all-solid-state' methodology, as there is no need to dip the protein-modified electrode in a liquid electrolyte (like the conventional electrochemical methods), it offers an easier means to study a number of proteins in a variety of polymer matrices (even biomimetic assemblies). In addition, the results of the present investigation could find interesting application in a variety of research disciplines, in addition to its fundamental scientific interest, including protein biotechnology, pharmaceutical and biomimetic chemistry.

  2. Measurements of water sorption enthalpy on polymer surfaces and its effect on protein adsorption

    NASA Astrophysics Data System (ADS)

    Kim, Joonyeong; Qian, Wei; Al-Saigh, Zeki Y.

    2011-02-01

    The molar enthalpy of sorption ( ΔHms`) of water vapor onto three polymer surfaces and its effect on nonspecific protein adsorption were investigated by inverse gas chromatography (IGC). The values of ΔHms measured by IGC were found to be -16.9 ± 1.2, -18.6 ± 1.3, and -29.9 ± 2.4 kJ/mole for polystyrene (PS), polymethylmethacrylate (PMMA), and poly(2-hydroxyethyl methacrylate) (PHEMA), respectively, over a temperature range of 333-423 K. Protein adsorption to three polymer-coated substrates was conducted as a function of the bulk protein concentration using lysozyme, fibrinogen, and bovine serum albumin (BSA), and the amount of adsorbed protein was measured by the solution depletion method. For a given bulk protein concentration, a larger amount of protein is adsorbed on PS and PMMA surfaces which have greater ΔHms than that of PHEMA surfaces. Although ΔHms for PS and PMMA are close to each other, PS surfaces were found to exhibit a higher adsorption affinity than PMMA surfaces over the proteins and concentrations investigated. Our results indicate that the strength of water-polymer interactions and the functional groups on the polymer surface are important factors for controlling the amount of nonspecifically adsorbed protein.

  3. Capillary electrophoresis-mass spectrometry of basic proteins using a new physically adsorbed polymer coating. Some applications in food analysis.

    PubMed

    Simó, Carolina; Elvira, Carlos; González, Nieves; San Román, J; Barbas, Coral; Cifuentes, Alejandro

    2004-07-01

    A new physically adsorbed capillary coating for capillary electrophoresis-mass spectrometry (CE-MS) of basic proteins is presented, which is easily obtained by flushing the capillary with a polymer aqueous solution for two min. This coating significantly reduces the electrostatic adsorption of a group of basic proteins (i.e., cytochrome c, lysozyme, and ribonuclease A) onto the capillary wall allowing their analysis by CE-MS. The coating protocol is compatible with electrospray inonization (ESI)-MS via the reproducible separation of the standard basic proteins (%RSD values (n = 5) < 1% for analysis time reproducibility and < 5% for peak heights, measured from the total ion electropherograms (TIEs) within the same day). The LODs determined using cytochrome c with total ion current and extracted ion current defection were 24.5 and 2.9 fmol, respectively. Using this new coating lysozymes from chicken and turkey egg white could be easily distinguished by CE-MS, demonstrating the usefulness of this method to differentiate animal species. Even after sterilization at 120 degrees C for 30 min, lysozyme could be detected, as well as in wines at concentrations much lower than the limit marked by the EC Commission Regulation. Adulteration of minced meat with 5% of egg-white could also be analysed by our CE-MS protocol. PMID:15237406

  4. An Experimental-Theoretical Analysis of Protein Adsorption on Peptidomimetic Polymer Brushes

    PubMed Central

    Lau, K.H. Aaron; Ren, Chunlai; Park, Sung Hyun; Szleifer, Igal; Messersmith, Phillip B.

    2012-01-01

    Surface-grafted water soluble polymer brushes are being intensely investigated for preventing protein adsorption to improve biomedical device function, prevent marine fouling, and enable applications in biosensing and tissue engineering. In this contribution, we present an experimental-theoretical analysis of a peptidomimetic polymer brush system with regard to the critical brush density required for preventing protein adsorption at varying chain lengths. A mussel adhesive-inspired DOPA-Lys pentapeptide surface grafting motif enabled aqueous deposition of our peptidomimetic polypeptoid brushes over a wide range of chain densities. Critical densities of 0.88 nm−2 for a relatively short polypeptoid 10-mer to 0.42 nm−2 for a 50-mer were identified from measurements of protein adsorption. The experiments were also compared with the protein adsorption isotherms predicted by a molecular theory. Excellent agreements in terms of both the polymer brush structure and the critical chain density were obtained. Furthermore, atomic force microscopy (AFM) imaging is shown to be useful in verifying the critical brush density for preventing protein adsorption. The present co-analysis of experimental and theoretical results demonstrates the significance of characterizing the critical brush density in evaluating the performance of an anti-fouling polymer brush system. The high fidelity of the agreement between the experiments and molecular theory also indicate that the theoretical approach presented can aid in the practical design of antifouling polymer brush systems. PMID:22107438

  5. Functional Polymers in Protein Detection Platforms: Optical, Electrochemical, Electrical, Mass-Sensitive, and Magnetic Biosensors

    PubMed Central

    Hahm, Jong-in

    2011-01-01

    The rapidly growing field of proteomics and related applied sectors in the life sciences demands convenient methodologies for detecting and measuring the levels of specific proteins as well as for screening and analyzing for interacting protein systems. Materials utilized for such protein detection and measurement platforms should meet particular specifications which include ease-of-mass manufacture, biological stability, chemical functionality, cost effectiveness, and portability. Polymers can satisfy many of these requirements and are often considered as choice materials in various biological detection platforms. Therefore, tremendous research efforts have been made for developing new polymers both in macroscopic and nanoscopic length scales as well as applying existing polymeric materials for protein measurements. In this review article, both conventional and alternative techniques for protein detection are overviewed while focusing on the use of various polymeric materials in different protein sensing technologies. Among many available detection mechanisms, most common approaches such as optical, electrochemical, electrical, mass-sensitive, and magnetic methods are comprehensively discussed in this article. Desired properties of polymers exploited for each type of protein detection approach are summarized. Current challenges associated with the application of polymeric materials are examined in each protein detection category. Difficulties facing both quantitative and qualitative protein measurements are also identified. The latest efforts on the development and evaluation of nanoscale polymeric systems for improved protein detection are also discussed from the standpoint of quantitative and qualitative measurements. Finally, future research directions towards further advancements in the field are considered. PMID:21691441

  6. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    PubMed

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  7. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.

    PubMed

    Quiroz, Felipe García; Chilkoti, Ashutosh

    2015-11-01

    Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level. PMID:26390327

  8. Bio-Organic Nanotechnology: Using Proteins and Synthetic Polymers for Nanoscale Devices

    NASA Technical Reports Server (NTRS)

    Molnar, Linda K.; Xu, Ting; Trent, Jonathan D.; Russell, Thomas P.

    2003-01-01

    While the ability of proteins to self-assemble makes them powerful tools in nanotechnology, in biological systems protein-based structures ultimately depend on the context in which they form. We combine the self-assembling properties of synthetic diblock copolymers and proteins to construct intricately ordered, three-dimensional polymer protein structures with the ultimate goal of forming nano-scale devices. This hybrid approach takes advantage of the capabilities of organic polymer chemistry to build ordered structures and the capabilities of genetic engineering to create proteins that are selective for inorganic or organic substrates. Here, microphase-separated block copolymers coupled with genetically engineered heat shock proteins are used to produce nano-scale patterning that maximizes the potential for both increased structural complexity and integrity.

  9. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

    NASA Astrophysics Data System (ADS)

    Quiroz, Felipe García; Chilkoti, Ashutosh

    2015-11-01

    Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level.

  10. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

    PubMed Central

    Quiroz, Felipe García; Chilkoti, Ashutosh

    2015-01-01

    Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level. PMID:26390327

  11. High-molecular-weight polymers for protein crystallization: poly-γ-glutamic acid-based precipitants

    SciTech Connect

    Hu, Ting-Chou; Korczyńska, Justyna; Smith, David K.; Brzozowski, Andrzej Marek

    2008-09-01

    High-molecular-weight poly-γ-glutamic acid-based polymers have been synthesized, tested and adopted for protein crystallization. Protein crystallization has been revolutionized by the introduction of high-throughput technologies, which have led to a speeding up of the process while simultaneously reducing the amount of protein sample necessary. Nonetheless, the chemistry dimension of protein crystallization has remained relatively undeveloped. Most crystallization screens are based on the same set of precipitants. To address this shortcoming, the development of new protein precipitants based on poly-γ-glutamic acid (PGA) polymers with different molecular-weight ranges is reported here: PGA-LM (low molecular weight) of ∼400 kDa and PGA-HM (high molecular weight) of >1000 kDa. It is also demonstrated that protein precipitants can be expanded further to polymers with much higher molecular weight than those that are currently in use. Furthermore, the modification of PGA-like polymers by covalent attachments of glucosamine substantially improved their solubility without affecting their crystallization properties. Some preliminary PGA-based screens are presented here.

  12. Polymers.

    ERIC Educational Resources Information Center

    Tucker, David C.

    1986-01-01

    Presents an open-ended experiment which has students exploring polymer chemistry and reverse osmosis. This activity involves construction of a polymer membrane, use of it in a simple osmosis experiment, and application of its principles in solving a science-technology-society problem. (ML)

  13. Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

    SciTech Connect

    Backues, Steven K.; Bednarek, Sebastian Y.

    2010-03-19

    The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.

  14. Protein PEGylation attenuates adsorption and aggregation on a negatively charged and moderately hydrophobic polymer surface.

    PubMed

    Pai, Sheetal S; Przybycien, Todd M; Tilton, Robert D

    2010-12-01

    Covalent grafting of poly(ethylene glycol) chains to proteins ("PEGylation") is emerging as an effective technique to increase the in vivo circulation time and efficacy of protein drugs. PEGylated protein adsorption at a variety of solid/aqueous interfaces is a critical aspect of their manufacture, storage, and delivery. A special category of block copolymer, PEGylated proteins have one or more water-soluble linear polymer (PEG) blocks and a single globular protein block that each exert distinct intermolecular and surface interaction forces. We report the impact of PEGylation on protein adsorption at the interface between aqueous solutions and solid films of poly(lactide-co-glycolide) (PLG), a moderately hydrophobic and negatively charged polymer. Using the model protein lysozyme with controlled degrees of PEGylation, we employ total internal reflection fluorescence techniques to measure adsorption isotherms, adsorption reversibility, and the extent of surface-induced aggregation. Lysozyme PEGylation reduces the extent of protein adsorption and surface-induced aggregation and increases the reversibility of adsorption compared to the unconjugated protein. Results are interpreted in terms of steric forces among grafted PEG chains and their effects on protein-protein interactions and protein orientation on the surface. PMID:21067142

  15. The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization and hydrophilic polymer grafting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein membrane separation is prone to fouling on the membrane surface resulting from protein adsorption onto the surface. Surface modification of synthetic membranes is one way to reduce fouling. We investigated surface modification of polyethersulfone (PES) as a way of improving hydrophilicity ...

  16. Protein displacement in dye-ligand chromatography using neutral and charged polymers.

    PubMed

    Galaev IYu; Arvidsson, P; Mattiasson, B

    1998-01-01

    Displacement chromatography was demonstrated to perform separations efficiently under mass-overloaded conditions, offering advantages such as increased product recovery and purity, superior resolving power, and concentration and purification in a single processing step. The use of water-soluble polymers for protein displacement in dye-ligand chromatography was initiated in our laboratory. The polymers for displacement were selected using differences spectroscopy to monitor their interactions with a dye-ligand in solution. Non-charged polymers such as poly(N-vinyl pyrrolidone) and poly(N-vinyl caprolactam) efficiently displaced lactate dehydrogenase from porcine muscle from a Blue Sepahrose column. The latter polymer, being thermosensitive, could be easily removed from the eluate and recovered by precipitation at 45 degrees C and low-speed centrifugation. The positively charged polymer poly(ethylene imine) proved to be an even more efficient displacer. The dye-ligand column could be regenerated after application of displacer either by washing with a solution of the soluble ligand Cibacron Blue (in the case of non-charged polymers) or by washing with highly alkaline solutions containing polyanions (in the case of poly(ethylene imine)) The latter formed a soluble complex with poly(ethylene imine) and stripped the column from the polymer. PMID:10076852

  17. Origins of Structural Flexibility in Protein-Based Supramolecular Polymers Revealed by DEER Spectroscopy

    PubMed Central

    2015-01-01

    Modular assembly of bio-inspired supramolecular polymers is a powerful technique to develop new soft nanomaterials, and protein folding is a versatile basis for preparing such materials. Previous work demonstrated a significant difference in the physical properties of closely related supramolecular polymers composed of building blocks in which identical coiled-coil-forming peptides are cross-linked by one of two subtly different organic linkers (one flexible and the other rigid). Herein, we investigate the molecular basis for this observation by isolating a single subunit of the supramolecular polymer chain and probing its structure and conformational flexibility by double electron–electron resonance (DEER) spectroscopy. Experimental spin–spin distance distributions for two different labeling sites coupled with molecular dynamics simulations provide insights into how the linker structure impacts chain dynamics in the coiled-coil supramolecular polymer. PMID:25060334

  18. Origins of structural flexibility in protein-based supramolecular polymers revealed by DEER spectroscopy.

    PubMed

    Tavenor, Nathan A; Silva, K Ishara; Saxena, Sunil; Horne, W Seth

    2014-08-21

    Modular assembly of bio-inspired supramolecular polymers is a powerful technique to develop new soft nanomaterials, and protein folding is a versatile basis for preparing such materials. Previous work demonstrated a significant difference in the physical properties of closely related supramolecular polymers composed of building blocks in which identical coiled-coil-forming peptides are cross-linked by one of two subtly different organic linkers (one flexible and the other rigid). Herein, we investigate the molecular basis for this observation by isolating a single subunit of the supramolecular polymer chain and probing its structure and conformational flexibility by double electron-electron resonance (DEER) spectroscopy. Experimental spin-spin distance distributions for two different labeling sites coupled with molecular dynamics simulations provide insights into how the linker structure impacts chain dynamics in the coiled-coil supramolecular polymer. PMID:25060334

  19. Biological and protein-binding studies of newly synthesized polymer-cobalt(III) complexes.

    PubMed

    Vignesh, G; Pradeep, I; Arunachalam, S; Vignesh, S; Arthur James, R; Arun, R; Premkumar, K

    2016-03-01

    The polymer-cobalt(III) complexes, [Co(bpy)(dien)BPEI]Cl3 · 4H2O (bpy = 2,2'-bipyridine, dien = diethylentriamine, BPEI = branched polyethyleneimine) were synthesized and characterized. The interaction of these complexes with human serum albumin (HSA) and bovine serum albumin (BSA) was investigated under physiological conditions using various physico-chemical techniques. The results reveal that the fluorescence quenching of serum albumins by polymer-cobalt(III) complexes took place through static quenching. The binding of these complexes changed the molecular conformation of the protein considerably. The polymer-cobalt(III) complex with x = 0.365 shows antimicrobial activity against several human pathogens. This complex also induces cytotoxicity against MCF-7 through apoptotic induction. However, further studies are needed to decipher the molecular mode of action of polymer-cobalt(III) complex and for its possible utilization in anticancer therapy. PMID:26278128

  20. Transiently responsive protein-polymer conjugates via a 'grafting-from' RAFT approach for intracellular co-delivery of proteins and immune-modulators.

    PubMed

    Vanparijs, N; De Coen, R; Laplace, D; Louage, B; Maji, S; Lybaert, L; Hoogenboom, R; De Geest, B G

    2015-09-21

    We report on transiently responsive protein-polymer conjugates that temporarily change their protein conformation from the soluble to the particle-like state. 'Grafting-from' RAFT polymerization of a dioxolane-containing acrylamide with a protein macroCTA is used to design polymer-protein conjugates that self-assemble into nanoparticles at physiological temperature and pH. Acid triggered hydrolysis of the dioxolane units into diol moeities rendered the conjugates fully water soluble irrespective of temperature. PMID:26242974

  1. Elastic Properties of Protein Functionalized Nanoporous Polymer Films.

    PubMed

    Wang, Haoyu; Black, Charles T; Akcora, Pinar

    2016-01-12

    Retaining the conformational structure and bioactivity of immobilized proteins is important for biosensor designs and drug delivery systems. Confined environments often lead to changes in conformation and functions of proteins. In this study, lysozyme is chemically tethered into nanopores of polystyrene thin films, and submicron pores in poly(methyl methacrylate) films are functionalized with streptavidin. Nanoindentation experiments show that stiffness of streptavidin increases with decreasing submicron pore sizes. Lysozymes in polystyrene nanopores are found to behave stiffer than the submicron pore sizes and still retain their specific bioactivity relative to the proteins on flat surfaces. Our results show that protein functionalized ordered nanoporous polystyrene/poly(methyl methacrylate) films present heterogeneous elasticity and can be used to study interactions between free proteins and designed surfaces. PMID:26672623

  2. Elastic properties of protein functionalized nanoporous polymer films

    SciTech Connect

    Charles T. Black; Wang, Haoyu; Akcora, Pinar

    2015-12-16

    Retaining the conformational structure and bioactivity of immobilized proteins is important for biosensor designs and drug delivery systems. Confined environments often lead to changes in conformation and functions of proteins. In this study, lysozyme is chemically tethered into nanopores of polystyrene thin films, and submicron pores in poly(methyl methacrylate) films are functionalized with streptavidin. Nanoindentation experiments show that stiffness of streptavidin increases with decreasing submicron pore sizes. Lysozymes in polystyrene nanopores are found to behave stiffer than the submicron pore sizes and still retain their specific bioactivity relative to the proteins on flat surfaces. Lastly, our results show that protein functionalized ordered nanoporous polystyrene/poly(methyl methacrylate) films present heterogeneous elasticity and can be used to study interactions between free proteins and designed surfaces.

  3. Compatible solutes

    PubMed Central

    Hill, Colin

    2010-01-01

    Recently we reported a role for compatible solute uptake in mediating bile tolerance and increased gastrointestinal persistence in the foodborne pathogen Listeria monocytogenes.1 Herein, we review the evolution in our understanding of how these low molecular weight molecules contribute to growth and survival of the pathogen both inside and outside the body, and how this stress survival mechanism may ultimately be used to target and kill the pathogen. PMID:21326913

  4. Elastic properties of protein functionalized nanoporous polymer films

    DOE PAGESBeta

    Charles T. Black; Wang, Haoyu; Akcora, Pinar

    2015-12-16

    Retaining the conformational structure and bioactivity of immobilized proteins is important for biosensor designs and drug delivery systems. Confined environments often lead to changes in conformation and functions of proteins. In this study, lysozyme is chemically tethered into nanopores of polystyrene thin films, and submicron pores in poly(methyl methacrylate) films are functionalized with streptavidin. Nanoindentation experiments show that stiffness of streptavidin increases with decreasing submicron pore sizes. Lysozymes in polystyrene nanopores are found to behave stiffer than the submicron pore sizes and still retain their specific bioactivity relative to the proteins on flat surfaces. Lastly, our results show that proteinmore » functionalized ordered nanoporous polystyrene/poly(methyl methacrylate) films present heterogeneous elasticity and can be used to study interactions between free proteins and designed surfaces.« less

  5. Kinetics of protein adsorption/desorption mediated by pH-responsive polymer layer

    NASA Astrophysics Data System (ADS)

    Su, Xiao-Hang; Lei, Qun-Li; Ren, Chun-Lai

    2015-11-01

    We propose a new way of regulating protein adsorption by using a pH-responsive polymer. According to the theoretical results obtained from the molecular theory and kinetic approaches, both thermodynamics and kinetics of protein adsorption are verified to be well controlled by the solution pH. The kinetics and the amount of adsorbed proteins at equilibrium are greatly increased when the solution environment changes from acid to neutral. The reason is that the increased pH promotes the dissociation of the weak polyelectrolyte, resulting in more charged monomers and more stretched chains. Thus the steric repulsion within the polymer layer is weakened, which effectively lowers the barrier felt by the protein during the process of adsorption. Interestingly, we also find that the kinetics of protein desorption is almost unchanged with the variation of pH. It is because although the barrier formed by the polymer layer changes along with the change of pH, the potential at contact with the surface varies equally. Our results may provide useful insights into controllable protein adsorption/desorption in practical applications. Project supported by the National Natural Science Foundation of China (Grant Nos. 21274062, 11474155, and 91027040).

  6. Multivalent protein polymers with controlled chemical and physical properties.

    PubMed

    Top, Ayben; Kiick, Kristi L

    2010-12-30

    In this review, we describe our work on the design, characterization, and modification of a series of alanine-rich helical polypeptides with novel functions. Glycosylation of the polypeptides has permitted investigation of polymer architecture effects on multivalent interactions. One of the members of this polypeptide family exhibits polymorphological behavior that is easily manipulated via simple changes in solution pH and temperature. Polypeptide-based fibrils formed at acidic pH and high temperature were shown to direct the one-dimensional organization of gold nanoparticles via electrostatic interactions. As a precursor to fibrils, aggregates likely comprising alanine-rich cores form at low temperatures and acidic pH and reversibly dissociate into monomers upon deprotonation. PEGylation of these polypeptides does not alter the self-association or conformational behavior of the polypeptide, suggesting potential applications in the development of assembled delivery vehicles, as modification of the polypeptides should be a useful strategy for controlling assembly. PMID:20562016

  7. Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

    PubMed

    Adams, Justin R; Haughney, Shannon L; Mallapragada, Surya K

    2015-03-01

    We have synthesized thermogelling cationic amphiphilic pentablock copolymers that have the potential to act as injectable vaccine carriers and adjuvants that can simultaneously provide sustained delivery and enhance the immunogenicity of released antigen. While these pentablock copolymers have shown efficacy in DNA delivery in past studies, the ability to deliver both DNA and protein for subunit vaccines using the same polymeric carrier can provide greater flexibility and efficacy. We demonstrate the ability of these pentablock copolymers, and the parent triblock Pluronic copolymers to slowly release structurally intact and antigenically stable protein antigens in vitro, create an antigen depot through long-term injection-site persistence and enhance the in vivo immune response to these antigens. We show release of the model protein antigen ovalbumin in vitro from the thermogelling block copolymers with the primary, secondary and tertiary structures of the released protein unchanged compared to the native protein, and its antigenicity preserved upon release. The block copolymers form a gel at physiological temperatures that serves as an antigenic depot and persists in vivo at the site of injection for over 50days. The pentablock copolymers show a significant fivefold enhancement in the immune response compared to soluble protein alone, even 6weeks after the administration, based on measurement of antibody titers. These results demonstrate the potential of these block copolymers hydrogels to persist for several weeks and sustain the release of antigen with minimal effects on protein stability and antigenicity; and their ability to be used simultaneously as a sustained delivery device as well as a subunit vaccine adjuvant platform. PMID:25484331

  8. Coil fraction-dependent phase behaviour of a model globular protein-polymer diblock copolymer.

    PubMed

    Thomas, Carla S; Olsen, Bradley D

    2014-05-01

    The self-assembly of the model globular protein-polymer block copolymer mCherry-b-poly(N-isopropyl acrylamide) is explored across a range of polymer coil fractions from 0.21 to 0.82 to produce a phase diagram for these materials as a function of molecular composition. Overall, four types of morphologies were observed: hexagonally packed cylinders, perforated lamellae, lamellae, and disordered nanostructures. Across all coil fractions and morphologies, a lyotropic re-entrant order-disorder transition in water was observed, with disordered structures below 30 wt% and above 70 wt% and well-ordered morphologies at intermediate concentrations. Solid state samples prepared by solvent evaporation show moderately ordered structures similar to those observed in 60 wt% solutions, suggesting that bulk structures result from kinetic trapping of morphologies which appear at lower concentrations. While highly ordered cylindrical nanostructures are observed around a bioconjugate polymer volume fraction of 0.3 and well-ordered lamellae are seen near a volume fraction of 0.6, materials at lower or higher coil fractions become increasingly disordered. Notable differences between the phase behaviour of globular protein-polymer block copolymers and coil-coil diblock copolymers include the lack of spherical nanostructures at either high or low polymer coil fractions as well as shifted phase boundaries between morphologies which result in an asymmetric phase diagram. PMID:24695642

  9. Nonfouling tunable βCD dextran polymer films for protein applications.

    PubMed

    Städe, Lars W; Nielsen, Thorbjørn T; Duroux, Laurent; Hinge, Mogens; Shimizu, Kyoko; Gurevich, Leonid; Kristensen, Peter K; Wingren, Christer; Larsen, Kim L

    2015-02-25

    Polymeric β-cyclodextrin (βCD) films tunable with respect to thickness and βCD content were prepared in order to develop a suitable platform, allowing for inclusion of nonpolar guest molecules in the βCD cavity, while suppressing nonspecific protein adsorption. The βCD films were synthesized from linear βCD dextran polymers, and grafted onto silicon oxide surfaces by "click" chemistry. Topographic and morphological characteristics are controllable by reaction conditions and polymer type, with average film heights from 2.5 to 12.5 nm. Reversible introduction of electrostatic charges in the βCD dextran by complex formation with 1-adamantanecarboxylic acid prior to surface grafting resulted in a thinner and denser film, presumably by decompaction of the polymers. Total internal reflection fluorescence spectroscopy (TIRF) was employed to evaluate the accessibility of βCD cavities to the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid. Only a minor fraction of the βCD cavities was accessible in the thicker and less dense films; however, accessibility was largely improved with increased ionic strength using NaCl up to 1 M. Antifouling properties of the βCD dextran polymer films were assessed by TIRF real-time monitoring, using bovine serum albumin as a model protein, and showed a 5- to 10-fold reduction in nonspecific adsorption as compared to a bare quartz surface with the degree of reduction reflecting film thickness and interfacial polymer density. PMID:25639169

  10. Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs).

    PubMed

    EL-Sharif, Hazim F; Hawkins, Daniel M; Stevenson, Derek; Reddy, Subrayal M

    2014-08-01

    Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 μM, 44 ± 3 μM, 17 ± 2 μM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface. PMID:24950144

  11. SIMS depth profiling of polymer blends with protein based drugs

    NASA Astrophysics Data System (ADS)

    Mahoney, Christine M.; Yu, Jinxiang; Fahey, Albert; Gardella, Joseph A.

    2006-07-01

    We report the results of the surface and in-depth characterization of two component blend films of poly( L-lactic acid) (PLLA) and Pluronic surfactant [poly(ethylene oxide) (A) poly(propylene oxide) (B) ABA block copolymer]. These blend systems are of particular importance for protein drug delivery, where it is expected that the Pluronic surfactant will retain the activity of the protein drug and enhance the biocompatibility of the device. Angle dependant X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) employing an SF 5+ polyatomic primary ion source were both used for monitoring the surfactant's concentration as a function of depth. The results show an increased concentration of surfactant at the surface, where the surface segregation initially increases with increasing bulk concentration and then remains constant above 5% (w/w) Pluronic. This surface segregated region is immediately followed by a depletion region with a homogeneous mixture in the bulk of the film. These results suggest the selection of the surfactant bulk concentration of the thin film matrices for drugs/proteins delivery should achieve a relatively homogeneous distribution of stabilizer/protein in the PLLA matrix. Analysis of three component blends of PLLA, Pluronic and insulin are also investigated. In the three component blends, ToF-SIMS imaging shows the spatial distribution of surfactant/protein mixtures. These data are reported also as depth profiles.

  12. Novel Thermogelling Dispersions of Polymer Nanoparticles for Controlled Protein Release

    PubMed Central

    Cai, Tong; Hu, Peter D.; Sun, Manwu; Zhou, Jun; Tsai, Yi-Ting; Baker, David; Tang, Liping

    2012-01-01

    A novel poly(oligo(ethylene glycol) methyl ether methacrylate-co-oligo(ethylene glycol) ethyl ether methacrylate)/ poly(acrylic acid) interpenetrating network (IPN) nanoparticle was synthesized. The temperature-responsive properties of the IPN nanoparticles were investigated by dynamic light scattering method. Atomic force microscopic images confirm the homogenous and mono-disperse morphology of the IPN nanoparticles. Both visual observation and viscosity testing demonstrated that the IPN nanoparticles exhibit thermogelling properties at body temperature, 37°C. Subsequent studies verified that such temperature sensitive properties of IPN nanoparticles allow their ease of injection and then slow release of model proteins, both in vitro and in vivo. Histological analysis showed that our IPN implants exerted minimal inflammation following subcutaneous implantation. Our results support that, by simply mixing with proteins of interest, the novel IPN nanoparticles can be used to form in situ thermogelling devices for controlled protein release. PMID:22349097

  13. Self-assembly of silk-elastinlike protein polymers into three-dimensional scaffolds for biomedical applications

    NASA Astrophysics Data System (ADS)

    Zeng, Like

    Production of brand new protein-based materials with precise control over the amino acid sequences at single residue level has been made possible by genetic engineering, through which artificial genes can be developed that encode protein-based materials with desired features. As an example, silk-elastinlike protein polymers (SELPs), composed of tandem repeats of amino acid sequence motifs from Bombyx mori (silkworm) silk and mammalian elastin, have been produced in this approach. SELPs have been studied extensively in the past two decades, however, the fundamental mechanism governing the self-assembly process to date still remains largely unresolved. Further, regardless of the unprecedented success when exploited in areas including drug delivery, gene therapy, and tissue augmentation, SELPs scaffolds as a three-dimensional cell culture model system are complicated by the inability of SELPs to provide the embedded tissue cells with appropriate biochemical stimuli essential for cell survival and function. In this dissertation, it is reported that the self-assembly of silk-elastinlike protein polymers (SELPs) into nanofibers in aqueous solutions can be modulated by tuning the curing temperature, the size of the silk blocks, and the charge of the elastin blocks. A core-sheath model was proposed for nanofiber formation, with the silk blocks in the cores and the hydrated elastin blocks in the sheaths. The folding of the silk blocks into stable cores -- affected by the size of the silk blocks and the charge of the elastin blocks -- plays a critical role in the assembly of silk-elastin nanofibers. The assembled nanofibers further form nanofiber clusters on the microscale, and the nanofiber clusters then coalesce into nanofiber micro-assemblies, interconnection of which eventually leads to the formation of three-dimensional scaffolds with distinct nanoscale and microscale features. SELP-Collagen hybrid scaffolds were also fabricated to enable independent control over the

  14. Thermal property and processability of elastomeric polymer alloy composed of segmented polyurethane and phospholipid polymer.

    PubMed

    Ogawa, Ryo; Iwasaki, Yasuhiko; Ishihara, Kazuhiko

    2002-11-01

    To develop a thermoplastic elastomer with high blood compatibility, a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer was blended with the segmented polyurethane (SPU) for preparing the polymer alloy. A tensile strength measurement was carried out to evaluate its mechanical strength. The mechanical strength of the SPU/MPC polymer alloy is the same as that of the original SPU and sufficient for use in medical applications. The thermal properties of the polymer alloy were evaluated by differential scanning calorimetry (DSC). The DSC curves indicated that the MPC polymer blended into the SPU did not affect the hard segment domain of the SPU. The SPU/MPC polymer alloy can be processed by heat treatment at 150 degrees C. Even after heat treatment, the SPU/MPC polymer alloy showed good mechanical properties, and MPC units were observed on the surface. Protein adsorption from human plasma was observed to evaluate the blood compatibility of the polymer alloy. The SPU/MPC polymer alloy suppressed protein adsorption on the surface before and after the heat treatment. Based on these results, it is concluded that the SPU/MPC polymer alloy has an excellent potential for application in various medical devices. PMID:12209941

  15. Water-compatible molecularly imprinted polymer as a sorbent for the selective extraction and purification of adefovir from human serum and urine.

    PubMed

    Pourfarzib, Mojgan; Dinarvand, Rasoul; Akbari-Adergani, Behrouz; Mehramizi, Ali; Rastegar, Hossein; Shekarchi, Maryam

    2015-05-01

    A molecularly imprinted polymer has been synthesized to specifically extract adefovir, an antiviral drug, from serum and urine by dispersive solid-phase extraction before high-performance liquid chromatography with UV analysis. The imprinted polymers were prepared by bulk polymerization by a noncovalent imprinting method that involved the use of adefovir (template molecule) and functional monomer (methacrylic acid) complex prior to polymerization, ethylene glycol dimethacrylate as cross-linker, and chloroform as porogen. Molecular recognition properties, binding capacity, and selectivity of the molecularly imprinted polymers were evaluated and the results show that the obtained polymers have high specific retention and enrichment for adefovir in aqueous medium. The new imprinted polymer was utilized as a molecular sorbent for the separation of adefovir from human serum and urine. The serum and urine extraction of adefovir by the molecularly imprinted polymer followed by high-performance liquid chromatography showed a linear calibration curve in the range of 20-100 μg/L with excellent precisions (2.5 and 2.8% for 50 μg/L), respectively. The limit of detection and limit of quantization were determined in serum (7.62 and 15.1 μg/L), and urine (5.45 and 16 μg/L). The recoveries for serum and urine samples were found to be 88.2-93.5 and 84.3-90.2%, respectively. PMID:25763883

  16. Polymer adhesion at surfaces: biological adhesive proteins and their synthetic mimics

    NASA Astrophysics Data System (ADS)

    Messersmith, Phillip

    2008-03-01

    Mussels are famous for their ability to permanently adhere to a wide variety of wet surfaces, such as rocks, metal and polymer ship hulls, and wood structures. They accomplish this through specialized proteins collectively referred to as mussel adhesive proteins (MAPs). The biophysical aspects of MAP adhesion is being revealed through the use of single molecule force measurements. The results provide insight into the adhesive roles of key amino acids found in these proteins, including the magnitude of adhesive forces, cooperative effects, and their self-healing properties. This molecular-level information is being incorporated into designs of biomimetic polymer coatings for a variety of applications. Our biomimetic approach to polymer design will be illustrated by a few examples where adhesive constituents found in MAPs are exploited to make wet-adhesive polymer coatings. In addition, small molecule analogs of MAPs can be used to apply thin functional films onto virtually any material surface using a facile approach. These coatings have a variety of potential uses in microelectronics, water treatment, prevention of environmental biofouling, and for control of biointerfacial phenomena at the surfaces of medical/diagnostic devices.

  17. Self-Assembly of Temperature-Responsive Protein-Polymer Bioconjugates.

    PubMed

    Moatsou, Dafni; Li, Jian; Ranji, Arnaz; Pitto-Barry, Anaïs; Ntai, Ioanna; Jewett, Michael C; O'Reilly, Rachel K

    2015-09-16

    We report a simple temperature-responsive bioconjugate system comprising superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used amber suppression to site-specifically incorporate the non-canonical azide-functional amino acid p-azidophenylalanine (pAzF) into sfGFP at different positions. The azide moiety on modified sfGFP was then coupled using copper-catalyzed "click" chemistry with the alkyne terminus of a PEGMA synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The protein in the resulting bioconjugate was found to remain functionally active (i.e., fluorescent) after conjugation. Turbidity measurements revealed that the point of attachment of the polymer onto the protein scaffold has an impact on the thermoresponsive behavior of the resultant bioconjugate. Furthermore, small-angle X-ray scattering analysis showed the wrapping of the polymer around the protein in a temperature-dependent fashion. Our work demonstrates that standard genetic manipulation combined with an expanded genetic code provides an easy way to construct functional hybrid biomaterials where the location of the conjugation site on the protein plays an important role in determining material properties. We anticipate that our approach could be generalized for the synthesis of complex functional materials with precisely defined domain orientation, connectivity, and composition. PMID:26083370

  18. Protein Analysis by Ambient Ionization Mass Spectrometry Using Trypsin-Immobilized Organosiloxane Polymer Surfaces.

    PubMed

    Dulay, Maria T; Eberlin, Livia S; Zare, Richard N

    2015-12-15

    In the growing field of proteomic research, rapid and simple protein analysis is a crucial component of protein identification. We report the use of immobilized trypsin on hybrid organic-inorganic organosiloxane (T-OSX) polymers for the on-surface, in situ digestion of four model proteins: melittin, cytochrome c, myoglobin, and bovine serum albumin. Tryptic digestion products were sampled, detected, and identified using desorption electrospray ionization mass spectrometry (DESI-MS) and nanoDESI-MS. These novel, reusable T-OSX arrays on glass slides allow for protein digestion in methanol:water solvents (1:1, v/v) and analysis directly from the same polymer surface without the need for sample preparation, high temperature, and pH conditions typically required for in-solution trypsin digestions. Digestion reactions were conducted with 2 μL protein sample droplets (0.35 mM) at incubation temperatures of 4, 25, 37, and 65 °C and digestion reaction times between 2 and 24 h. Sequence coverages were dependent on the hydrophobicity of the OSX polymer support and varied by temperature and digestion time. Under the best conditions, the sequence coverages, determined by DESI-MS, were 100% for melittin, 100% for cytochrome c, 90% for myoglobin, and 65% for bovine serum albumin. PMID:26567450

  19. Grafting zwitterionic polymer onto cryogel surface enhances protein retention in steric exclusion chromatography on cryogel monolith.

    PubMed

    Tao, Shi-Peng; Zheng, Jie; Sun, Yan

    2015-04-10

    Cryogel monoliths with interconnected macropores (10-100μm) and hydrophilic surfaces can be employed as chromatography media for protein retention in steric exclusion chromatography (SXC). SXC is based on the principle that the exclusion of polyethylene glycol (PEG) on both a hydrophilic chromatography surface and a protein favors their association, leading to the protein retention on the chromatography surface. Elution of the retained protein can be achieved by reducing PEG concentration. In this work, the surface of polyacrylamide-based cryogel monolith was modified by grafting zwitterionic poly(carboxybetaine methacrylate) (pCBMA), leading the increase in the surface hydrophilicity. Observation by scanning electron microscopy revealed the presence of the grafted pCBMA chain clusters on the cryogel surface, but pCBMA grafting did not result in the changes of the physical properties of the monolith column, and the columns maintained good recyclability in SXC. The effect of the surface grafting on the SXC behavior of γ-globulin was investigated in a wide flow rate range (0.6-12cm/min). It was found that the dynamic retention capacity increased 1.4-1.8 times by the zwitterionic polymer grafting in the flow rate range of 1.5-12cm/min. The mechanism of enhanced protein retention on the zwitterionic polymer-grafted surface was proposed. The research proved that zwitterionic polymer modification was promising for the development of new materials for SXC applications. PMID:25757821

  20. Bifunctional polyacrylamide based polymers for the specific binding of hexahistidine tagged proteins on gold surfaces.

    PubMed

    Thompson, Lucas B; Mack, Nathan H; Nuzzo, Ralph G

    2010-05-01

    We describe a modified bifunctional analogue of polyacrylamide that spontaneously forms self-assembled polymeric thin films on Au surfaces. The film is engineered to specifically bind histidine tagged proteins (6His), while simultaneously remaining inherently resistant to the non-specific adsorption of proteins in solution. The backbone of a polyacrylamide-co-n-acryloxysuccinimide copolymer is functionalized via tandem active ester (NHS) couplings with 3-(methylthio)propylamine (MTP) and nitrilotriacetic acid (NTA). The resulting functionalized polymers form stable and exceptionally hydrophilic thin films that are approximately 2-5 nm thick, a mass coverage that varies with the MTP graft density. These films are characterized using a variety of techniques (X-ray photoelectron spectroscopy (XPS), reflection absorption infrared spectroscopy (RAIRS), ellipsometry, surface plasmon resonance (SPR), and matrix assisted laser desorption ionization (MALDI)) to establish their structure and function. The protein resistance of the films, as demonstrated by their exposure to solutions of bovine serum albumin (BSA), can be modulated by the amount of MTP grafted to the polymer, which in turn, affects their mass coverage. We show that it is possible to specifically capture hexahistidine tagged proteins with low incidences of nonspecific adsorption using these materials, a discrimination quantified using surface plasmon resonance (SPR) at concentrations down to approximately 20 nM. These polymers also bind strongly to the surfaces of Au nanoparticles, stabilizing them against aggregation, providing them with a similar capacity to selectively bind 6His tagged proteins that can then be speciated using MALDI. PMID:20407699

  1. Trypsin immobilization in ordered porous polymer membranes for effective protein digestion.

    PubMed

    Qiao, Juan; Kim, Jin Yong; Wang, Yuan Yuan; Qi, Li; Wang, Fu Yi; Moon, Myeong Hee

    2016-02-01

    Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores. PMID:26772135

  2. Nanoparticle-Templated Formation and Growth Mechanism of Curved Protein Polymer Fibrils.

    PubMed

    Pham, Thao T H; Rombouts, Wolf H; Fokkink, Remco; Stuart, Marc C A; Cohen Stuart, Martien A; Kleijn, J Mieke

    2016-07-11

    We investigated the growth of biosynthetic protein polymers with templated curvature on pluronic nanospheres. The protein has a central silk-like block containing glutamic residues (S(E)) and collagen-like end-blocks (C). The S(E) blocks stack into filaments when their charge is removed (pH <5). Indeed, at low pH curved and circular fibers are formed at the surface of the nanospheres, which keep their shape after removal of the pluronics. The data reveal the mechanism of the templated fibril-growth: The growth of protein assemblies is nucleated in solution; small protein fibrils adsorb on the nanospheres, presumably due to hydrogen bond formation between the silk-like blocks and the pluronic PEO blocks. The surface of the pluronic particles templates further growth. At relatively low protein/pluronic weight ratios, only a fraction of the nanospheres bears protein fibers, pointing to a limiting amount of nuclei in solution. Because the nanospheres capture fibrils at an early stage of growth, they can be used to separate growth and nucleation rates in protein fibril formation. Moreover, the nanoparticle-templated growth of stable curved fibers opens ways to build proteinaceous nanocapsules from designed protein polymers. PMID:27250876

  3. The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Membrane separation is an important processing technology used for separating food ingredients and fractionating value-added components from food processing by-products. Long-term performance of polymeric membranes in food protein processing is impeded by formation of fouled layers on the membrane ...

  4. Development, characterization and applications of electrodes modified with conductive polymers, ionic liquids and proteins

    NASA Astrophysics Data System (ADS)

    Tang, Yijun

    My research involves both fundamental studies and applications of the electrodes whose surfaces are chemically modified. Conductive polymers are one of the major materials that are used to modify electrode surfaces. The thorough understanding of the behavior of conductive polymers in ionic liquids is interesting and important as the ionic liquids are becoming promising solvents. With poly(vinyl ferrocene) as the model conductive polymer, electrochemical studies were performed in various ionic liquid electrolytes. A theoretical square model and dynamic equilibrium were proposed to describe the interaction between conductive polymers and ionic liquids when the electrons transferred between the electrode and electrolyte. These findings were applied to enable and accelerate the structure relaxation of conductive polymers so that the conductive polymers were capable of delivering peptides efficiently. Incorporation of metallic nanoparticles to the conductive polymer matrix entitled new properties to the conductive polymer, increasing conductivity and providing catalytic abilities. This modification on electrode surface might bring potential uses in gas sensing, energy storage, energy conversion, etc. Conductive polymer coated electrodes produced unique double layer in ionic liquids and a fundamental study of quantum charging help to understand the double layer properties. I also studied the application of surface modified electrodes in chemo- and biosensing. A nonregeneration protocol was created to save the cost and the time in analyzing interfacial binding activities and to prevent the potential of deterioration caused to biological ligands by the conventional regeneration. In the study of carbohydrate/protein interactions, a "click" chemical reaction was first used in constructing a carbohydrate-based biosensor, which was capable of detecting and analyzing proteins specifically and accurately. In another biosensor design, the hydrogen bonding between the template and

  5. Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy

    PubMed Central

    Hofmann, Hagen; Soranno, Andrea; Borgia, Alessandro; Gast, Klaus; Nettels, Daniel; Schuler, Benjamin

    2012-01-01

    The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 ± 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 ± 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the Θ-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution. PMID:22984159

  6. Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum.

    PubMed

    Lu, Karen W; Pérez-Gil, Jesús; Echaide, Mercedes; Taeusch, H William

    2011-10-01

    Acute respiratory distress syndrome (ARDS) is an inflammatory condition that can be associated with capillary leak of serum into alveoli causing inactivation of surfactant. Resistance to inactivation is affected by types and concentrations of surfactant proteins, lipids, and polymers. Our aim was to investigate the effects of different combinations of these three components. A simple lipid mixture (DPPC/POPG) or a more complex lipid mixture (DPPC/POPC/POPG/cholesterol) was used. Native surfactant proteins SP-B and SP-C obtained from pig lung lavage were added either singly or combined at two concentrations. Also, non-ionic polymers polyethylene glycol and dextran and the anionic polymer hyaluronan were added either singly or in pairs with hyaluronan included. Non-ionic polymers work by different mechanisms than anionic polymers, thus the purpose of placing them together in the same surfactant mixture was to evaluate if the combination would show enhanced beneficial effects. The resulting surfactant mixtures were studied in the presence or absence of serum. A modified bubble surfactometer was used to evaluate surface activities. Mixtures that included both SP-B and SP-C plus hyaluronan and either dextran or polyethylene glycol were found to be the most resistant to inhibition by serum. These mixtures, as well as some with either SP-B or SP-C with combined polymers were as or more resistant to inactivation than native surfactant. These results suggest that improved formulations of lung surfactants are possible and may be useful in reducing some types of surfactant inactivation in treating lung injuries. PMID:21741354

  7. Influence of the Compatible Solute Ectoine on the Local Water Structure: Implications for the Binding of the Protein G5P to DNA.

    PubMed

    Hahn, Marc Benjamin; Solomun, Tihomir; Wellhausen, Robert; Hermann, Sabrina; Seitz, Harald; Meyer, Susann; Kunte, Hans-Jörg; Zeman, Johannes; Uhlig, Frank; Smiatek, Jens; Sturm, Heinz

    2015-12-10

    Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-5-protein (G5P) to a single-stranded DNA (dT25). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration-dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonucleotide, which has important consequences for osmotic regulation mechanisms. PMID:26555929

  8. Protein Adsorption and Transport in Polymer-Functionalized Ion-Exchangers

    PubMed Central

    Lenhoff, Abraham M.

    2012-01-01

    A wide variety of stationary phases is available for use in preparative chromatography of proteins, covering different base matrices, pore structures and modes of chromatography. There has recently been significant growth in the number of such materials in which the base matrix is derivatized to add a covalently attached or grafted polymer layer or, in some cases, a hydrogel that fills the pore space. This review summarizes the main structural and functional features of ion exchangers of this kind, which represent the largest class of such materials. Although the adsorption and transport properties may generally be used operationally and modeled phenomenologically using the same methods as are used for proteins in conventional media, there are noteworthy mechanistic differences in protein behavior in these adsorbents. A fundamental difference in protein retention is that it may be portrayed as partitioning into a three-dimensional polymer phase rather than adsorption at an extended two-dimensional surface, as applies in more conventional media. Beyond this partitioning behavior, however, the polymer-functionalized media often display rapid intraparticle transport that, while qualitatively comparable to that in conventional media, is sufficiently rapid quantitatively under certain conditions that it can lead to clear benefits in key measures of performance such as the dynamic binding capacity. Although possible mechanistic bases for the retention and transport properties are discussed, appreciable areas of uncertainty make detailed mechanistic modeling very challenging, and more detailed experimental characterization is likely to be more productive. PMID:21752388

  9. Microelectrospotting as a new method for electrosynthesis of surface-imprinted polymer microarrays for protein recognition.

    PubMed

    Bosserdt, Maria; Erdőssy, Júlia; Lautner, Gergely; Witt, Julia; Köhler, Katja; Gajovic-Eichelmann, Nenad; Yarman, Aysu; Wittstock, Gunther; Scheller, Frieder W; Gyurcsányi, Róbert E

    2015-11-15

    Here we introduce microelectrospotting as a new approach for preparation of protein-selective molecularly imprinted polymer microarrays on bare gold SPR imaging chips. During electrospotting both the gold chip and the spotting tip are electrically connected to a potentiostat as working and counter electrodes, respectively. The spotting pin encloses the monomer-template protein cocktail that upon contacting the gold surface is in-situ electropolymerized resulting in surface confined polymer spots of ca. 500 µm diameter. By repeating this procedure at preprogrammed locations for various composition monomer-template mixtures microarrays of nanometer-thin surface-imprinted films are generated in a controlled manner. We show that the removal and rebinding kinetics of the template and various potential interferents to such microarrays can be monitored in real-time and multiplexed manner by SPR imaging. The proof of principle for microelectrospotting of electrically insulating surface-imprinted films is made by using scopoletin as monomer and ferritin as protein template. It is shown that microelectrospotting in combination with SPR imaging can offer a versatile platform for label-free and enhanced throughput optimization of the molecularly imprinted polymers for protein recognition and for their analytical application. PMID:26056955

  10. Design of molecularly imprinted conducting polymer protein-sensing films via substrate-dopant binding.

    PubMed

    Komarova, Elena; Aldissi, Matt; Bogomolova, Anastasia

    2015-02-21

    Addressing the challenge of protein biosensing using molecularly imprinted polymers (MIP), we have developed and tested a novel approach to creating sensing conducive polymer films imprinted with a protein substrate, ricin toxin chain A (RTA). Our approach for creating MIP protein sensing films is based on a concept of substrate-guided dopant immobilization with subsequent conducting polymer film formation. In this proof-of-concept work we have tested three macromolecular dopants with strong protein affinity, Ponceau S, Coomassie BB R250 and ι-Carrageenan. The films were formed using sequential interactions of the substrate, dopant and pyrrole, followed by electrochemical polymerization. The films were formed on gold array electrodes allowing for extensive data acquisition. The thickness of the films was optimized to allow for efficient substrate extraction, which was removed by a combination of protease and detergent treatment. The MIP films were tested for substrate rebinding using electrochemical impedance spectroscopy (EIS). The presence of macromolecular dopants was essential for MIP film specificity. Out of three dopants tested, RTA-imprinted polypyrrole films doped with Coomassie BB performed with highest specificity towards detection of RTA with a level of detection (LOD) of 0.1 ng ml(-1). PMID:25574520

  11. Polymer-drug conjugates for intracellar molecule-targeted photoinduced inactivation of protein and growth inhibition of cancer cells

    NASA Astrophysics Data System (ADS)

    Wang, Bing; Yuan, Huanxiang; Zhu, Chunlei; Yang, Qiong; Lv, Fengting; Liu, Libing; Wang, Shu

    2012-10-01

    For most molecule-targeted anticancer systems, intracellular protein targets are very difficult to be accessed by antibodies, and also most efforts are made to inhibit protein activity temporarily rather than inactivate them permanently. In this work we firstly designed and synthesized multifunctional polymer-drug conjugates (polythiophene-tamoxifen) for intracellular molecule-targeted binding and inactivation of protein (estrogen receptor α, ERα) for growth inhibition of MCF-7 cancer cells. Small molecule drug was conjugated to polymer side chain for intracellular signal protein targeting, and simultaneously the fluorescent characteristic of polymer for tracing the cellular uptake and localization of polythiophene-drug conjugates by cell imaging. Under light irradiation, the conjugated polymer can sensitize oxygen to produce reactive oxygen species (ROS) that specifically inactivate the targeted protein, and thus inhibit the growth of tumor cells. The conjugates showed selective growth inhibition of ERα positive cancer cells, which exhibits low side effect for our intracellular molecule-targeted therapy system.

  12. One-step polymer surface modification for minimizing drug, protein, and DNA adsorption in microanalytical systems.

    PubMed

    Larsen, Esben Kjær Unmack; Larsen, Niels B

    2013-02-21

    The non-specific adsorption of dissolved analytes strongly reduces the sensitivity and reliability in polymer microanalytical systems. Here, a one-step aqueous phase procedure modifies polymer material surfaces to strongly reduce their non-specific adsorption of a broad range of organic analytes including hydrophobic and hydrophilic drugs (0.23 < ClogP < 8.95), small and large proteins (insulin, albumin, IgG), and DNA. The coating is shown to limit the adsorption of even highly hydrophobic drugs (ClogP > 8) in their pharmaceutically relevant concentration range ≤100 nM. The low adsorption is mediated by photochemical conjugation, where polyethylene glycol (PEG) polymers in aqueous solution are covalently bound to the surface by UV illumination of dissolved benzophenone and a functionalized PEG. The method can coat the interior of polymer systems made from a range of materials commonly used in microanalytical systems, including polystyrene (PS), cyclic olefin copolymer (COC), liquid crystalline polymer (LCP), and polyimide (PI). PMID:23254780

  13. Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads.

    PubMed

    Wong, Chin Ken; Laos, Alistair J; Soeriyadi, Alexander H; Wiedenmann, Jörg; Curmi, Paul M G; Gooding, J Justin; Marquis, Christopher P; Stenzel, Martina H; Thordarson, Pall

    2015-04-27

    Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane. PMID:25736460

  14. Biomimetic polymer brushes containing tethered acetylcholine analogs for protein and hippocampal neuronal cell patterning.

    PubMed

    Zhou, Zhaoli; Yu, Panpan; Geller, Herbert M; Ober, Christopher K

    2013-02-11

    This paper describes a method to control neuronal cell adhesion and differentiation with both chemical and topographic cues by using a spatially defined polymer brush pattern. First, biomimetic methacrylate polymer brushes containing tethered neurotransmitter acetylcholine functionalities in the form of dimethylaminoethyl methacrylate or free hydroxyl-terminated poly(ethylene glycol) units were prepared using the "grown from" method through surface-initiated atom transfer radical polymerization reactions. The surface properties of the resulting brushes were thoroughly characterized with various techniques and hippocampal neuronal cell culture on the brush surfaces exhibit cell viability and differentiation comparable to, or even better than, those on commonly used poly-l-lysine coated glass coverslips. The polymer brushes were then patterned via UV photolithography techniques to provide specially designed surface features with different sizes (varying from 2 to 200 μm) and orientations (horizontal and vertical). Protein absorption experiments and hippocampal neuronal cell culture tests on the brush patterns showed that both protein and neurons can adhere to the patterns and therefore be guided by such patterns. These results also demonstrate that, because of their unique chemical composition and well-defined nature, the developed polymer brushes may find many potential applications in cell-material interactions studies and neural tissue engineering. PMID:23336729

  15. Protein imprinted ionic liquid polymer on the surface of multiwall carbon nanotubes with high binding capacity for lysozyme.

    PubMed

    Yuan, Shifang; Deng, Qiliang; Fang, Guozhen; Wu, Jianhua; Li, Wangwang; Wang, Shuo

    2014-06-01

    In this research, ionic liquid as functional monomer to prepare molecularly imprinted polymers for protein recognition was for the first time demonstrated, in which, 1-vinyl-3-butylimidazolium chloride was selected as functional monomer, acrylamide as co-functional monomer and lysozyme (Lyz) as template protein to synthesize imprinted polymers on the surface of multiwall carbon nanotubes in aqueous medium. The results indicated that ionic liquid was helpful to improve binding capacity of imprinted polymers, which had a maximum binding capacity of 763.36 mg/g in the optimum adsorption conditions. The prepared imprinted polymers had a fast adsorption rate and a much higher adsorption capacity than the corresponding non-imprinted polymers, with the difference in adsorption capacity up to 258.31 mg/g. The obtained polymer was evaluated by Lyz, bovine serum albumin (BSA), bovine hemoglobin (BHb), equine myoglobin (MB) and cytochrome c (Cyt c). The selectivity factor (β) for Lyz/BSA, Lyz/Mb, Lyz/BHb, and Lyz/Cyt c were 7.17, 2.12, 1.76 and 1.57, respectively, indicating the imprinted polymers had a good selectivity. Easy preparation of the imprinted polymers as well as high ability and selectivity to adsorb template proteins makes this polymer attractive and broadly applicable in biomacromolecular separation, biotechnology and sensors. PMID:24835511

  16. Artificial Organelles: Reactions inside Protein-Polymer Supramolecular Assemblies.

    PubMed

    Garni, Martina; Einfalt, TomaŽ; Lomora, Mihai; Car, Anja; Meier, Wolfgang; Palivan, Cornelia G

    2016-01-01

    Reactions inside confined compartments at the nanoscale represent an essential step in the development of complex multifunctional systems to serve as molecular factories. In this respect, the biomimetic approach of combining biomolecules (proteins, enzymes, mimics) with synthetic membranes is an elegant way to create functional nanoreactors, or even simple artificial organelles, that function inside cells after uptake. Functionality is provided by the specificity of the biomolecule(s), whilst the synthetic compartment provides mechanical stability and robustness. The availability of a large variety of biomolecules and synthetic membranes allows the properties and functionality of these reaction spaces to be tailored and adjusted for building complex self-organized systems as the basis for molecular factories. PMID:27363371

  17. Advanced biomolecular materials based on membrane-protein/polymer complexation

    SciTech Connect

    Smith, G.S.; Nowak, A.; Safinya, C.

    1998-12-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The goal of this project was to apply neutron reflectometry and atomic force microscopy to the study of lipid membranes containing proteins. Standard sample preparation techniques were used to produce thin films of these materials appropriate for these techniques. However, these films were not stable, and a new sample preparation technique was required. Toward this goal, the authors have developed a new capability to produce large, freely suspended films of lipid multi-bilayers appropriate for these studies. This system includes a controlled temperature/humidity oven in which the films 5-cm x 5-cm are remotely drawn. The first neutron scattering experiments were then performed using this oven.

  18. Control of biomimetic hydroxyapatite deposition on polymer substrates using different protein adsorption abilities.

    PubMed

    Iijima, Kazutoshi; Sakai, Atsushi; Komori, Akinori; Sakamoto, Yuri; Matsuno, Hisao; Serizawa, Takeshi; Hashizume, Mineo

    2015-06-01

    We recently developed a system for coating polystyrene (PS) substrates with hydroxyapatite (HAp) by utilizing serum protein adsorption layers as mediators to induce the heterogeneous nucleation of HAp in simulated body fluids (SBFs). In this study, the selective deposition of HAp on polymer substrate surfaces with different protein adsorption abilities was investigated using PS and poly(methyl methacrylate) (PMMA). Atomic force microscopic observations and the results of a quantitative analysis using a quartz-crystal microbalance (QCM) revealed that the amounts of proteins such as human serum albumin (HSA) and human immunoglobulin G (hIgG) adsorbed on PS substrate surfaces were markedly greater than those on PMMA substrate surfaces. A markedly larger amount of HAp was deposited on protein-treated PS substrate surfaces than on PMMA substrate surfaces, reflecting protein adsorption to polymers. We also revealed that the deposition of HAp on protein-adsorbed PS substrate surfaces was enhanced by aqueous calcium chloride treatments before immersion in 1.5SBF. In the case of 2.5 M calcium chloride treatment, these surfaces were completely covered with deposits. PMID:25909182

  19. ELISA-mimic screen for synthetic polymer nanoparticles with high affinity to target proteins.

    PubMed

    Yonamine, Yusuke; Hoshino, Yu; Shea, Kenneth J

    2012-09-10

    Synthetic polymer nanoparticles (NPs) that display high affinity to protein targets have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). In this study, we modified an immunological assay (enzyme-linked immunosorbent assay: ELISA) into a high-throughput screening method to select nanoparticles with high affinity to target proteins. Histone and fibrinogen were chosen as target proteins to demonstrate this concept. The selection process utilized a biotinylated NP library constructed with combinations of functional monomers. The screen identified NPs with distinctive functional group compositions that exhibited high affinity to either histone or fibrinogen. The variation of protein affinity with changes in the nature and amount of functional groups in the NP provided chemical insight into the principle determinants of protein-NP binding. The NP affinity was semiquantified using the ELISA-mimic assay by varying the NP concentrations. The screening results were found to correlate with solution-based assay results. This screening system utilizing a biotinylated NP is a general approach to optimize functional monomer compositions and can be used to rapidly search for synthetic polymers with high (or low) affinity for target biological macromolecules. PMID:22813352

  20. Engineered Protein Polymer-Gold Nanoparticle Hybrid Materials for Small Molecule Delivery

    PubMed Central

    Dai, Min; Frezzo, JA; Sharma, E; Chen, R; Singh, N; Yuvienco, C; Caglar, E; Xiao, S; Saxena, A; Montclare, JK

    2016-01-01

    We have fabricated protein polymer-gold nanoparticle (P-GNP) nanocomposites that exhibit enhanced binding and delivery properties of the small hydrophobic molecule drug, curcumin, to the model breast cancer cell line, MCF-7. These hybrid biomaterials are constructed via in situ GNP templated-synthesis with genetically engineered histidine tags. The P-GNP nanocomposites exhibit enhanced small molecule loading, sustained release and increased uptake by MCF-7 cells. When compared to the proteins polymers alone, the P-GNPs demonstrate a greater than 7-fold increase in curcumin binding, a nearly 50% slower release profile and more than 2-fold increase in cellular uptake of curcumin. These results suggest that P-GNP nanocomposites serve as promising candidates for drug delivery vehicles. PMID:27081576

  1. The Role of Electrostatics in the Partitioning Behavior of Proteins into Polymer Hydrogels

    NASA Astrophysics Data System (ADS)

    Sharma, Upma; Carbeck, Jeffrey

    2000-03-01

    The goal of this work is to quantify the role of electrostatic interactions in the partitioning behavior of proteins into polymer hydrogels using charge ladders of proteins and capillary electrophoresis. Previous attempts to study electrostatic interactions between polyelectrolytes and proteins have been performed by conducting experiments in which the pH of the system is varied. This method does alter the charge of the protein; it also affects the degree of ionization of the polyelectrolytes. The partitioning behavior will vary as a combination of these effects. Protein charge ladders offer a superior approach relative to changing solution pH as this approach allows for isolation of charge as the independent variable. Partitioning experiments conducted using neutral, anionic, and cationic gels show that partitioning behavior for proteins was independent of the molecular weight of the protein for myoglobin, a-lactalbumin, lysozyme, and bovine carbonic anhydrase. Partitioning varied with the protein charge in a way not expected from simple electrostatic arguments. For example, in gels with a low charge density, the partitioning behavior varied linearly with protein charge; in high charge density gels, the parititioning behavior was independent of the protein charge.

  2. Materials compatibility.

    SciTech Connect

    Somerday, Brian P.

    2010-04-01

    Objectives are to enable development and implementation of codes and standards for H{sub 2} containment components: (1) Evaluate data on mechanical properties of materials in H{sub 2} gas - Technical Reference on Hydrogen Compatibility of Materials; (2) Generate new benchmark data on high-priority materials - Pressure vessel steels, stainless steels; and (3) Establish procedures for reliable materials testing - Sustained-load cracking, fatigue crack propagation. Summary of this presentation are: (1) Completed measurement of cracking thresholds (K{sub TH}) for Ni-Cr-Mo pressure vessel steels in high-pressure H{sub 2} gas - K{sub TH} measurements required in ASME Article KD-10 (2) Crack arrest test methods appear to yield non-conservative results compared to crack initiation test methods - (a) Proposal to insert crack initiation test methods in Article KD-10 will be presented to ASME Project Team on Hydrogen Tanks, and (b) Crack initiation methods require test apparatus designed for dynamic loading of specimens in H{sub 2} gas; and (3) Demonstrated ability to measure fatigue crack growth of pressure vessel steels in high-pressure H{sub 2} gas - (a) Fatigue crack growth data in H{sub 2} required in ASME Article KD-10, and (b) Test apparatus is one of few in U.S. or abroad for measuring fatigue crack growth in >100 MPa H{sub 2} gas.

  3. Production in Pichia pastoris of protein-based polymers with small heterodimer-forming blocks.

    PubMed

    Domeradzka, Natalia E; Werten, Marc W T; de Vries, Renko; de Wolf, Frits A

    2016-05-01

    Some combinations of leucine zipper peptides are capable of forming α-helical heterodimeric coiled coils with very high affinity. These can be used as physical cross-linkers in the design of protein-based polymers that form supramolecular structures, for example hydrogels, upon mixing solutions containing the complementary blocks. Such two-component physical networks are of interest for many applications in biomedicine, pharmaceutics, and diagnostics. This article describes the efficient secretory production of A and B type leucine zipper peptides fused to protein-based polymers in Pichia pastoris. By adjusting the fermentation conditions, we were able to significantly reduce undesirable proteolytic degradation. The formation of A-B heterodimers in mixtures of the purified products was confirmed by size exclusion chromatography. Our results demonstrate that protein-based polymers incorporating functional heterodimer-forming blocks can be produced with P. pastoris in sufficient quantities for use in future supramolecular self-assembly studies and in various applications. Biotechnol. Bioeng. 2016;113: 953-960. © 2015 Wiley Periodicals, Inc. PMID:26479855

  4. Protein-polymer functionalized aqueous ferrofluids showing high T2 relaxivity.

    PubMed

    Bhattacharya, S; Sheikh, L; Tiwari, V; Ghosh, M; Patel, J N; Patel, A B; Nayar, S

    2014-05-01

    Controlled size, shape and dispersibility of superparamagnetic iron oxide nanoparticles (SPIONs), has been achieved in a protein-polymer colloidal dispersion. Stable ferrofluid (FF) is synthesized in an aqueous medium of collagen, bovine serum albumin and poly(vinyl) alcohol that equilibrates with time, at ambient conditions, into an organized matrix with iron oxide particles sterically caged at defined sites. It mimics a biomineralization system; hence the process is termed biomimetics. Though the exact mechanism is not understood at this stage, we have established, with serial dilution of the protein-polymer solution that the SPIONs are formed inside the self-contained clusters of the two proteins and the polymer, which show a tendency to self assemble. More than the interparticle dipolar attractions of magnetic particles, electrostatic interactions play a role in cluster formation and collagen is responsible for the overall stability, supported by systematic dynamic light scattering data. The basic aim of this study was to increase magnetization of a previously synthesized ferrofluid without hampering stability, by reducing the total macromolecular concentration. Thrice the magnetization was achieved and in addition, the synthesized FFs exhibited very high transverse relaxivity and showed good contrast in mice liver, in the in vivo studies. PMID:24734534

  5. Partitioning and diffusion of proteins and linear polymers in polyacrylamide gels.

    PubMed Central

    Tong, J; Anderson, J L

    1996-01-01

    The equilibrium partition coefficient (K) and diffusion coefficient (Dgel) of two proteins and two linear polymers were measured as a function of polymer content of a 2.7% cross-linked polyacrylamide (PA) gel. The gel concentration, expressed as a volume percentage of PA in the gel (phi), varied between 0 and 14%. The measurements were made by fluorescence spectroscopy; fluorescent dyes were covalently attached to the macromolecules. The dependence of K on phi for the proteins agrees with a model of the gel network as randomly placed, impenetrable rods. The diffusion data are interpreted in terms of an effective medium theory for the mobility of a sphere in a Brinkman fluid. Using values of the Brinkman parameter in the literature, the effective medium model with no adjustable parameters fits the diffusion data for the proteins very well but underpredicts Dgel for the linear polymers. The gel effect on partitioning is significantly greater than that on diffusion. The permeability (KDgel) of bovine serum albumin decreased by 10(3) over the range phi = 0 --> 8%, and the ratio of permeabilities for ribonuclease compared to BSA increased from 2 to 30. Images FIGURE 1 PMID:8785307

  6. Immobilization of proteins on glow discharge treated polymers

    NASA Astrophysics Data System (ADS)

    Kiaei, D.; Safranj, A.; Chen, J. P.; Johnston, A. B.; Zavala, F.; Deelder, A.; Castelino, J. B.; Markovic, V.; Hoffman, A. S.

    Certain glow discharge-treated surfaces have been shown to enhance retention of adsorbed proteins. On the basis of this phenomenon, we have investigated the possibility of immobilizing (a) albumin for developing thromboresistant and non-fouling surfaces, (b) antibodies for immuno-diagnostic assays and (c) enzymes for various biosensors and industrial bioprocesses. Albumin retention was highest on surfaces treated with tetrafluoroethylene (TFE) compared to untreated surfaces or other glow discharge treatments studied. Preadsorption of albumin on TFE-treated surfaces resulted in low fibrinogen adsorption and platelet adhesion. IgG retention was also highest on TFE-treated surfaces. The lower detection limits of both malaria antigen and circulating anodic antigen of the schistosomiasis worm were enhanced following glow discharge treatment of the assay plates with TFE. Both TFE and tetrachloroethylene (TCE) glow discharge treated surfaces showed high retention of adsorbed horseradish peroxidase (HRP). However, the retained specific activity of HRP after adsorption on TCE-treated surfaces was remarkably higher than on TFE-treated surfaces.

  7. Multiplexed detection of protein cancer markers on Au/Ag-barcoded nanorods using fluorescent-conjugated polymers.

    PubMed

    Zheng, Weiming; He, Lin

    2010-07-01

    Integration of fluorescent-conjugated polymers as detection moiety with metallic striped nanorods for multiplexed detection of clinically important cancer marker proteins in an immunoassay format was demonstrated in this report. Specifically, cationic conjugated polymers were introduced to protein complexes through electrostatic binding to negatively charged double-stranded DNA, which was tagged on detection antibodies prior to antigen recognition. The intense fluorescence emission of conjugated polymers resulted in highly sensitive detection of cancer marker proteins wherein an undiluted bovine serum sample as low as approximately 25 target molecules captured on each particle was detectable. Meanwhile, the use of polymer molecules as the detection probe did not obscure the optical pattern of underlying nanorods, i.e., the encoding capability of barcoded nanorods was preserved, which allowed simultaneous detection of three cancer marker proteins with good specificity. PMID:20496173

  8. Integrated reactive ion etching to pattern cross-linked hydrophilic polymer structures for protein immobilization

    NASA Astrophysics Data System (ADS)

    Bhatnagar, Parijat; Strickland, Aaron D.; Kim, Il; Malliaras, George G.; Batt, Carl A.

    2007-04-01

    Patterning of cross-linked hydrophilic polymer features using reactive ion etching (RIE) capable of covalently immobilizing proteins has been achieved. Projection photolithography was used to pattern photoresist to create micromolds. Vapor phase molecular self-assembly of polymerizable monolayer in molds allowed covalent binding of hydrogel on surface during free-radical polymerization. Excess hydrogel blanket film was consumed with oxygen RIE resulting into hydrogel pattern of 1μm size aligned to prefabricated silicon oxide structures. Proteins were finally coupled through their primary amine groups selectively to acid functionalized hydrogel features through stable amide linkages using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide.

  9. Fundamentals of nanoscale polymer-protein interactions and potential contributions to solid-state nanobioarrays.

    PubMed

    Hahm, Jong-in

    2014-08-26

    Protein adsorption onto polymer surfaces is a very complex, ubiquitous, and integrated process, impacting essential areas of food processing and packaging, health devices, diagnostic tools, and medical products. The nature of protein-surface interactions is becoming much more complicated with continuous efforts toward miniaturization, especially for the development of highly compact protein detection and diagnostic devices. A large body of literature reports on protein adsorption from the perspective of ensemble-averaged behavior on macroscopic, chemically homogeneous, polymeric surfaces. However, protein-surface interactions governing the nanoscale size regime may not be effectively inferred from their macroscopic and microscopic characteristics. Recently, research efforts have been made to produce periodically arranged, nanoscopic protein patterns on diblock copolymer surfaces solely through self-assembly. Intriguing protein adsorption phenomena are directly probed on the individual biomolecule level for a fundamental understanding of protein adsorption on nanoscale surfaces exhibiting varying degrees of chemical heterogeneity. Insight gained from protein assembly on diblock copolymers can be effectively used to control the surface density, conformation, orientation, and biofunctionality of prebound proteins in highly miniaturized applications, now approaching the nanoscale. This feature article will highlight recent experimental and theoretical advances made on these fronts while focusing on single-biomolecule-level investigations of protein adsorption behavior combined with surface chemical heterogeneity on the length scale commensurate with a single protein. This article will also address advantages and challenges of the self-assembly-driven patterning technology used to produce protein nanoarrays and its implications for ultrahigh density, functional, and quantifiable protein detection in a highly miniaturized format. PMID:24456577

  10. Well-Defined Protein/Peptide-Polymer Conjugates by Aqueous Cu-LRP: Synthesis and Controlled Self-Assembly.

    PubMed

    Zhang, Qiang; Li, Muxiu; Zhu, Chongyu; Nurumbetov, Gabit; Li, Zaidong; Wilson, Paul; Kempe, Kristian; Haddleton, David M

    2015-07-29

    The synthesis of well-defined protein/peptide-polymer conjugates with interesting self-assembly behavior via single electron transfer living radical polymerization in water is described. A range of protein/peptides with different physical and chemical properties have been modified to macroinitiators and optimized polymerization conditions ensure successful polymerization from soluble, insoluble, and dispersed protein/peptide molecules or protein aggregates. This powerful strategy tolerates a range of functional monomers and mediates efficient homo or block copolymerization to generate hydrophilic polymers with controlled molecular weight (MW) and narrow MW distribution. The polymerizations from bovine insulin macroinitiators follow surface-initiated "grafting from" polymerization mechanism and may involve a series of self-assembly and disassembly processes. Synthesized insulin-polymer conjugates form spheres in water, and the self-assembly behavior could be controlled via thermal control, carbohydrate-protein interaction, and protein denaturation. PMID:26149497

  11. Direct prediction of profiles of sequences compatible with a protein structure by neural networks with fragment-based local and energy-based nonlocal profiles.

    PubMed

    Li, Zhixiu; Yang, Yuedong; Faraggi, Eshel; Zhan, Jian; Zhou, Yaoqi

    2014-10-01

    Locating sequences compatible with a protein structural fold is the well-known inverse protein-folding problem. While significant progress has been made, the success rate of protein design remains low. As a result, a library of designed sequences or profile of sequences is currently employed for guiding experimental screening or directed evolution. Sequence profiles can be computationally predicted by iterative mutations of a random sequence to produce energy-optimized sequences, or by combining sequences of structurally similar fragments in a template library. The latter approach is computationally more efficient but yields less accurate profiles than the former because of lacking tertiary structural information. Here we present a method called SPIN that predicts Sequence Profiles by Integrated Neural network based on fragment-derived sequence profiles and structure-derived energy profiles. SPIN improves over the fragment-derived profile by 6.7% (from 23.6 to 30.3%) in sequence identity between predicted and wild-type sequences. The method also reduces the number of residues in low complex regions by 15.7% and has a significantly better balance of hydrophilic and hydrophobic residues at protein surface. The accuracy of sequence profiles obtained is comparable to those generated from the protein design program RosettaDesign 3.5. This highly efficient method for predicting sequence profiles from structures will be useful as a single-body scoring term for improving scoring functions used in protein design and fold recognition. It also complements protein design programs in guiding experimental design of the sequence library for screening and directed evolution of designed sequences. The SPIN server is available at http://sparks-lab.org. PMID:24898915

  12. Direct prediction of profiles of sequences compatible to a protein structure by neural networks with fragment-based local and energy-based nonlocal profiles

    PubMed Central

    Li, Zhixiu; Yang, Yuedong; Faraggi, Eshel; Zhan, Jian; Zhou, Yaoqi

    2014-01-01

    Locating sequences compatible to a protein structural fold is the well-known inverse protein-folding problem. While significant progress has been made, the success rate of protein design remains low. As a result, a library of designed sequences or profile of sequences is currently employed for guiding experimental screening or directed evolution. Sequence profiles can be computationally predicted by iterative mutations of a random sequence to produce energy-optimized sequences, or by combining sequences of structurally similar fragments in a template library. The latter approach is computationally more efficient but yields less accurate profiles than the former because of lacking tertiary structural information. Here we present a method called SPIN that predicts Sequence Profiles by Integrated Neural network based on fragment-derived sequence profiles and structure-derived energy profiles. SPIN improves over the fragment-derived profile by 6.7% (from 23.6% to 30.3%) in sequence identity between predicted and wild-type sequences. The method also reduces the number of residues in low complex regions by 15.7% and has a significant better balance of hydrophilic and hydrophobic residues at protein surfaces. The accuracy of sequence profiles obtained is comparable to those generated from the protein design program RosettaDesign 3.5. This highly efficient method for predicting sequence profiles from structures will be useful as a single-body scoring term for improving scoring functions used in protein design and fold recognition. It also complements protein design programs in guiding experimental design of the sequence library for screening and directed evolution of designed sequences. The SPIN server is available at http://sparks-lab.org. PMID:24898915

  13. Self-Assembly of Differently Shaped Protein-Polymer Conjugates through Modification of the Bioconjugation Site.

    PubMed

    Huang, Aaron; Olsen, Bradley D

    2016-08-01

    Self-assembly of protein-polymer block copolymers is an attractive route for preparing biocatalytic materials. To clarify the effect of bioconjugate shape on self-assembly without changing the chemical details of either block, four different conjugation sites are selected on the surface of the model globular protein mCherry at residues 3, 108, 131, and 222 to alter the colloidal shape of the bioconjugate. All four mCherry-b-poly(N-isopropylacrylamide) bioconjugates show qualitatively similar phase diagrams, indicating that self-assembly is robust with respect to changes in conjugation site. However, protein orientation has an effect on the location of the order-disorder transition concentration, and the stability of the disordered micellar phase is shown to be different for each conjugate. Differences in domain spacing also suggest changes in protein orientation within the lamellae. PMID:27322114

  14. Proteometabolomic Study of Compatible Interaction in Tomato Fruit Challenged with Sclerotinia rolfsii Illustrates Novel Protein Network during Disease Progression

    PubMed Central

    Ghosh, Sudip; Narula, Kanika; Sinha, Arunima; Ghosh, Rajgourab; Jawa, Priyanka; Chakraborty, Niranjan; Chakraborty, Subhra

    2016-01-01

    Fruit is an assimilator of metabolites, nutrients, and signaling molecules, thus considered as potential target for pathogen attack. In response to patho-stress, such as fungal invasion, plants reorganize their proteome, and reconfigure their physiology in the infected organ. This remodeling is coordinated by a poorly understood signal transduction network, hormonal cascades, and metabolite reallocation. The aim of the study was to explore organ-based proteomic alterations in the susceptibility of heterotrophic fruit to necrotrophic fungal attack. We conducted time-series protein profiling of Sclerotinia rolfsii invaded tomato (Solanum lycopersicum) fruit. The differential display of proteome revealed 216 patho-stress responsive proteins (PSRPs) that change their abundance by more than 2.5-fold. Mass spectrometric analyses led to the identification of 56 PSRPs presumably involved in disease progression; regulating diverse functions viz. metabolism, signaling, redox homeostasis, transport, stress-response, protein folding, modification and degradation, development. Metabolome study indicated differential regulation of organic acid, amino acids, and carbohydrates paralleling with the proteomics analysis. Further, we interrogated the proteome data using network analysis that identified two significant functional protein hubs centered around malate dehydrogenase, T-complex protein 1 subunit gamma, and ATP synthase beta. This study reports, for the first-time, kinetically controlled patho-stress responsive protein network during post-harvest storage in a sink tissue, particularly fruit and constitute the basis toward understanding the onset and context of disease signaling and metabolic pathway alterations. The network representation may facilitate the prioritization of candidate proteins for quality improvement in storage organ. PMID:27507973

  15. Proteometabolomic Study of Compatible Interaction in Tomato Fruit Challenged with Sclerotinia rolfsii Illustrates Novel Protein Network during Disease Progression.

    PubMed

    Ghosh, Sudip; Narula, Kanika; Sinha, Arunima; Ghosh, Rajgourab; Jawa, Priyanka; Chakraborty, Niranjan; Chakraborty, Subhra

    2016-01-01

    Fruit is an assimilator of metabolites, nutrients, and signaling molecules, thus considered as potential target for pathogen attack. In response to patho-stress, such as fungal invasion, plants reorganize their proteome, and reconfigure their physiology in the infected organ. This remodeling is coordinated by a poorly understood signal transduction network, hormonal cascades, and metabolite reallocation. The aim of the study was to explore organ-based proteomic alterations in the susceptibility of heterotrophic fruit to necrotrophic fungal attack. We conducted time-series protein profiling of Sclerotinia rolfsii invaded tomato (Solanum lycopersicum) fruit. The differential display of proteome revealed 216 patho-stress responsive proteins (PSRPs) that change their abundance by more than 2.5-fold. Mass spectrometric analyses led to the identification of 56 PSRPs presumably involved in disease progression; regulating diverse functions viz. metabolism, signaling, redox homeostasis, transport, stress-response, protein folding, modification and degradation, development. Metabolome study indicated differential regulation of organic acid, amino acids, and carbohydrates paralleling with the proteomics analysis. Further, we interrogated the proteome data using network analysis that identified two significant functional protein hubs centered around malate dehydrogenase, T-complex protein 1 subunit gamma, and ATP synthase beta. This study reports, for the first-time, kinetically controlled patho-stress responsive protein network during post-harvest storage in a sink tissue, particularly fruit and constitute the basis toward understanding the onset and context of disease signaling and metabolic pathway alterations. The network representation may facilitate the prioritization of candidate proteins for quality improvement in storage organ. PMID:27507973

  16. A Modular Method for the High-Yield Synthesis of Site-Specific Protein-Polymer Therapeutics.

    PubMed

    Pang, Yan; Liu, Jinyao; Qi, Yizhi; Li, Xinghai; Chilkoti, Ashutosh

    2016-08-22

    A versatile method is described to engineer precisely defined protein/peptide-polymer therapeutics by a modular approach that consists of three steps: 1) fusion of a protein/peptide of interest with an elastin-like polypeptide that enables facile purification and high yields; 2) installation of a clickable group at the C terminus of the recombinant protein/peptide with almost complete conversion by enzyme-mediated ligation; and 3) attachment of a polymer by a click reaction with near-quantitative conversion. We demonstrate that this modular approach is applicable to various protein/peptide drugs and used it to conjugate them to structurally diverse water-soluble polymers that prolong the plasma circulation duration of these proteins. The protein/peptide-polymer conjugates exhibited significantly improved pharmacokinetics and therapeutic effects over the native protein/peptide upon administration to mice. The studies reported here provide a facile method for the synthesis of protein/peptide-polymer conjugates for therapeutic use and other applications. PMID:27439953

  17. Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds.

    PubMed

    Meese, Thomas M; Hu, Yunhua; Nowak, Richard W; Marra, Kacey G

    2002-01-01

    The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissue-engineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA. PMID:12022746

  18. Partitioning of a polymer into a nanoscopic protein pore obeys a simple scaling law

    PubMed Central

    Movileanu, Liviu; Bayley, Hagan

    2001-01-01

    The dependence of the rate on polymer mass was examined for the reaction of four sulfhydryl-directed poly(ethylene glycol) reagents with cysteine residues located in the lumen of the staphylococcal α-hemolysin pore. The logarithms of the apparent rate constants for a particular site in the lumen were proportional to N, the number of repeat units in a polymer chain. The proportionality constant was −(a/D)5/3, where a is the persistence length of the polymer (≈3.5Å) and D is the diameter of the pore. Despite some incongruencies with the assumptions of the derivation, the result suggests that the polymers partition into the lumen of the pore according to the simple scaling law of Daoud and de Gennes, cpore/csolution = exp(−N(a/D)5/3). Therefore, the measured reaction rates yield an estimate of the diameter of the pore and might be applied to determine the approximate dimensions of cavities within other similar proteins. PMID:11504913

  19. Solid state formulations composed by amphiphilic polymers for delivery of proteins: characterization and stability.

    PubMed

    Andrade, Fernanda; Fonte, Pedro; Oliva, Mireia; Videira, Mafalda; Ferreira, Domingos; Sarmento, Bruno

    2015-01-01

    Nanocomposite powders composed by polymeric micelles as vehicles for delivery proteins were developed in this work, using insulin as model protein. Results showed that size and polydispersity of micelles were dependent on the amphiphilic polymer used, being all lower than 300 nm, while all the formulations displayed spherical shape and surface charge close to neutrality. Percentages of association efficiency and loading capacity up to 94.15 ± 3.92 and 8.56 ± 0.36, respectively, were obtained. X-ray photoelectron spectroscopy (XPS) measurements confirmed that insulin was partially present at the hydrophilic shell of the micelles. Lyophilization did not significantly change the physical characteristics of micelles, further providing easily dispersion when in contact to aqueous medium. The native-like conformation of insulin was maintained at high percentages (around 80%) after lyophilization as indicated by Fourier transform infrared spectroscopy (FTIR) and far-UV circular dichroism (CD). Moreover, Raman spectroscopy did not evidenced significant interactions among the formulation components. The formulations shown to be physically stable upon storage up to 6 months both at room-temperature (20 °C) and fridge (4 °C), with only a slight loss (maximum of 15%) of the secondary structure of the protein. Among the polymers tested, Pluronic(®) F127 produced the carrier formulations more promising for delivery of proteins. PMID:25818062

  20. John H. Dillon Medal Talk: Protein Fibrils, Polymer Physics: Encounter at the Nanoscale

    NASA Astrophysics Data System (ADS)

    Mezzenga, Raffaele

    2011-03-01

    Aggregation of proteins is central to many aspects of daily life, ranging from blood coagulation, to eye cataract formation disease, food processing, or neurodegenerative infections. In particular, the physical mechanisms responsible for amyloidosis, the irreversible fibril formation of various proteins implicated in protein misfolding disorders such as Alzheimer, Creutzfeldt-Jakob or Huntington's diseases, have not yet been fully elucidated. In this talk I will discuss how polymer physics and colloidal science concepts can be used to reveal very useful information on the formation, structure and properties of amyloid protein fibrils. I will discuss their physical properties at various length scales, from their collective liquid crystalline behavior in solution to their structural features at the single molecule length scale and show how polymer science notions can shed a new light on these interesting systems. 1) ``Understanding amyloid aggregation by statistical analysis of atomic force microscopy images'' J. Adamcik, J.-M. Jung, J. Flakowski, P. De Los Rios, G. Dietler and R. Mezzenga, Nature nanotechnology, 5, 423 (2010)

  1. Integrin-mediated targeting of protein polymer nanoparticles carrying a cytostatic macrolide

    NASA Astrophysics Data System (ADS)

    Shi, Pu

    Cytotoxicity, low water solubility, rapid clearance from circulation, and offtarget side-effects are common drawbacks of conventional small-molecule drugs. To overcome these shortcomings, many multifunctional nanocarriers have been proposed to enhance drug delivery. In concept, multifunctional nanoparticles might carry multiple agents, control release rate, biodegrade, and utilize target-mediated drug delivery; however, the design of these particles presents many challenges at the stage of pharmaceutical development. An emerging solution to improve control over these particles is to turn to genetic engineering. Genetically engineered nanocarriers are precisely controlled in size and structure and can provide specific control over sites for chemical attachment of drugs. Genetically engineered drug carriers that assemble nanostructures including nanoparticles and nanofibers can be polymeric or nonpolymeric. This chapter summarizes the recent development of applications in drug and gene delivery utilizing nanostructures of polymeric genetically engineered drug carriers such as elastin-like polypeptides, silk-like polypeptides, and silk-elastin-like protein polymers, and non-polymeric genetically engineered drug carriers such as vault proteins and viral proteins. This chapter explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for

  2. Entropic interaction chromatography: separating proteins on the basis of size using end-grafted polymer brushes.

    PubMed

    Pang, Peter; Koska, Jürgen; Coad, Bryan R; Brooks, Donald E; Haynes, Charles A

    2005-04-01

    Partitioning of a macromolecule into the interfacial volume occupied by a grafted polymer brush decreases the configurational entropy (DeltaSbrush(c)) of the terminally attached linear polymer chains due to a loss of free volume. Self-consistent field theory (SCF) calculations are used to show that DeltaSbrush(c) is a strong function of both the size (MWp) of the partitioning macromolecule and the depth of penetration into the brush volume. We further demonstrate that the strong dependence of DeltaSbrush(c) on MWp provides a novel and powerful platform, which we call entropic interaction chromatography (EIC), for efficiently separating mixtures of proteins on the basis of size. Two EIC columns, differing primarily in polymer grafting density, were prepared by growing a brush of poly(methoxyethyl acrylamide) chains on the surface of a wide-pore (1,000-A pores, 64-microm diameter rigid beads) resin (Toyopearl AF-650M) bearing surface aldehyde groups. Semipreparative 0.1-L columns packed with either EIC resin provide reduced-plate heights of 2 or less for efficient separation of globular protein mixtures over at least three molecular-weight decades. Protein partitioning within these wide-pore EIC columns is shown to be effectively modeled as a thermodynamically controlled process, allowing partition coefficients (K(P)) and elution chromatograms to be accurately predicted using a column model that combines SCF calculation of K(P) values with an equilibrium-dispersion type model of solute transport through the column. This model is used to explore the dependence of column separation efficiency on brush properties, predicting that optimal separation of proteins over a broad MWp range is achieved at low to moderate grafting densities and intermediate chain lengths. PMID:15706591

  3. An amphipathic alpha-helical peptide from apolipoprotein A1 stabilizes protein polymer vesicles.

    PubMed

    Pastuszka, Martha K; Wang, Xiangdong; Lock, Lye Lin; Janib, Siti Mohd; Cui, Honggang; DeLeve, Laurie D; MacKay, J Andrew

    2014-10-10

    L4F, an alpha helical peptide inspired by the lipid-binding domain of the ApoA1 protein, has potential applications in the reduction of inflammation involved with cardiovascular disease as well as an antioxidant effect that inhibits liver fibrosis. In addition to its biological activity, amphipathic peptides such as L4F are likely candidates to direct the molecular assembly of peptide nanostructures. Here we describe the stabilization of the amphipathic L4F peptide through fusion to a high molecular weight protein polymer. Comprised of multiple pentameric repeats, elastin-like polypeptides (ELPs) are biodegradable protein polymers inspired from the human gene for tropoelastin. Dynamic light scattering confirmed that the fusion peptide forms nanoparticles with a hydrodynamic radius of approximately 50nm, which is unexpectedly above that observed for the free ELP (~5.1nm). To further investigate their morphology, conventional and cryogenic transmission electron microscopy were used to reveal that they are unilamellar vesicles. On average, these vesicles are 49nm in radius with lamellae 8nm in thickness. To evaluate their therapeutic potential, the L4F nanoparticles were incubated with hepatic stellate cells. Stellate cell activation leads to hepatic fibrosis; furthermore, their activation is suppressed by anti-oxidant activity of ApoA1 mimetic peptides. Consistent with this observation, L4F nanoparticles were found to suppress hepatic stellate cell activation in vitro. To evaluate the in vivo potential for these nanostructures, their plasma pharmacokinetics were evaluated in rats. Despite the assembly of nanostructures, both free L4F and L4F nanoparticles exhibited similar half-lives of approximately 1h in plasma. This is the first study reporting the stabilization of peptide-based vesicles using ApoA1 mimetic peptides fused to a protein polymer; furthermore, this platform for peptide-vesicle assembly may have utility in the design of biodegradable nanostructures

  4. Core-shell molecularly imprinted polymer nanoparticles with assistant recognition polymer chains for effective recognition and enrichment of natural low-abundance protein.

    PubMed

    Liu, Dejing; Yang, Qian; Jin, Susu; Song, Yingying; Gao, Junfei; Wang, Ying; Mi, Huaifeng

    2014-02-01

    Core-shell molecular imprinting of nanomaterials overcomes difficulties with template transfer and achieves higher binding capacities for macromolecular imprinting, which are more important to the imprinting of natural low-abundance proteins from cell extracts. In the present study, a novel strategy of preparing core-shell nanostructured molecularly imprinted polymers (MIPs) was developed that combined the core-shell approach with assistant recognition polymer chains (ARPCs). Vinyl-modified silica nanoparticles were used as support and ARPCs were used as additional functional monomers. Immunoglobulin heavy chain binding protein (BiP) from the endoplasmic reticulum (ER) was chosen as the model protein. The cloned template protein BiP was selectively assembled with ARPCs from their library, which contained numerous limited-length polymer chains with randomly distributed recognition and immobilization sites. The resulting complex was copolymerized onto the surface of vinyl-modified silica nanoparticles under low concentrations of the monomers. After template removal, core-shell-structured nanoparticles with a thin imprinted polymer layer were produced. The particles demonstrated considerably high adsorption capacity, fast adsorption kinetics and selective binding affinities toward the template BiP. Furthermore, the synthesized MIP nanoparticles successfully isolated cloned protein BiP from protein mixtures and highly enriched BiP from an ER extract containing thousands of kinds of proteins. The enrichment reached 115-fold and the binding capacity was 5.4 μg g(-1), which were higher than those achieved by using traditional MIP microspheres. The advantageous properties of MIP nanoparticles hold promise for further practical applications in biology, such as protein analysis and purification. PMID:24140608

  5. Electrophoresis of DNA-protein complexes in polymer solutions: from free-flow to gels

    NASA Astrophysics Data System (ADS)

    Slater, Gary W.; Desruisseaux, Claude; Drouin, Guy

    2000-03-01

    We previously showed that labeling one of the ends of single-stranded DNA molecules with a neutral label like the protein streptavidin increases the interband separation of these hybrid molecules when they are electrophoresed in gels because of strong steric trapping effects. In 1999, we also demonstrated that these labeled DNA molecules can be sequenced in free-solution, a novel separation process that we called ELFSE. Here, we examine the fascinating intermediate regime where the streptavidin-DNA molecules are electrophoresed in polymer solutions of increasing concentrations, from ultra-dilute to fully entangled conditions. Our capillary electrophoresis results clarify the respective roles of friction, polymer capture,reptation and steric trapping. In some cases, two separation regimes coexist and the mobility becomes a non-monotonic function of the DNA size. A universal relationship is found to relate the mobility of labeled and unlabeled DNA molecules for all systems.

  6. H2O2-treated actin: assembly and polymer interactions with cross-linking proteins.

    PubMed Central

    DalleDonne, I; Milzani, A; Colombo, R

    1995-01-01

    During inflammation, hydrogen peroxide, produced by polymorphonuclear leukocytes, provokes cell death mainly by disarranging filamentous (polymerized) actin (F-actin). To show the molecular mechanism(s) by which hydrogen peroxide could alter actin dynamics, we analyzed the ability of H2O2-treated actin samples to polymerize as well as the suitability of actin polymers (from oxidized monomers) to interact with cross-linking proteins. H2O2-treated monomeric (globular) actin (G-actin) shows an altered time course of polymerization. The increase in the lag phase and the lowering in both the polymerization rate and the polymerization extent have been evidenced. Furthermore, steady-state actin polymers, from oxidized monomers, are more fragmented than control polymers. This seems to be ascribable to the enhanced fragility of oxidized filaments rather than to the increase in the nucleation activity, which markedly falls. These facts; along with the unsuitability of actin polymers from oxidized monomers to interact with both filamin and alpha-actinin, suggest that hydrogen peroxide influences actin dynamics mainly by changing the F-actin structure. H2O2, via the oxidation of actin thiols (in particular, the sulfhydryl group of Cys-374), likely alters the actin C-terminus, influencing both subunit/subunit interactions and the spatial structure of the binding sites for cross-linking proteins in F-actin. We suggest that most of the effects of hydrogen peroxide on actin could be explained in the light of the "structural connectivity," demonstrated previously in actin. Images FIGURE 3 FIGURE 9 PMID:8599677

  7. In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth.

    PubMed

    Wang, Dong-an; Ji, Jian; Sun, Yong-hong; Shen, Jia-cong; Feng, Lin-xian; Elisseeff, Jennifer H

    2002-01-01

    A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces. PMID:12425667

  8. Compatibility of poly(bisAEA4)-LiTFSI-MPPipTFSI ionic liquid gel polymer electrolyte with Li4Ti5O12 lithium ion battery anode

    NASA Astrophysics Data System (ADS)

    Stepniak, Izabela

    2014-02-01

    This paper presents the use of Li4Ti5O12 (LTO) as anode with ionic liquid gel polymer electrolyte (IL-GPE) for application in lithium ion batteries. IL-GPE was obtained by in situ photopolymerization method of a mixture of ethoxylated bisphenol A diacrylate (bis(AEA4) and 0.4 M solution of lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) in N-methyl-N-propylpiperidinium bis(trifluoromethanesulfonyl)imide (MPPipTFSI). The surface morphology of the IL-GPE was studied using scanning electron microscopy (SEM). Stable, porous and flexible gel polymer electrolyte characterized high ionic conductivity (0.64 mS cm-1 at 25 °C) and a wide electrochemical stability window (ESW) (4.8 V). The performances of LTO/IL-GPE/Li cell were tested by using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and galvanostatic charge/discharge. Good charge/discharge capacities and low capacity loss at medium C rates preliminary cycling was obtained.

  9. Characterization and Compatibility Studies of Different Rate Retardant Polymer Loaded Microspheres by Solvent Evaporation Technique: In Vitro-In Vivo Study of Vildagliptin as a Model Drug

    PubMed Central

    Dewan, Irin; Islam, Swarnali; Rana, Md. Sohel

    2015-01-01

    The present study has been performed to microencapsulate the antidiabetic drug of Vildagliptin to get sustained release of drug. The attempt of this study was to formulate and evaluate the Vildagliptin loaded microspheres by emulsion solvent evaporation technique using different polymers like Eudragit RL100, Eudragit RS100, Ethyl cellulose, and Methocel K100M. In vitro dissolution studies were carried out in 0.1 N HCl for 8 hours according to USP paddle method. The maximum and minimum drug release were observed as 92.5% and 68.5% from microspheres, respectively, after 8 hours. Release kinetics were studied in different mathematical release models to find out the linear relationship and release rate of drug. The SEM, DSC, and FTIR studies have been done to confirm good spheres and smooth surface as well as interaction along with drug and polymer. In this experiment, it is difficult to explain the exact mechanism of drug release. But the drug might be released by both diffusion and erosion as the correlation coefficient (R2) best fitted with Korsmeyer model and release exponent (n) was 0.45–0.89. At last it can be concluded that all in vitro and in vivo experiments exhibited promising result to treat type II diabetes mellitus with Vildagliptin microspheres. PMID:26640713

  10. Processing-Dependent Self-Assembly of Protein-Polymer Diblock Copolymers

    NASA Astrophysics Data System (ADS)

    Thomas, C. S.; Xu, L.; Olsen, B. D.

    2012-02-01

    Self-assembly of globular protein-polymer diblock copolymers is a novel method for nanopatterning protein-based materials which maintains a high fraction of protein activity as well as the folded protein structure. By subjecting these copolymers to different processing conditions, long range ordering and the fraction of active protein can be controlled. Here, self-assembly of model mCherry-b-poly(N-isopropyl acrylamide) (PNIPAM) block copolymers is induced by water evaporation from dilute aqueous solutions of conjugate material, and followed by solvent annealing of the resulting nanostructures. Different pathways towards self-assembly are accessed by orthogonally manipulating the solvent quality for each block of the copolymer using temperature and pH. Small-angle scattering and transmission electron microscopy show nanostructure depends heavily on PNIPAM coil fraction and solvent annealing condition, with solution self-assembly reflected in the solid state structure under certain conditions. Protein structure is unaffected by the processing pathway, while protein activity levels in the nanodomains depend strongly on processing conditions and can retain up to 80% of the initial activity.

  11. Nanoparticle-based capillary electroseparation of proteins in polymer capillaries under physiological conditions.

    PubMed

    Nilsson, Christian; Harwigsson, Ian; Becker, Kristian; Kutter, Jörg P; Birnbaum, Staffan; Nilsson, Staffan

    2010-01-01

    Totally porous lipid-based liquid crystalline nanoparticles were used as pseudostationary phase for capillary electroseparation with LIF detection of proteins at physiological conditions using unmodified cyclic olefin copolymer capillaries (Topas, 6.7 cm effective length). In the absence of nanoparticles, i.e. in CE mode, the protein samples adsorbed completely to the capillary walls and could not be recovered. In contrast, nanoparticle-based capillary electroseparation resolved green fluorescent protein from several of its impurities within 1 min. Furthermore, a mixture of native green fluorescent protein and two of its single-amino-acid-substituted variants was separated within 2.5 min with efficiencies of 400 000 plates/m. The nanoparticles prevent adsorption by introducing a large interacting surface and by obstructing the attachment of the protein to the capillary wall. A one-step procedure based on self-assembly of lipids was used to prepare the nanoparticles, which benefit from their biocompatibility and suspension stability at high concentrations. An aqueous tricine buffer at pH 7.5 containing lipid-based nanoparticles (2% w/w) was used as electrolyte, enabling separation at protein friendly conditions. The developed capillary-based method facilitates future electrochromatography of proteins on polymer-based microchips under physiological conditions and enables the initial optimization of separation conditions in parallel to the chip development. PMID:20119954

  12. Exploring the role of internal friction in the dynamics of unfolded proteins using simple polymer models

    NASA Astrophysics Data System (ADS)

    Cheng, Ryan R.; Hawk, Alexander T.; Makarov, Dmitrii E.

    2013-02-01

    Recent experiments showed that the reconfiguration dynamics of unfolded proteins are often adequately described by simple polymer models. In particular, the Rouse model with internal friction (RIF) captures internal friction effects as observed in single-molecule fluorescence correlation spectroscopy (FCS) studies of a number of proteins. Here we use RIF, and its non-free draining analog, Zimm model with internal friction, to explore the effect of internal friction on the rate with which intramolecular contacts can be formed within the unfolded chain. Unlike the reconfiguration times inferred from FCS experiments, which depend linearly on the solvent viscosity, the first passage times to form intramolecular contacts are shown to display a more complex viscosity dependence. We further describe scaling relationships obeyed by contact formation times in the limits of high and low internal friction. Our findings provide experimentally testable predictions that can serve as a framework for the analysis of future studies of contact formation in proteins.

  13. Supramolecular Ensembles Formed between Charged Conjugated Polymers and Glycoprobes for the Fluorogenic Recognition of Receptor Proteins.

    PubMed

    Dou, Wei-Tao; Zeng, Ya-Li; Lv, Ying; Wu, Jiatao; He, Xiao-Peng; Chen, Guo-Rong; Tan, Chunyan

    2016-06-01

    This paper describes the simple construction of a unique class of supramolecular ensembles formed by electrostatic self-assembly between charged conjugated polymers and fluorophore-coupled glycoligands (glycoprobes) for the selective fluorogenic detection of receptor proteins at both the molecular and cellular levels. We show that positively and negatively charged diazobenzene-containing poly(p-phenylethynylenes) (PPEs) can be used to form stable fluorogenic probes with fluorescein-based (negatively charged) and rhodamine B based (positively charged) glycoprobes by electrostatic interaction. The structures of the ensembles have been characterized by spectroscopic and microscopic techniques. The supramolecular probes formed show quenched fluorescence in an aqueous buffer solution, which can be specifically recovered, in a concentration-dependent manner, through competitive complexation with a selective protein receptor, over a range of other unselective proteins. The ensembles also show selective fluorescence enhancement with a live cell that expresses the glycoligand receptor but not a control cell without receptor expression. PMID:27159586

  14. Bromovirus RNA replication and transcription require compatibility between the polymerase- and helicase-like viral RNA synthesis proteins.

    PubMed Central

    Dinant, S; Janda, M; Kroner, P A; Ahlquist, P

    1993-01-01

    The positive-strand RNA bromoviruses encode two nonstructural proteins, 1a and 2a, involved in RNA-dependent RNA replication. These proteins have extensive sequence similarities with methyltransferase, helicase, and polymerase proteins of other plant and animal viruses. 1a and 2a can also form a complex in vitro. To explore whether 1a-2a interaction is required for RNA replication in vivo, we reassorted the 1a and 2a genes from two different bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV). 1a and 2a were expressed independently of viral replication by using RNA- or DNA-based transient expression, and their in vivo RNA replication activities were tested in protoplasts with BMV and CCMV RNA3 templates. RNA-based transient expression confirmed prior indications that bromovirus RNA replication is more sensitive to reductions in 1a expression than to reductions in 2a expression. DNA-based expression of the homologous combinations of 1a and 2a supported high levels of RNA synthesis, but both 1a-2a heterologous combinations exhibited RNA synthesis defects. The combination of CCMV 1a and BMV 2a did not support detectable synthesis of negative-strand, positive-strand, or subgenomic RNA. The converse combination of BMV 1a and CCMV 2a was preferentially defective in positive-strand and subgenomic RNA accumulation, showing that 1a-2a interaction is involved in these processes in ways distinct from negative-strand RNA synthesis, which was only slightly affected. These results indicate that at least some functions of 1a and 2a operate in a mutually dependent manner in vivo and that the mechanisms of positive- and negative-strand RNA synthesis are differentiated in part by features of such interactions. Images PMID:8230440

  15. A hybrid protein-polymer nanoworm potentiates apoptosis better than a monoclonal antibody.

    PubMed

    Aluri, Suhaas Rayudu; Shi, Pu; Gustafson, Joshua A; Wang, Wan; Lin, Yi-An; Cui, Honggang; Liu, Shuanglong; Conti, Peter S; Li, Zibo; Hu, Peisheng; Epstein, Alan L; MacKay, John Andrew

    2014-03-25

    B-cell lymphomas continue to occur with a high incidence. The chimeric antibody known as Rituximab (Rituxan) has become a vital therapy for these patients. Rituximab induces cell death via binding and clustering of the CD20 receptor by Fcγ expressing effector cells. Because of the limited mobility of effector cells, it may be advantageous to cluster CD20 directly using multivalent nanostructures. To explore this strategy, this manuscript introduces a nanoparticle that assembles from a fusion between a single chain antibody and a soluble protein polymer. These hybrid proteins express in Escherichia coli and do not require bioconjugation between the antibody and a substrate. Surprisingly a fusion between an anti-CD20 single chain antibody and a soluble protein polymer assemble worm-like nanostructures, which were characterized using light scattering and cryogenic transmission electron microscopy. These nanoworms competitively bind CD20 on two B-cell lymphoma cell lines, exhibit concentration-dependent induction of apoptosis, and induce apoptosis better than Rituximab alone. Similar activity was observed in vivo using a non-Hodgkin lymphoma xenograft model. In comparison to Rituximab, systemic nanoworms significantly slowed tumor growth. These findings suggest that hybrid nanoworms targeted at CD20 may be useful treatments for B-cell related malignancies. Because of the ubiquity of antibody therapeutics, related nanoworms may have uses against other molecular targets. PMID:24484356

  16. Magnetic deep eutectic solvents molecularly imprinted polymers for the selective recognition and separation of protein.

    PubMed

    Liu, Yanjin; Wang, Yuzhi; Dai, Qingzhou; Zhou, Yigang

    2016-09-14

    A novel and facile magnetic deep eutectic solvents (DES) molecularly imprinted polymers (MIPs) for the selective recognition and separation of Bovine hemoglobin (BHb) was prepared. The new-type DES was adopted as the functional monomer which would bring molecular imprinted technology to a new direction. The amounts of DES were optimized. The obtained magnetic DES-MIPs were characterized with fourier transform infrared spectrometry (FT-IR), thermogravimetric analysis (TGA), field emission scanning electron microscope (FESEM), dynamic light scattering (DLS), elemental analysis and vibrating sample magnetometer (VSM). The results suggested that the imprinted polymers were successfully formed and possessed a charming magnetism. The maximum adsorption capability (Qmax) and dissociation constant (KL) were analyzed by Langmuir isotherms (R(2) = 0.9983) and the value were estimated to be 175.44 mg/g and 0.035 mg/mL for the imprinted particles. And the imprinted particles showed a high imprinting factor of 4.77. In addition, the magnetic DES-MIPs presented outstanding recognition specificity and selectivity so that it can be utilized to separate template protein from the mixture of proteins and real samples. Last but not least, the combination of deep eutectic solvents and molecular imprinted technology in this paper provides a new perspective for the recognition and separation of proteins. PMID:27566352

  17. Autoclaving as a chemical-free process to stabilize recombinant silk-elastinlike protein polymer nanofibers

    NASA Astrophysics Data System (ADS)

    Qiu, Weiguo; Cappello, Joseph; Wu, Xiaoyi

    2011-06-01

    We report here that autoclaving is a chemical-free, physical crosslinking strategy capable of stabilizing electrospun recombinant silk-elastinlike protein (SELP) polymer nanofibers. Fourier transform infrared spectroscopy showed that the autoclaving of SELP nanofibers induced a conformational conversion of β-turns and unordered structures to ordered β-sheets. Tensile stress-strain analysis of the autoclaved SELP nanofibrous scaffolds in phosphate buffered saline at 37 °C revealed a Young's modulus of 1.02 ± 0.28 MPa, an ultimate tensile strength of 0.34 ± 0.04 MPa, and a strain at failure of 29% ± 3%.

  18. Protein encapsulation in and release from monodisperse double-wall polymer microspheres

    PubMed Central

    Xia, Yujie; Xu, Qingxing; Wang, Chi-Hwa; Pack, Daniel W.

    2014-01-01

    Biodegradable polymer double-wall microspheres (DWMS) are promising vehicles for macromolecular therapeutics such as proteins and peptides. Using precision particle fabrication (PPF) technology, uniform DWMS with outer diameter ~55 μm were fabricated comprising poly(lactide-co-glycolide) cores encapsulating bovine serum albumin (BSA) and ~10 μm thick, drug-free, poly(lactic acid) shells of varying PLA molecular weight. Also, monolithic single-wall microspheres (SWMS) were fabricated to mimic the BSA-loaded core. The use of relatively fast extracting ethyl acetate and slowly extracting dichloromethane as shell- and core-phase solvents, respectively, was found to produce DWMS with well-defined core-shell structure, high BSA encapsulation efficiency, and the desired localization of protein in the particle core. Initial protein distribution, particle erosion, and in vitro protein release from DWMS and SWMS were examined. The presence of a BSA-free shell in DWMS decreased the protein release rate and extended the duration of release from ~50 days to 70-80 days, demonstrating the capacity of such DWMS to provide enhanced control of protein delivery rates. PMID:23529836

  19. Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

    PubMed

    Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

    2008-12-01

    A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity. PMID:18991420

  20. Suppression of apoptosis by enhanced protein adsorption on polymer/hydroxyapatite composite scaffolds

    PubMed Central

    Woo, Kyung Mi; Seo, Jihye; Zhang, Ruiyun; Ma, Peter X.

    2007-01-01

    Bone tissue engineering is a promising alternative to bone grafting. Scaffolds play a critical role in tissue engineering. Composite scaffolds made of biodegradable polymers and bone mineral-like inorganic compounds have been reported to be advantageous over plain polymer scaffolds by our group and others. In this study, we compared cellular and molecular events during the early periods of osteoblastic cell culture on poly(l-lactic acid)/hydroxyapatite (PLLA/HAP) composite scaffolds with those on plain PLLA scaffolds, and showed that PLLA/HAP scaffolds improved cell survival over plain PLLA scaffolds. Most cells (MC3T3-E1) on PLLA/HAP scaffolds survived the early culture. In contrast, about 50% of the cells initially adhered to the plain PLLA scaffolds were detached within the first 12 h and showed characteristics of apoptotic cell death, which was confirmed by TUNEL staining and caspase-3 activation. To investigate the mechanisms, we examined the adsorption of serum protein and adhesion molecules to the scaffolds. The PLLA/HAP scaffold adsorbed more than 1.4 times of total serum protein and much greater amounts of serum fibronectin and vitronectin than pure PLLA scaffolds. Similarly, significantly larger amounts of individual adhesion proteins and peptides (fibronectin, vitronectin, RGD, and KRSR) were adsorbed on the PLLA/HAP scaffolds than on the PLLA scaffolds, which resulted in higher cell density on the PLLA/HAP scaffolds. Furthermore, β1 and β3 integrins and phosphorylation of Fak and Akt proteins in the cells on the PLLA/HAP scaffolds were significantly more abundent than those on PLLA scaffolds, which suggest that enhanced adsorption of serum adhesion proteins to PLLA/HAP scaffolds protect the cells from apoptosis possibly through the integrin-Fak-Akt pathway. These results demonstrate that biomimetic composite scaffolds are advantageous for bone tissue engineering. PMID:17320948

  1. Temperature-Triggered Protein Adsorption on Polymer-Coated Nanoparticles in Serum.

    PubMed

    Koshkina, Olga; Lang, Thomas; Thiermann, Raphael; Docter, Dominic; Stauber, Roland H; Secker, Christian; Schlaad, Helmut; Weidner, Steffen; Mohr, Benjamin; Maskos, Michael; Bertin, Annabelle

    2015-08-18

    The protein corona, which forms on the nanoparticle's surface in most biological media, determines the nanoparticle's physicochemical characteristics. The formation of the protein corona has a significant impact on the biodistribution and clearance of nanoparticles in vivo. Therefore, the ability to influence the formation of the protein corona is essential to most biomedical applications, including drug delivery and imaging. In this study, we investigate the protein adsorption on nanoparticles with a hydrodynamic radius of 30 nm and a coating of thermoresponsive poly(2-isopropyl-2-oxazoline) in serum. Using multiangle dynamic light scattering (DLS) we demonstrate that heating of the nanoparticles above their phase separation temperature induces the formation of agglomerates, with a hydrodynamic radius of 1 μm. In serum, noticeably stronger agglomeration occurs at lower temperatures compared to serum-free conditions. Cryogenic transmission electron microscopy (cryo-TEM) revealed a high packing density of agglomerates when serum was not present. In contrast, in the presence of serum, agglomerated nanoparticles were loosely packed, indicating that proteins are intercalated between them. Moreover, an increase in protein content is observed upon heating, confirming that protein adsorption is induced by the alteration of the surface during phase separation. After cooling and switching the surface back, most of the agglomerates were dissolved and the main fraction returned to the original size of approximately 30 nm as shown by asymmetrical flow-field flow fractionation (AF-FFF) and DLS. Furthermore, the amounts of adsorbed proteins are similar before and after heating the nanoparticles to above their phase-separation temperature. Overall, our results demonstrate that the thermoresponsivity of the polymer coating enables turning the corona formation on nanoparticles on and off in situ. As the local heating of body areas can be easily done in vivo, the thermoresponsive

  2. Fabrication of Highly Uniform Nanoparticles from Recombinant Silk-Elastinlike Protein Polymers for Therapeutic Agent Delivery

    PubMed Central

    Anumolu, Rajasekhar; Gustafson, Joshua A.; Magda, Jules J.; Cappello, Joseph; Ghandehari, Hamidreza; Pease, Leonard F.

    2011-01-01

    Here we generate silk-elastinlike protein (SELP) polymeric nanoparticles and demonstrate precise control over their dimensions using an electrospray differential mobility analyzer (ES-DMA). Electrospray produces droplets encompassing several polymer strands. Evaporation ensues, leading polymer strands to accumulate at the droplet interface forming a hollow nanoparticle. The resulting nanoparticle size distributions which govern particle yield, depend on buffer concentration to the −1/3 power, polymer concentration to the 1/3 power, and ratio of silk to elastin blocks. Three recombinantly tuned ratios of silk to elastin blocks, 8:16, 4:8, and 4:16, respectively named SELP-815K, SELP-47K, and SELP-415K, are employed with the latter ratio resulting in a thinner shell and larger diameter for the nanoparticles than the former. The DMA narrows the size distribution by electrostatically classifying the aerosolized nanoparticles. These highly uniform nanoparticles have variations of 1.2 nm and 1.4 nm for 24.0 nm and 36.0 nm particles, respectively. Transmission electron microscopy reveals the nanoparticles to be faceted, as a buckling instability releases compression energy arising from evaporation after the shell has formed by bending it. A thermodynamic equilibrium exists between compression and bending energies, where the facet length is 1/2 the particle diameter, in agreement with experiments. Rod-like particles also formed from polymer stabilized filaments when the viscous length exceeds the jet radius at higher solution viscosities. The unusual uniformity in composition and dimension indicates the potential of these nanoparticles to deliver bioactive and imaging agents. PMID:21696150

  3. Integrin-mediated targeting of protein polymer nanoparticles carrying a cytostatic macrolide

    NASA Astrophysics Data System (ADS)

    Shi, Pu

    Cytotoxicity, low water solubility, rapid clearance from circulation, and offtarget side-effects are common drawbacks of conventional small-molecule drugs. To overcome these shortcomings, many multifunctional nanocarriers have been proposed to enhance drug delivery. In concept, multifunctional nanoparticles might carry multiple agents, control release rate, biodegrade, and utilize target-mediated drug delivery; however, the design of these particles presents many challenges at the stage of pharmaceutical development. An emerging solution to improve control over these particles is to turn to genetic engineering. Genetically engineered nanocarriers are precisely controlled in size and structure and can provide specific control over sites for chemical attachment of drugs. Genetically engineered drug carriers that assemble nanostructures including nanoparticles and nanofibers can be polymeric or nonpolymeric. This chapter summarizes the recent development of applications in drug and gene delivery utilizing nanostructures of polymeric genetically engineered drug carriers such as elastin-like polypeptides, silk-like polypeptides, and silk-elastin-like protein polymers, and non-polymeric genetically engineered drug carriers such as vault proteins and viral proteins. This chapter explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for

  4. EPR spectroscopy of protein microcrystals oriented in a liquid crystalline polymer medium

    NASA Astrophysics Data System (ADS)

    Caldeira, Jorge; Figueirinhas, João Luis; Santos, Celina; Godinho, Maria Helena

    2004-10-01

    Correlation of the g-tensor of a paramagnetic active center of a protein with its structure provides a unique experimental information on the electronic structure of the metal site. To address this problem, we made solid films containing metalloprotein ( Desulfovibrio gigas cytochrome c3) microcrystals. The microcrystals in a liquid crystalline polymer medium (water/hydroxypropylcellulose) were partially aligned by a shear flow. A strong orientation effect of the metalloprotein was observed by EPR spectroscopy and polarizing optical microscopy. The EPR spectra of partially oriented samples were simulated, allowing for molecular orientation distribution function determination. The observed effect results in enhanced sensitivity and resolution of the EPR spectra and provides a new approach towards the correlation of spectroscopic data, obtained by EPR or some other technique, with the three-dimensional structure of a protein or a model compound.

  5. BioC-compatible full-text passage detection for protein–protein interactions using extended dependency graph

    PubMed Central

    Arighi, Cecilia; Wu, Cathy H.; Vijay-Shanker, K.

    2016-01-01

    There has been a large growth in the number of biomedical publications that report experimental results. Many of these results concern detection of protein–protein interactions (PPI). In BioCreative V, we participated in the BioC task and developed a PPI system to detect text passages with PPIs in the full-text articles. By adopting the BioC format, the output of the system can be seamlessly added to the biocuration pipeline with little effort required for the system integration. A distinctive feature of our PPI system is that it utilizes extended dependency graph, an intermediate level of representation that attempts to abstract away syntactic variations in text. As a result, we are able to use only a limited set of rules to extract PPI pairs in the sentences, and additional rules to detect additional passages for PPI pairs. For evaluation, we used the 95 articles that were provided for the BioC annotation task. We retrieved the unique PPIs from the BioGRID database for these articles and show that our system achieves a recall of 83.5%. In order to evaluate the detection of passages with PPIs, we further annotated Abstract and Results sections of 20 documents from the dataset and show that an f-value of 80.5% was obtained. To evaluate the generalizability of the system, we also conducted experiments on AIMed, a well-known PPI corpus. We achieved an f-value of 76.1% for sentence detection and an f-value of 64.7% for unique PPI detection. Database URL: http://proteininformationresource.org/iprolink/corpora PMID:27170286

  6. Interfacial assembly of protein-polymer nano-conjugates into stimulus-responsive biomimetic protocells.

    PubMed

    Huang, Xin; Li, Mei; Green, David C; Williams, David S; Patil, Avinash J; Mann, Stephen

    2013-01-01

    The mechanism of spontaneous assembly of microscale compartments is a central question for the origin of life, and has technological repercussions in diverse areas such as materials science, catalysis, biotechnology and biomedicine. Such compartments need to be semi-permeable, structurally robust and capable of housing assemblages of functional components for internalized chemical transformations. In principle, proteins should be ideal building blocks for the construction of membrane-bound compartments but protein vesicles with cell-like properties are extremely rare. Here we present an approach to the interfacial assembly of protein-based micro-compartments (proteinosomes) that are delineated by a semi-permeable, stimulus-responsive, enzymatically active, elastic membrane consisting of a closely packed monolayer of conjugated protein-polymer building blocks. The proteinosomes can be dispersed in oil or water, thermally cycled to temperatures of 70 °C, and partially dried and re-inflated without loss of structural integrity. As a consequence, they exhibit protocellular properties such as guest molecule encapsulation, selective permeability, gene-directed protein synthesis and membrane-gated internalized enzyme catalysis. PMID:23896993

  7. [Construction, fermentation and purification of high polymer spider dragline silk protein containing RGD peptide].

    PubMed

    Ruan, Chao-Ran; Huang, Jing-Xing; Wei, Mei-Hong; Li, Min

    2007-09-01

    Spider silk is a natural protein fibroin with excellent character as it is light and tenacious. It has a wild potential applications in the biomedical field due to its good biocompatibility and degradation. Arginine-glycine-aspartic acid (RGD) is a highly conserved amino acid sequence of many adhesion protein. Biological materials binding with RGD peptide in the surface can promote cells adhesion, migration and proliferation. Our lab had constructed the 16 muhimers with the introduced RGD peptide codons which involve cell adhesion for the first time. It was found that the mechanical capability of the 16 mulimer protein was very limited because of the big gap in molecular weight with nature spider proteins when it was used to made biomaterial scaffold.In this paper,based on the 16 multimers of the highly, repetitive sequence of spider dragline silk and with RGD peptide condons which has been constructed by our lab forestall, it was used to construct the 32 and 64 multimers sequence of spider dragline silk by the strategy of "head to tail". The 32 and 64 multimers were ligated into prokaryotic expression vector pET-30a, and then the B121 (DE3) pLysS. The fragments were in agreement with the desired through digestion, agarose gel electrophoresis respectively. By registration into the GenBank data-base, the serial numbers of DQ469929 and DQ837297 were gained respectively. The expression of recombinant protein was introduced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 102 kD and 196.6kD in agreement with the desired respectively. It was the first time for the high polymer spider dragline silk protein expressed in prokaryotic biology. Furthermore, a larger quantity of synthetical proteins with high density fermentation were searched after, and a suit of high efficient purification methods for 32 multimers protein were established. PMID:18051865

  8. Exploring the low temperature thermodynamics of lattice proteins and polymers with chain lengths > 1000

    NASA Astrophysics Data System (ADS)

    Wuest, Thomas

    2012-02-01

    Coarse-grained (lattice-) models have a long tradition in aiding to decipher the physical or biological complexity of polymers and proteins. Despite their simplicity however, numerical simulations of such models are often computationally very demanding and the quest for efficient algorithms is as old as the models themselves. I present a computational method based on Wang-Landau sampling in combination with suitable trial move sets which is particularly effective to study models such as the hydrophobic-polar (HP) lattice model of protein folding or its counterpart in polymer physics, the interactive self-avoiding walk (ISAW) at low temperatures. The approach provides a versatile and powerful mean for both the ground state search and the determination of the entire energy density of states (DOS) yielding reliable estimates of thermodynamic quantities for chain lengths > 4000 (ISAW) even in the very dense collapsed phase. The appearance of multiple low temperature pseudo-transitions for ISAWs will be elucidated. Further methodological improvements will be discussed.

  9. Protein Biosensors Based on Polymer Nanowires, Carbon Nanotubes and Zinc Oxide Nanorods

    PubMed Central

    M., Anish Kumar; Jung, Soyoun; Ji, Taeksoo

    2011-01-01

    The development of biosensors using electrochemical methods is a promising application in the field of biotechnology. High sensitivity sensors for the bio-detection of proteins have been developed using several kinds of nanomaterials. The performance of the sensors depends on the type of nanostructures with which the biomaterials interact. One dimensional (1-D) structures such as nanowires, nanotubes and nanorods are proven to have high potential for bio-applications. In this paper we review these three different kinds of nanostructures that have attracted much attention at recent times with their great performance as biosensors. Materials such as polymers, carbon and zinc oxide have been widely used for the fabrication of nanostructures because of their enhanced performance in terms of sensitivity, biocompatibility, and ease of preparation. Thus we consider polymer nanowires, carbon nanotubes and zinc oxide nanorods for discussion in this paper. We consider three stages in the development of biosensors: (a) fabrication of biomaterials into nanostructures, (b) alignment of the nanostructures and (c) immobilization of proteins. Two different methods by which the biosensors can be developed at each stage for all the three nanostructures are examined. Finally, we conclude by mentioning some of the major challenges faced by many researchers who seek to fabricate biosensors for real time applications. PMID:22163892

  10. Rapid and serum-insensitive endocytotic delivery of proteins using biotinylated polymers attached via multivalent hydrophobic anchors.

    PubMed

    Tobinaga, Kyohei; Li, Cuicui; Takeo, Masafumi; Matsuda, Masayoshi; Nagai, Hiroko; Niidome, Takuro; Yamamoto, Tatsuhiro; Kishimura, Akihiro; Mori, Takeshi; Katayama, Yoshiki

    2014-03-10

    We have designed biotinylated polymers as synthetic receptors that have multiple alkyl groups for endocytotic delivery of target proteins. The polymers were stably attached to a cell surface via multivalent anchoring. The presented biotin was bound to streptavidin (SA) on the cell surface, and, via an endocytotic pathway, the cell rapidly internalized the biotinylated polymer/SA complex. The cell's uptake of the complex was not inhibited by the presence of 10% fetal bovine serum, and its efficacy for the uptake of SA was the highest when compared with commercial reagents and single-anchored-type synthetic receptors. The synthetic receptor-mediated endocytosis can be used generally for other kind of protein by using SA as an adaptor molecule between a target protein and the cell-surface presented biotin. PMID:24389131

  11. Impact of glucose polymer chain length on heat and physical stability of milk protein-carbohydrate nutritional beverages.

    PubMed

    Chen, Biye; O'Mahony, James A

    2016-11-15

    This study investigated the impact of glucose polymer chain length on heat and physical stability of milk protein isolate (MPI)-carbohydrate nutritional beverages containing 8.5% w/w total protein and 5% w/w carbohydrate. The maltodextrin and corn syrup solids glucose polymers used had dextrose equivalent (DE) values of 17 or 38, respectively. Increasing DE value of the glucose polymers resulted in a greater increase in brown colour development, ionic calcium, protein particle size, apparent viscosity and pseudoplastic rheological behaviour, and greater reduction in pH, hydration and heat stability on sterilisation at 120°C. Incorporation of glucose polymers with MPI retarded sedimentation of protein during accelerated physical stability testing, with maltodextrin DE17 causing a greater reduction in sedimentation velocity and compressibility of sediment formed than corn syrup solids DE38. The results demonstrate that chain length of the glucose polymer used strongly impacts heat and physical stability of MPI-carbohydrate nutritional beverages. PMID:27283657

  12. Neuroserpin Polymers Activate NF-κB by a Calcium Signaling Pathway That Is Independent of the Unfolded Protein Response*

    PubMed Central

    Davies, Mark J.; Miranda, Elena; Roussel, Benoit D.; Kaufman, Randal J.; Marciniak, Stefan J.; Lomas, David A.

    2009-01-01

    The autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies is characterized by the accumulation of ordered polymers of mutant neuroserpin within the endoplasmic reticulum of neurones. We show here that intracellular neuroserpin polymers activate NF-κB by a pathway that is independent of the IRE1, ATF6, and PERK limbs of the canonical unfolded protein response but is dependent on intracellular calcium. This pathway provides a mechanism for cells to sense and react to the accumulation of folded structures of mutant serpins within the endoplasmic reticulum. Our results provide strong support for the endoplasmic reticulum overload response being independent of the unfolded protein response. PMID:19423713

  13. The amino-terminal structure of human fragile X mental retardation protein obtained using precipitant-immobilized imprinted polymers

    NASA Astrophysics Data System (ADS)

    Hu, Yufeng; Chen, Zhenhang; Fu, Yanjun; He, Qingzhong; Jiang, Lun; Zheng, Jiangge; Gao, Yina; Mei, Pinchao; Chen, Zhongzhou; Ren, Xueqin

    2015-03-01

    Flexibility is an intrinsic property of proteins and essential for their biological functions. However, because of structural flexibility, obtaining high-quality crystals of proteins with heterogeneous conformations remain challenging. Here, we show a novel approach to immobilize traditional precipitants onto molecularly imprinted polymers (MIPs) to facilitate protein crystallization, especially for flexible proteins. By applying this method, high-quality crystals of the flexible N-terminus of human fragile X mental retardation protein are obtained, whose absence causes the most common inherited mental retardation. A novel KH domain and an intermolecular disulfide bond are discovered, and several types of dimers are found in solution, thus providing insights into the function of this protein. Furthermore, the precipitant-immobilized MIPs (piMIPs) successfully facilitate flexible protein crystal formation for five model proteins with increased diffraction resolution. This highlights the potential of piMIPs for the crystallization of flexible proteins.

  14. Salicylic Acid-Based Polymers for Guided Bone Regeneration Using Bone Morphogenetic Protein-2

    PubMed Central

    Subramanian, Sangeeta; Mitchell, Ashley; Yu, Weiling; Snyder, Sabrina; Uhrich, Kathryn

    2015-01-01

    Bone morphogenetic protein-2 (BMP-2) is used clinically to promote spinal fusion, treat complex tibia fractures, and to promote bone formation in craniomaxillofacial surgery. Excessive bone formation at sites where BMP-2 has been applied is an established complication and one that could be corrected by guided tissue regeneration methods. In this study, anti-inflammatory polymers containing salicylic acid [salicylic acid-based poly(anhydride-ester), SAPAE] were electrospun with polycaprolactone (PCL) to create thin flexible matrices for use as guided bone regeneration membranes. SAPAE polymers hydrolyze to release salicylic acid, which is a nonsteroidal anti-inflammatory drug. PCL was used to enhance the mechanical integrity of the matrices. Two different SAPAE-containing membranes were produced and compared: fast-degrading (FD-SAPAE) and slow-degrading (SD-SAPAE) membranes that release salicylic acid at a faster and slower rate, respectively. Rat femur defects were treated with BMP-2 and wrapped with FD-SAPAE, SD-SAPAE, or PCL membrane or were left unwrapped. The effects of different membranes on bone formation within and outside of the femur defects were measured by histomorphometry and microcomputed tomography. Bone formation within the defect was not affected by membrane wrapping at BMP-2 doses of 12 μg or more. In contrast, the FD-SAPAE membrane significantly reduced bone formation outside the defect compared with all other treatments. The rapid release of salicylic acid from the FD-SAPAE membrane suggests that localized salicylic acid treatment during the first few days of BMP-2 treatment can limit ectopic bone formation. The data support development of SAPAE polymer membranes for guided bone regeneration applications as well as barriers to excessive bone formation. PMID:25813520

  15. Highly Stable Trypsin-Aggregate Coatings on Polymer Nanofibers for Repeated Protein Digestion

    SciTech Connect

    Kim, Byoung Chan; Lopez-Ferrer, Daniel; Lee, Sang-mok; Ahn, Hye-kyung; Nair, Sujith; Kim, Seong H.; Kim, Beom S.; Petritis, Konstantinos; Camp, David G.; Grate, Jay W.; Smith, Richard D.; Koo, Yoon-mo; Gu, Man Bock; Kim, Jungbae

    2009-04-01

    A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This new process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was also resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with enzyme aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

  16. Characterization of the molecular structure and mechanical properties of polymer surfaces and protein/polymer interfaces by sum frequency generation vibrational spectroscopy and atomic force microscopy

    SciTech Connect

    Koffas, Telly Stelianos

    2004-05-15

    Sum frequency generation (SFG) vibrational spectroscopy, atomic force microscopy (AFM), and other complementary surface-sensitive techniques have been used to study the surface molecular structure and surface mechanical behavior of biologically-relevant polymer systems. SFG and AFM have emerged as powerful analytical tools to deduce structure/property relationships, in situ, for polymers at air, liquid and solid interfaces. The experiments described in this dissertation have been performed to understand how polymer surface properties are linked to polymer bulk composition, substrate hydrophobicity, changes in the ambient environment (e.g., humidity and temperature), or the adsorption of macromolecules. The correlation of spectroscopic and mechanical data by SFG and AFM can become a powerful methodology to study and engineer materials with tailored surface properties. The overarching theme of this research is the interrogation of systems of increasing structural complexity, which allows us to extend conclusions made on simpler model systems. We begin by systematically describing the surface molecular composition and mechanical properties of polymers, copolymers, and blends having simple linear architectures. Subsequent chapters focus on networked hydrogel materials used as soft contact lenses and the adsorption of protein and surfactant at the polymer/liquid interface. The power of SFG is immediately demonstrated in experiments which identify the chemical parameters that influence the molecular composition and ordering of a polymer chain's side groups at the polymer/air and polymer/liquid interfaces. In general, side groups with increasingly greater hydrophobic character will be more surface active in air. Larger side groups impose steric restrictions, thus they will tend to be more randomly ordered than smaller hydrophobic groups. If exposed to a hydrophilic environment, such as water, the polymer chain will attempt to orient more of its hydrophilic groups to the

  17. Electrochemical deposition and surface-initiated RAFT polymerization: protein and cell-resistant PPEGMEMA polymer brushes.

    PubMed

    Tria, Maria Celeste R; Grande, Carlos David T; Ponnapati, Ramakrishna R; Advincula, Rigoberto C

    2010-12-13

    This paper introduces a novel and versatile method of grafting protein and cell-resistant poly(poly ethylene glycol methyl ether methacrylate) (PPEGMEMA) brushes on conducting Au surface. The process started with the electrochemical deposition and full characterization of an electro-active chain transfer agent (CTA) on the Au surface. The electrochemical behavior of the CTA was investigated by cyclic voltammetry (CV) while the deposition and stability of the CTA on the surface were confirmed by ellipsometry, contact angle, and X-ray photoelectron spectroscopy (XPS). The capability of the electrodeposited CTA to mediate surface-initiated reversible addition-fragmentation chain transfer (SI-RAFT) polymerization on both the polymethyl methacrylate (PMMA; model polymer) and PPEGMEMA brushes was demonstrated by the increase in thicknesses of the films after polymerization. Contact angles also decreased with the incorporation of the more hydrophilic brushes. Significant changes in the morphologies of the films before and after polymerization were also observed with atomic force microscopy (AFM) analyses. Furthermore, XPS results showed an increase in the O 1s peak intensity relative to C 1s after polymerizations, which confirmed the grafting of polyethyleneglycol (PEG) bearing brushes. The ability of the PPEGMEMA-modified Au surface to resist nonspecific adhesion of proteins and cells was monitored and confirmed by XPS, ellipsometry, contact angle, AFM, and fluorescence imaging. The new method presented has potential application as robust protein and cell-resistant coatings for electrically conducting electrodes and biomedical devices. PMID:21028799

  18. Development of porous polymer monoliths for reverse-phase chromatography of proteins.

    SciTech Connect

    Shepodd, Timothy J.; Stephens, Christopher P.

    2003-09-01

    The polymers developed in this project are intended for use as a stationary phase in reverse-phase chromatography of proteins, where the mobile phase is a solution of acetonitrile and a phosphate buffer, 6.6 pH. A full library of pore sizes have been developed ranging from 0.41{micro}m to 4.09 {micro}m; these pore sizes can be determined by the solvent ratio of tetrahydrofuran:methoxyethanol during polymerization. A column that can separate proteins in an isocratic mode would be a vast improvement from the common method of separating proteins through gradient chromatography using multiple solvents. In the stationary phase, the main monomers have hydrophobic tails, lauryl acrylate and steryl acrylate. Separations of small hydrophobic molecules and peptides (trial molecules) have efficiencies of 24,000-33,000 theoretical plates m{sup -1}. The combination of a highly non-polar stationary phase and a mobile phase where the polarity can be controlled provide for excellent separation.

  19. Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

    PubMed

    Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

    2013-01-01

    Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB. PMID:24145242

  20. Ratiometric Array of Conjugated Polymers-Fluorescent Protein Provides a Robust Mammalian Cell Sensor.

    PubMed

    Rana, Subinoy; Elci, S Gokhan; Mout, Rubul; Singla, Arvind K; Yazdani, Mahdieh; Bender, Markus; Bajaj, Avinash; Saha, Krishnendu; Bunz, Uwe H F; Jirik, Frank R; Rotello, Vincent M

    2016-04-01

    Supramolecular complexes of a family of positively charged conjugated polymers (CPs) and green fluorescent protein (GFP) create a fluorescence resonance energy transfer (FRET)-based ratiometric biosensor array. Selective multivalent interactions of the CPs with mammalian cell surfaces caused differential change in FRET signals, providing a fingerprint signature for each cell type. The resulting fluorescence signatures allowed the identification of 16 different cell types and discrimination between healthy, cancerous, and metastatic cells, with the same genetic background. While the CP-GFP sensor array completely differentiated between the cell types, only partial classification was achieved for the CPs alone, validating the effectiveness of the ratiometric sensor. The utility of the biosensor was further demonstrated in the detection of blinded unknown samples, where 121 of 128 samples were correctly identified. Notably, this selectivity-based sensor stratified diverse cell types in minutes, using only 2000 cells, without requiring specific biomarkers or cell labeling. PMID:26967961

  1. Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering

    NASA Astrophysics Data System (ADS)

    Kasálková, Nikola Slepičková; Slepička, Petr; Kolská, Zdeňka; Hodačová, Petra; Kučková, Štěpánka; Švorčík, Václav

    2014-04-01

    In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly- l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly- l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation.

  2. Nanoparticle-Loaded Protein-Polymer Nanodroplets for Improved Stability and Conversion Efficiency in Ultrasound Imaging and Drug Delivery.

    PubMed

    Lee, Jeong Yu; Carugo, Dario; Crake, Calum; Owen, Joshua; de Saint Victor, Marie; Seth, Anjali; Coussios, Constantin; Stride, Eleanor

    2015-10-01

    A new formulation of volatile nanodroplets stabilized by a protein and polymer coating and loaded with magnetic nanoparticles is developed. The droplets show enhanced stability and phase conversion efficiency upon ultrasound exposure compared with existing formulations. Magnetic targeting, encapsulation, and release of an anticancer drug are demonstrated in vitro with a 40% improvement in cytotoxicity compared with free drug. PMID:26265592

  3. Creeping proteins in microporous structures: polymer brush-assisted fabrication of 3D gradients for tissue engineering.

    PubMed

    Gunnewiek, Michel Klein; Di Luca, Andrea; Bollemaat, Hermannes Z; van Blitterswijk, Clemens A; Vancso, G Julius; Moroni, Lorenzo; Benetti, Edmondo M

    2015-06-01

    Coupling of rapid prototyping techniques and surface-confined polymerizations allows the fabrication of 3D multidirectional gradients of biomolecules within microporous scaffolds. The compositional gradients can be tailored by polymer-brush-assisted diffusion of protein solutions. This technique allows spatial control over stem cells manipulation within 3D environments. PMID:25676461

  4. Plasticization and crosslinking effects of acetone-formaldehyde and tannin resins on wheat protein-based natural polymers.

    PubMed

    Zhang, Xiaoqing; Do, My Dieu

    2009-07-01

    Efficient plasticization and sufficient crosslinking were achieved by using an acetone-formaldehyde (AF) resin as an additive in the thermal processing of wheat protein-based natural polymers. The mobile AF resin and its strong intermolecular interactions with a wheat protein matrix produced sufficient flexibility for the plastics, while the covalent bonds formed between AF and the protein chains also caused the water-soluble resin to be retained in the materials under wet conditions. The mechanical properties of the materials were also enhanced as an additional benefit due to the formation of crosslinked networks through the polymer matrix. Tensile strength was further enhanced when using AF in conjunction with tannin resin (AFTR) in the systems as rigid aromatic structures were formed in the crosslinking segments. Different components in wheat proteins (WPs) or wheat gluten (WG) (e.g., proteins, residual starch and lipids) displayed different capabilities in interaction and reaction with the AFTR additives, and thus resulted in different performances when the ratio of these components varied in the materials. The application of the AFTR additives provides a feasible methodology to thermally process wheat protein-based natural polymers with improved mechanical performance and water-resistant properties. PMID:19447383

  5. Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer.

    PubMed

    Podust, Vladimir N; Sim, Bee-Cheng; Kothari, Dharti; Henthorn, Lana; Gu, Chen; Wang, Chia-wei; McLaughlin, Bryant; Schellenberger, Volker

    2013-11-01

    XTEN, unstructured biodegradable proteins, have been used to extend the in vivo half-life of genetically fused therapeutic proteins and peptides. To expand the applications of XTEN technology to half-life extension of other classes of molecules, XTEN protein polymers and methods for chemical XTENylation were developed. Two XTEN precursors were engineered to contain enzymatically removable purification tags. The proteins were readily expressed in bacteria and purified to homogeneity by chromatography techniques. As proof-of-principle, GLP2-2G peptide was chemically conjugated to each of the two XTEN protein polymers using maleimide-thiol chemistry. The monodisperse nature of XTEN protein polymer enabled reaction monitoring as well as the detection of peptide modifications in the conjugated state using reverse phase-high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry. The resulting GLP2-2G-XTEN conjugates were purified by preparative RP-HPLC to homogeneity. In comparison with recombinantly fused GLP2-2G-XTEN, chemically conjugated GLP2-2G-XTEN molecules exhibited comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats. These data suggest that chemical XTENylation could effectively extend the half-life of a wide spectrum of biologically active molecules, therefore broadening its applicability. PMID:24133142

  6. Studies on the application of temperature-responsive ion exchange polymers with whey proteins.

    PubMed

    Maharjan, Pankaj; Campi, Eva M; De Silva, Kirthi; Woonton, Brad W; Jackson, W Roy; Hearn, Milton T W

    2016-03-18

    Several new types of temperature-responsive ion exchange resins of different polymer composition have been prepared by grafting the products from the co-polymerisation of N-phenylacrylamide, N-iso-propylacrylamide and acrylic acid derivatives onto cross-linked agarose. Analysis of the binding isotherms for these different resins obtained under batch adsorption conditions indicated that the resin based on N-iso-propylacrylamide containing 5% (w/w) N-phenylacrylamide and 5% (w/w) acrylic acid resulted in the highest adsorption capacity, Bmax, for the whey protein, bovine lactoferrin, e.g. 14 mg bovine lactoferrin/mL resin at 4 °C and 62 mg bovine lactoferrin/mL resin at 40 °C, respectively. Under dynamic loading conditions at 40 °C, 94% of the loaded bovine lactoferrin on a normalised mg protein per mL resin basis was adsorbed by this new temperature-responsive ion-exchanger, and 76% was eluted by a single cycle temperature shift to 4 °C without varying the composition of the 10mM sodium dihydrogen phosphate buffer, pH 6.5, or the flow rate. The binding characteristics of these different ion exchange resins with bovine lactoferrin were also compared to results obtained using other resins based on N-isopropylacrylamide but contained N-tert-butylacrylamide rather than N-phenylacrylamide, where the corresponding dynamic capture and release properties for bovine lactoferrin required different temperature conditions of 20 °C and 50 °C, respectively for optimal desorption/adsorption. The cationic protein, bovine lactoperoxidase, was also adsorbed and desorbed with these temperature-responsive resins under similar conditions of changing temperature, whereas the anionic protein, bovine β-lactoglobulin, was not adsorbed under this regime of temperature conditions but instead eluted in the flow-through. PMID:26905884

  7. Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion

    SciTech Connect

    Ahn, Hye-Kyung; Kim, Byoung Chan; Jun, Seung-Hyun; Chang, Mun Seock; Lopez-Ferrer, Daniel; Smith, Richard D.; Gu, Man Bock; Lee, Sang-Won; Kim, Beom S.; Kim, Jungbae

    2010-12-15

    An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently-attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 °C and 50 °C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 0.24 and 163.20 hours at 40 ºC, respectively. The improved stability of EC-TR can be explained by covalent-linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40 °C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of one week. In the same condition, CA-TR showed poor performance from the beginning, and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis, but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.

  8. Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB

    PubMed Central

    Roussel, Benoit D.; Newton, Timothy M.; Malzer, Elke; Simecek, Nikol; Haq, Imran; Thomas, Sally E.; Burr, Marian L.; Lehner, Paul J.; Crowther, Damian C.; Marciniak, Stefan J.; Lomas, David A.

    2013-01-01

    Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR. PMID:23814041

  9. Active surfaces engineered by immobilizing protein-polymer nanoreactors for selectively detecting sugar alcohols.

    PubMed

    Zhang, Xiaoyan; Lomora, Mihai; Einfalt, Tomaz; Meier, Wolfgang; Klein, Noreen; Schneider, Dirk; Palivan, Cornelia G

    2016-05-01

    We introduce active surfaces generated by immobilizing protein-polymer nanoreactors on a solid support for sensitive sugar alcohols detection. First, such selective nanoreactors were engineered in solution by simultaneous encapsulation of specific enzymes in copolymer polymersomes, and insertion of membrane proteins for selective conduct of sugar alcohols. Despite the artificial surroundings, and the thickness of the copolymer membrane, functionality of reconstituted Escherichia coli glycerol facilitator (GlpF) was preserved, and allowed selective diffusion of sugar alcohols to the inner cavity of the polymersome, where encapsulated ribitol dehydrogenase (RDH) enzymes served as biosensing entities. Ribitol, selected as a model sugar alcohol, was detected quantitatively by the RDH-nanoreactors with GlpF-mediated permeability in a concentration range of 1.5-9 mM. To obtain "active surfaces" for detecting sugar alcohols, the nanoreactors optimized in solution were then immobilized on a solid support: aldehyde groups exposed at the compartment external surface reacted via an aldehyde-amino reaction with glass surfaces chemically modified with amino groups. The nanoreactors preserved their architecture and activity after immobilization on the glass surface, and represent active biosensing surfaces for selective detection of sugar alcohols, with high sensitivity. PMID:26950167

  10. Long-term biostability of self-assembling protein polymers in the absence of covalent crosslinking.

    PubMed

    Sallach, Rory E; Cui, Wanxing; Balderrama, Fanor; Martinez, Adam W; Wen, Jing; Haller, Carolyn A; Taylor, Jeannette V; Wright, Elizabeth R; Long, Robert C; Chaikof, Elliot L

    2010-02-01

    Unless chemically crosslinked, matrix proteins, such as collagen or silk, display a limited lifetime in vivo with significant degradation observed over a period of weeks. Likewise, amphiphilic peptides, lipopeptides, or glycolipids that self-assemble through hydrophobic interactions to form thin films, fiber networks, or vesicles do not demonstrate in vivo biostability beyond a few days. We report herein that a self-assembling, recombinant elastin-mimetic triblock copolymer elicited minimal inflammatory response and displayed robust in vivo stability for periods exceeding 1 year, in the absence of either chemical or ionic crosslinking. Specifically, neither a significant inflammatory response nor calcification was observed upon implantation of test materials into the peritoneal cavity or subcutaneous space of a mouse model. Moreover, serial quantitative magnetic resonance imaging, evaluation of pre- and post-explant ultrastructure by cryo-high resolution scanning electron microscopy, and an examination of implant mechanical responses revealed substantial preservation of form, material architecture, and biomechanical properties, providing convincing evidence of a non-chemically or ionically crosslinked protein polymer system that exhibits long-term stability in vivo. PMID:19854505