WorldWideScience

Sample records for antifreeze protein prepared

  1. Protein-water dynamics in antifreeze protein III activity

    Science.gov (United States)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  2. Plant Antifreeze Proteins and Their Expression Regulatory Mechanism

    Institute of Scientific and Technical Information of China (English)

    Lin Yuan-zhen; Lin Shan-zhi; Zhang Zhi-yi; Zhang Wei; Liu Wen-feng

    2005-01-01

    Low temperature is one of the major limiting environmental factors which constitutes the growth, development,productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identification and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.

  3. Antivirulence Properties of an Antifreeze Protein

    Directory of Open Access Journals (Sweden)

    Martin Heisig

    2014-10-01

    Full Text Available As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.

  4. Coarse grained simulation reveals antifreeze properties of hyperactive antifreeze protein from Antarctic bacterium Colwellia sp.

    Science.gov (United States)

    Nguyen, Hung; Van, Thanh Dac; Le, Ly

    2015-10-01

    The novel hyperactive antifreeze protein (AFP) of Antarctic sea ice bacterium Colwellia sp. provides a target for studying the protection of psychrophilic microgoranisms against freezing environment. Interestingly, the Colwellia sp. hyperactive antifreeze protein (ColAFP) was crystallized without the structural dynamic characteristics. Here, the result indicated, through coarse grained simulation of ColAFP under various subfreezing temperature, that ColAFP remains active at temperature of equal and greater than 275 K (∼2 °C). Extensive simulation analyses also revealed the adaptive mechanism of ColAFP in subfreezing environment. Our result provides a structural dynamic understanding of the ColAFP.

  5. Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax.

    Science.gov (United States)

    Friis, Dennis Steven; Kristiansen, Erlend; von Solms, Nicolas; Ramløv, Hans

    2014-05-01

    The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active at concentrations above 270 μmol.

  6. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    Ravi Gupta; Renu Deswal

    2014-12-01

    Overwintering plants secrete antifreeze proteins (AFPs) to provide freezing tolerance. These proteins bind to and inhibit the growth of ice crystals that are formed in the apoplast during subzero temperatures. Antifreeze activity has been detected in more than 60 plants and AFPs have been purified from 15 of these, including gymnosperms, dicots and monocots. Biochemical characterization of plant antifreeze activity, as determined by the high ice recrystallization inhibition (IRI) activities and low thermal hysteresis (TH) of AFPs, showed that their main function is inhibition of ice crystal growth rather than the lowering of freezing temperatures. However, recent studies showed that antifreeze activity with higher TH also exists in plants. Calcium and hormones like ethylene and jasmonic acid have been shown to regulate plant antifreeze activity. Recent studies have shown that plant AFPs bind to both prism planes and basal planes of ice crystals by means of two flat ice binding sites. Plant AFPs have been postulated to evolve from the OsLRR-PSR gene nearly 36 million years ago. In this review, we present the current scenario of plant AFP research in order to understand the possible potential of plant AFPs in generation of freezing-tolerant crops.

  7. Expression of a Carrot Antifreeze Protein Gene in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Ma Xinyu; Shen Xin; Lu Cunfu

    2003-01-01

    The recombinant expression vectorpET43. lb-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol. L-1 IPTG (isopropyl-β-D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein.The analysis of product solubility revealed that pET43. 1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.

  8. Dynamical mechanism of antifreeze proteins to prevent ice growth

    CERN Document Server

    Kutschan, B; Thoms, S

    2014-01-01

    The fascinating ability of algae, insects and fishes to survive at temperatures below normal freezing is realized by antifreeze proteins (AFPs). Antifreeze proteins (AFPs) are surface-active molecules and interact with the diffusive water/ice interface preventing a complete solidification. A new dynamical mechanism is proposed how these proteins inhibit the freezing of water. We apply a Ginzburg-Landau type approach to describe the phase separation in the two-component system (ice, AFP). The free energy density involves two fields: one for the ice phase with low AFP concentration, and one for the liquid water with high AFP concentration. The time evolution of the ice reveals microstructures as a result of phase separation in the presence of AFPs. We observe a faster clustering of pre-ice structure connected with a locking of grain size by the action of AFP which is an essentially dynamical process. The adsorption of additional water molecules are inhibited and the further growth of ice grains are stopped. The...

  9. Preparation and application of antifreeze proteins extracted from winter wheat bran%冬小麦抗冻蛋白制备及其在汤圆中的应用研究

    Institute of Scientific and Technical Information of China (English)

    夏露; 张超; 王立; 张晖

    2009-01-01

    研究了冬小麦麸皮抗冻蛋白的制备方法及其在速冻汤圆中的应用.研究确定冬小麦麸皮抗冻蛋白的提取工艺为:pH8.0,提取时间3h,液料比5:1,该条件下小麦麸皮水溶性蛋白质提取率达到38%,其中含抗冻蛋白1.6%.抗冻蛋白粗品在汤圆中的应用实验结果显示,2.5%的蛋白添加量对汤圆的品质有明显的改善效果.%The preparation and application of antifreeze proteins (AFPs) were studied.The extraction process of AFPs from winter wheat bran was optimized as following, water/material ratio 5:1, pH 8.0, extraction time 3h.The extraction rate of soluble protein from winter wheat bran was 38%, and the AFPs content in the extraction (crude AFPs) was 1.6%.The application experiment of AFP in rice dumpling showed that the quality of rice dumpling would be improved by adding with 2.5% crude AFPs.

  10. Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Kristiansen, Erlend; von Solms, Nicolas;

    2014-01-01

    of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active...

  11. Computational simulations on the fish-type-Ⅱ antifreeze protein-ice-solvent system

    Institute of Scientific and Technical Information of China (English)

    LIU Kai; WANG Yan; TAN Hongwei; CHEN Guangju; TONG Zhenhe

    2007-01-01

    Based on the computational simulation with the vacuum environment for the fish-type-Ⅱ antifreeze proteinice-solvent (water)system,the multi-complex system of the antifreeze protein-ice-water has been constructed and calculated.We have studied the interaction of such proteinice system with water solvent through the dynamics simulation with 350 ps.By employing the Molecular Dynamics simulation and semi-empirical method calculation,we have further investigated the interface properties of the antifreeze protein and ice crystal combined system.Consequently,a water solvent affects significantly the properties of this combined system.

  12. Towards a green hydrate inhibitor: imaging antifreeze proteins on clathrates.

    Directory of Open Access Journals (Sweden)

    Raimond Gordienko

    Full Text Available The formation of hydrate plugs in oil and gas pipelines is a serious industrial problem and recently there has been an increased interest in the use of alternative hydrate inhibitors as substitutes for thermodynamic inhibitors like methanol. We show here that antifreeze proteins (AFPs possess the ability to modify structure II (sII tetrahydrofuran (THF hydrate crystal morphologies by adhering to the hydrate surface and inhibiting growth in a similar fashion to the kinetic inhibitor poly-N-vinylpyrrolidone (PVP. The effects of AFPs on the formation and growth rate of high-pressure sII gas mix hydrate demonstrated that AFPs are superior hydrate inhibitors compared to PVP. These results indicate that AFPs may be suitable for the study of new inhibitor systems and represent an important step towards the development of biologically-based hydrate inhibitors.

  13. A root bond between ice and antifreeze protein.

    Science.gov (United States)

    Hawes, Timothy C

    2016-10-01

    It has always been assumed that a three-dimensional protein structure is essential to antifreeze protein (AFP) ice interactions. Using a 9 kDa AFP isolated from the springtail, Gomphiocephalus hodgsoni, it was found that the bond between ice and protein is maintained independent of higher order protein structure. GomplyAFP9 remained bound to ice after denaturing by a range of agents (boiling, extreme pH, DTT, ethanol, urea). Thermal hysteresis was minimal (0.03-0.04 °C), but not lost. Crystal faceting and growth occurred normal to the c-axis, indicating the protein binds primarily to sites along the a-axis. These observations lend additional support to the hypothesis of irreversible binding. More significantly, they suggest that binding to ice and functional hysteresis may be achieved independently (i.e. are different operations). These results are consistent with the view that there is a root bond with ice and it is achieved via an amino acid derived interface that bonds to water molecules in aqueous solutions. PMID:27542583

  14. Antifreeze proteins in the Antarctic springtail, Gressittacantha terranova.

    Science.gov (United States)

    Hawes, T C; Marshall, C J; Wharton, D A

    2011-08-01

    Antarctic springtails are exemplars of extreme low temperature adaptation in terrestrial arthropods. This paper represents the first examination of such adaptation in the springtail, Gressittacantha terranova. Acclimatization state was measured in field-fresh samples over a 22-day period at the beginning of the austral summer. No evidence of temperature tracking was observed. Mean temperature of crystallization (T(c)) for all samples was -20.67 ± 0.32°C and the lowest T(c) recorded was -32.62°C. Ice affinity purification was used to collect antifreeze proteins (AFPs) from springtail homogenate. The purified ice fraction demonstrated both thermal hysteresis activity and recrystallisation inhibition. Growth-melt observations revealed that ice crystals grow normal to the c-axis (basal plane). Reverse-phased HPLC produce one clearly resolved peak (P1) and one compound peak (P2). Mass spectrometry identified the molecular mass of P1 as 8,599 Da. The P1 protein was also the most prominent in P2, although additional peptides of 6-7 KDa were also prominent. The main AFP of the Antarctic springtail, G. terranova has been isolated, although like other AFP-expressing arthropods, it shows evidence of expressing a family of AFPs. PMID:21399953

  15. Thermodynamic Properties of Linear Protein Solutions: an Application to Type Ⅰ Antifreeze Protein Solutions

    Institute of Scientific and Technical Information of China (English)

    LI Li-fen; LIANG Xi-xia; LI Qian-zhong

    2012-01-01

    A statistical thermodynamic theory of linear protein solutions was proposed with the aid of a lattice model and applied to type Ⅰ antifreeze protein(AFPI) solutions.The numerical results for several AFPI solutions show that the Gibbs function of the solution has a minimum at a certain protein concentration,but the protein chemical potential increases with increasing the concentration.The influences of temperature and protein chain length on the AFPI chemical potential were also discussed.The evaluation for the colligative depression of the freezing point confirms that the antifreeze action should be recognized as non-colligative.The theoretical deduction for the concentration dependence of the thermal hysteresis activity coincides qualitatively with the previous experimental and theoretical results.

  16. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

    International Nuclear Information System (INIS)

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 310-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins

  17. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yong-Geun [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of); Park, Chin-Ju [Gwangju Institute of Science and Technology, Division of Liberal Arts and Sciences and Department of Chemistry (Korea, Republic of); Kim, Hee-Eun; Seo, Yeo-Jin; Lee, Ae-Ree; Choi, Seo-Ree; Lee, Shim Sung; Lee, Joon-Hwa, E-mail: joonhwa@gnu.ac.kr [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of)

    2015-02-15

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3{sub 10}-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

  18. A study of the growth rates and growth habits of ice crystals in a solution of antifreeze (glyco) proteins

    Science.gov (United States)

    Li, Qianzhong; Luo, Liaofu

    1996-12-01

    The mechanism of the antifreeze glycoprotein/antifreeze protein interaction on the surface of ice is analyzed. The theory of ice crystal growth in an AF(G)P solution is presented. A quantitative calculation of the growth rates for gain growth has been obtained. The anisotropic growth habits and growth rates of ice crystals in an AF(G)P solution are explained.

  19. Snow-mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity

    Science.gov (United States)

    Hiilovaara-Teijo; Hannukkala; Griffith; Yu; Pihakaski-Maunsbach

    1999-10-01

    During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.

  20. Research Progress on Insect Antifreeze Proteins%昆虫抗冻蛋白研究进展

    Institute of Scientific and Technical Information of China (English)

    万军; 朱兴友; 张洋

    2012-01-01

    近年来昆虫抗冻蛋白(AFPs)的研究取得了较快的发展。本文综述了昆虫抗冻蛋白的发现过程、结构特点、表达规律、抗冻机制及相关的昆虫基因工程简况。%In recent years, the research of insect antifreeze proteins has developed rapidly. The discovery process, structural characteristics, expression laws and antifreeze mechanism of insect antifreeze proteins, as well as the related insect gene engineering research were reviewed in this paper.

  1. Interaction of Tenebrio Molitor Antifreeze Protein with Ice Crystal: Insights from Molecular Dynamics Simulations.

    Science.gov (United States)

    Ramya, L; Ramakrishnan, Vigneshwar

    2016-07-01

    Antifreeze proteins (AFP) observed in cold-adapting organisms bind to ice crystals and prevent further ice growth. However, the molecular mechanism of AFP-ice binding and AFP-inhibited ice growth remains unclear. Here we report the interaction of the insect antifreeze protein (Tenebrio molitor, TmAFP) with ice crystal by molecular dynamics simulation studies. Two sets of simulations were carried out at 263 K by placing the protein near the primary prism plane (PP) and basal plane (BL) of the ice crystal. To delineate the effect of temperatures, both the PP and BL simulations were carried out at 253 K as well. The analyses revealed that the protein interacts strongly with the ice crystal in BL simulation than in PP simulation both at 263 K and 253 K. Further, it was observed that the interactions are primarily mediated through the interface waters. We also observed that as the temperature decreases, the interaction between the protein and the ice increases which can be attributed to the decreased flexibility and the increased structuring of the protein at low temperature. In essence, our study has shed light on the interaction mechanism between the TmAFP antifreeze protein and the ice crystal. PMID:27492241

  2. An Investigation of Freezing of Supercooled Water on Anti-Freeze Protein Modified Surfaces

    Institute of Scientific and Technical Information of China (English)

    Thibaut V J Charpentier; Anne Neville; Paul Millner; Rob Hewson; Ardian Morina

    2013-01-01

    This work investigates how functionalization ofaluminium surfaces with natural type Ⅲ Anti-Freeze Protein (AFP) affects the mechanism of heterogeneous ice nucleation.First the bulk ice nucleation properties of distilled water and aqueous solution of AFP were evaluated by differential scanning calorimetry.Then the modified surface was characterized by Secondary Ions Mass Spectroscopy (SIMS),Fourier Transform InfraRed (FTIR) spectroscopy and contact angle measurement.Freezing experiments were then conducted in which water droplets underwent a slow controlled cooling.This study shows that compared to uncoated aluminium,the anti-freeze proteins functionalized surfaces exhibit a higher and narrower range of freezing temperature.It was found that these proteins that keep living organisms from freezing in cold environment act in the opposite way once immobilized on surfaces by promoting ice nucleation.Some suggestions regarding the mechanism of action of the observed phenomena were proposed based on the Classical Nucleation Theory (CNT).

  3. Adsorption thermodynamics of two-domain antifreeze proteins: theory and Monte Carlo simulations.

    Science.gov (United States)

    Narambuena, Claudio F; Sanchez Varretti, Fabricio O; Ramirez-Pastor, Antonio J

    2016-09-21

    In this paper we develop the statistical thermodynamics of two-domain antifreeze proteins adsorbed on ice. We use a coarse-grained model and a lattice network in order to represent the protein and ice, respectively. The theory is obtained by combining the exact analytical expression for the partition function of non-interacting linear k-mers adsorbed in one dimension, and its extension to higher dimensions. The total and partial adsorption isotherms, and the coverage and temperature dependence of the Helmholtz free energy and configurational entropy are given. The formalism reproduces the classical Langmuir equation, leads to the exact statistical thermodynamics of molecules adsorbed in one dimension, and provides a close approximation for two-dimensional systems. Comparisons with analytical data obtained using the modified Langmuir model (MLM) and Monte Carlo simulations in the grand canonical ensemble were performed in order to test the validity of the theoretical predictions. In the MC calculations, the different mechanisms proposed in the literature to describe the adsorption of two-domain antifreeze proteins on ice were analyzed. Indistinguishable results were obtained in all cases, which verifies the thermodynamic equivalence of these mechanisms and allows the choice of the most suitable mechanism for theoretical studies of equilibrium properties. Even though a good qualitative agreement is obtained between MLM and MC data, it is found that the new theoretical framework offers a more accurate description of the phenomenon of adsorption of two-domain antifreeze proteins. PMID:27539563

  4. The response of watercress (nasturtium officinale) to vacuum impregnation: Effect of an antifreeze protein type I

    OpenAIRE

    Rui M.S. Cruz; Vieira, Margarida C.; Silva, Cristina L. M.

    2009-01-01

    The setting up of methodologies that reduce the size of ice crystals and reduce or inhibit the recrystallisation phenomena could have an extraordinary significance in the final quality of frozen products and consequently bring out new market opportunities. In this work, the effect of an antifreeze protein type I (AFP-I), by vacuum impregnation (VI), on frozen watercress was studied. The VI pressure, samples’ weight, Hunter Lab colour, scanning electron microscopy (SEM), and a wilting test ...

  5. Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature

    OpenAIRE

    WEN, Xin; Wang, Sen; Duman, John G.; Arifin, Josh Fnu; Juwita, Vonny; Goddard, William A.; Rios, Alejandra; Liu, Fan; Kim, Soo-Kyung; Abrol, Ravinder; DeVries, Arthur L.; Henling, Lawrence M.

    2016-01-01

    The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding ...

  6. 几种昆虫抗冻蛋白的研究概况%Research overview on several insect antifreeze proteins

    Institute of Scientific and Technical Information of China (English)

    李亚平; 刘丽娟

    2012-01-01

    昆虫抗冻蛋白具有很强的抗冻活性,其结构与其他生物不同.本文分别从结构、基因克隆、抗冻活性等方面对几种昆虫的抗冻蛋白进行综述,并对昆虫抗冻蛋白的发展前景进行了展望.%Insect antifreeze proteins have special structures and have stronger antifreeze activity than antifreeze proteins of other organisms. In this paper, the special properties of insect antifreeze proteins are reviewed. New advances on gene cloning and antifreeze activity are introduced in detail and the development perspectives of insect antifreeze proteins are prospected.

  7. Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor

    DEFF Research Database (Denmark)

    Kristiansen, E; Ramløv, Hans; Højrup, Peter;

    2011-01-01

    Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn...... beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is...... structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part...

  8. A low molecular weight peptide from snow mold with epitopic homology to the winter flounder antifreeze protein.

    Science.gov (United States)

    Newsted, W J; Polvi, S; Papish, B; Kendall, E; Saleem, M; Koch, M; Hussain, A; Cutler, A J; Georges, F

    1994-01-01

    Evidence for a small size protein (ca. 3500 kDa) exhibiting epitopic homology to the Atlantic winter flounder antifreeze protein (AFP) is found in the snow molds Coprinus psychromorbidus, Myriosclerotinia borealis, and Typhula incarnata. The protein shows strong cross-reactivity with antisera specific for the flounder AFP. Preliminary studies suggest that the protein is synthesized in response to lowering the culture temperature, and that it is membrane associated and, therefore, may function in an analogous capacity to the fish AFP. Also, the protein is shown to have antifreeze properties as determined by nuclear magnetic resonance microimaging experiments.

  9. Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function.

    Science.gov (United States)

    Duman, John G

    2015-06-01

    Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization - a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones - and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants. PMID:26085662

  10. Unusual dynamic properties of water near the ice-binding plane of hyperactive antifreeze protein

    Energy Technology Data Exchange (ETDEWEB)

    Kuffel, Anna; Czapiewski, Dariusz; Zielkiewicz, Jan, E-mail: jaz@chem.pg.gda.pl [Department of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80–233 Gdansk (Poland)

    2015-10-07

    The dynamical properties of solvation water of hyperactive antifreeze protein from Choristoneura fumiferana (CfAFP) are analyzed and discussed in context of its antifreeze activity. The protein comprises of three well-defined planes and one of them binds to the surface of ice. The dynamical properties of solvation water around each of these planes were analyzed separately; the results are compared with the dynamical properties of solvation water of ice around its two crystallographic planes: basal and prism. Three main conclusions are inferred from our investigations. The first one is that the solvation shell of CfAFP does not seem to be particularly far-ranged, at least not beyond what is usually observed for proteins that do not interact with ice. Therefore, it does not appear to us that the antifreeze activity is enhanced by a long-ranged retardation of water mobility. Also the correlation between the collective mobility of water and the collective mobility of protein atoms highly resembles the one measured for the protein that does not interact with ice. Our second conclusion is that the dynamical properties of solvation water of CfAFP are non-uniform. The dynamics of solvation water of ice-binding plane is, in some respects, different from the dynamics of solvation water of the two remaining planes. The feature that distinguishes the dynamics of solvation water of the three planes is the activation energy of diffusion process. The third conclusion is that—from the three analyzed solvation shells of CfAFP—the dynamical properties of solvation water of the ice-binding plane resemble the most the properties of solvation water of ice; note, however, that these properties still clearly differ from the dynamic properties of solvation water of ice.

  11. Unusual dynamic properties of water near the ice-binding plane of hyperactive antifreeze protein

    International Nuclear Information System (INIS)

    The dynamical properties of solvation water of hyperactive antifreeze protein from Choristoneura fumiferana (CfAFP) are analyzed and discussed in context of its antifreeze activity. The protein comprises of three well-defined planes and one of them binds to the surface of ice. The dynamical properties of solvation water around each of these planes were analyzed separately; the results are compared with the dynamical properties of solvation water of ice around its two crystallographic planes: basal and prism. Three main conclusions are inferred from our investigations. The first one is that the solvation shell of CfAFP does not seem to be particularly far-ranged, at least not beyond what is usually observed for proteins that do not interact with ice. Therefore, it does not appear to us that the antifreeze activity is enhanced by a long-ranged retardation of water mobility. Also the correlation between the collective mobility of water and the collective mobility of protein atoms highly resembles the one measured for the protein that does not interact with ice. Our second conclusion is that the dynamical properties of solvation water of CfAFP are non-uniform. The dynamics of solvation water of ice-binding plane is, in some respects, different from the dynamics of solvation water of the two remaining planes. The feature that distinguishes the dynamics of solvation water of the three planes is the activation energy of diffusion process. The third conclusion is that—from the three analyzed solvation shells of CfAFP—the dynamical properties of solvation water of the ice-binding plane resemble the most the properties of solvation water of ice; note, however, that these properties still clearly differ from the dynamic properties of solvation water of ice

  12. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

  13. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters. PMID:26371748

  14. The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate.

    Science.gov (United States)

    Duman, J G

    2002-02-01

    Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators. PMID:11916110

  15. Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

    2013-08-01

    Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian. PMID:23915159

  16. Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

    2013-08-01

    Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

  17. Agrobacterium-mediated transformation of modified antifreeze protein gene in strawberry

    Directory of Open Access Journals (Sweden)

    Srisulak Dheeranupattana

    2005-07-01

    Full Text Available The optimum condition for shoot regeneration from leaf explants of strawberry cultivar Tiogar was investigated. It was found that the best regeneration condition was MS medium containing N6-Benzyladenine (BA and 2,4-Dichlorophenoxy acetic acid (2,4-D at concentrations of 1 mg.l-1 and 0.2 mg.l-1, respectively. Antibiotics sensitivity test found that shoot regeneration from leaf explant was inhibited more than 90% at the concentration of kanamycin (Km as low as 5 mg.l-1. The modified gene encoding antifreeze protein isoform HPLC 6 was successfully constructed using codons which were optimally expressed in the strawberry plant. The antifreeze protein genes, naturally in plasmid pSW1 and modified in plasmid BB, were transformed to strawberry leaf explants by Agrobacterium tumefaciens LBA 4404. The strawberry plants, transformed with both AFP genes, were able to root in MS media containing 50 mg.l-1 Km, while no roots grew from nontransformed plant in this condition. Polymerase chain reaction indicated that the transgenes were integrated in the genome of transformants.

  18. An Effective Antifreeze Protein Predictor with Ensemble Classifiers and Comprehensive Sequence Descriptors

    Directory of Open Access Journals (Sweden)

    Runtao Yang

    2015-09-01

    Full Text Available Antifreeze proteins (AFPs play a pivotal role in the antifreeze effect of overwintering organisms. They have a wide range of applications in numerous fields, such as improving the production of crops and the quality of frozen foods. Accurate identification of AFPs may provide important clues to decipher the underlying mechanisms of AFPs in ice-binding and to facilitate the selection of the most appropriate AFPs for several applications. Based on an ensemble learning technique, this study proposes an AFP identification system called AFP-Ensemble. In this system, random forest classifiers are trained by different training subsets and then aggregated into a consensus classifier by majority voting. The resulting predictor yields a sensitivity of 0.892, a specificity of 0.940, an accuracy of 0.938 and a balanced accuracy of 0.916 on an independent dataset, which are far better than the results obtained by previous methods. These results reveal that AFP-Ensemble is an effective and promising predictor for large-scale determination of AFPs. The detailed feature analysis in this study may give useful insights into the molecular mechanisms of AFP-ice interactions and provide guidance for the related experimental validation. A web server has been designed to implement the proposed method.

  19. A 9 kDa antifreeze protein from the Antarctic springtail, Gomphiocephalus hodgsoni.

    Science.gov (United States)

    Hawes, T C; Marshall, C J; Wharton, D A

    2014-08-01

    A 9 kDA antifreeze protein (AFP) was isolated and purified from the Antarctic springtail, Gomphiocephalus hodgsoni. By combining selective sampling procedures and a modified ice affinity purification protocol it was possible to directly isolate a single AFP protein without recourse to chromatographic separation techniques. Mass spectrometry identified a single 9 kDa component in the purified ice fraction. Intramolecular disulphide bonding was suggested by the presence of 12 cysteine residues. The specific amino acid composition is unique, particularly with regard to the presence of histidine (11.5%). But it also shows noticeable commonalities with insect AFPs in the abundance of cysteine (13.8%), while simultaneously hinting, through the presence of glycine (11.5%), that the metabolic building blocks of AFPs in Collembola may have a phylogenetically-determined component. PMID:25025820

  20. Improvement of texture properties and flavor of frozen dough by carrot (Daucus carota) antifreeze protein supplementation.

    Science.gov (United States)

    Zhang, Chao; Zhang, Hui; Wang, Li; Gao, Hong; Guo, Xiao Na; Yao, Hui Yuan

    2007-11-14

    The effects of concentrated carrot protein (CCP) containing 15.4% (w/w) carrot (Daucus carota) antifreeze protein on texture properties of frozen dough and volatile compounds of crumb were studied. CCP supplementation lowered the freezable water content of the dough, resulting in some beneficial effects including holding loaf volume steadily and making the dough softer and steadier during frozen storage. Furthermore, SPME-GC-MS analysis showed CCP supplementation did not give any negative influences on volatile compounds of crumb and gave a pleasant aroma felt like Michelia alba DC from trans-caryophyllene simultaneously. Combining our previous results that CCP supplementation improves the fermentation capacity of the frozen dough, CCP could be used as a beneficial additive for frozen dough processing. PMID:17935294

  1. Recent Advances in Research of Antifreeze Proteins%抗冻蛋白研究进展

    Institute of Scientific and Technical Information of China (English)

    李金耀; 马纪; 张富春

    2005-01-01

    Many overwintering organisms produce antifreeze proteins (AFPs) that can be adsorbed onto the surface of ice crystals and modify their growth. These proteins show great diversity in structures, and they have been found in a variety of organisms. AFPs from insects have higher thermal hysteresis activity than other organisms. Recent studies revealed the structures of AFPs and put forward different ice-binding models. No mechanism, however, can apply to all antifreeze proteins and the molecular interaction between AFPs and ice are not accurately resolved. AFPs can be applied extensively to agriculture, aquaculture and low temperature storage of organs, tissues, as well as cells. To confer transgenic plant cold resistance application of AFPs is essential, while the expression and regulation of antifreeze gene need to be elucidated.%很多越冬的生物会产生抗冻蛋白,这些抗冻蛋白能够吸附到冰晶的表面改变冰晶形态并抑制冰晶的生长.抗冻蛋白在很多生物体内都被发现,不同的抗冻蛋白结构差异非常大.目前的一些研究揭示了几种抗冻蛋白的结构,并提出了抗冻蛋白与冰晶的结合模型,但是还没有一种机制能解释所有抗冻蛋白的作用机理.抗冻蛋白能被广泛的应用到农业、水产业和低温储藏器官、组织和细胞,利用转基因技术提高植物的抗冻性具有重要应用价值.而抗冻蛋白基因的表达调控则有待进一步阐明.

  2. Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

    Directory of Open Access Journals (Sweden)

    Shuaiqi Guo

    Full Text Available A novel role for antifreeze proteins (AFPs may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII and region IV (RIV, divide MpAFP into five distinct regions, all of which require mM Ca(2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2 server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.

  3. The mysteries of memory effect and its elimination with antifreeze proteins

    Energy Technology Data Exchange (ETDEWEB)

    Walker, V.; Gordienko, R.; Kuiper, M.; Huva, E.; Wu, Z. [Queen' s Univ., Kingston, ON (Canada). Dept. of Biology; Zeng, H.; Ripmeester, J. [Queen' s Univ., Kingston, ON (Canada). Dept. of Biology]|[National Research Council of Canada, Ottawa, ON (Canada). Steacie Inst. for Molecular Sciences

    2008-07-01

    With the decline in easily accessible and conventional hydrocarbon supplies, exploration will focus on hydrocarbons in deep offshore waters, in permafrost or in crystalline water as gas hydrates. Crystallization of water or water-encaged gas molecules takes place when nuclei reach a critical size, but the crystal growth may be inhibited by certain antifreeze proteins (AFPs). In this study, the authors hypothesized that the crystal lattice of gas hydrates may act as an alternative for substrate antifreeze proteins (AFPs). AFP-mediated inhibition of ice and clathrate hydrate crystallization was examined. Since the AFPs had a notable ability to eliminate the memory effect (ME) or the faster reformation of clathrate hydrates after melting, the authors were prompted to examine heterogeneous nucleation. Silica, served as a model nucleator hydrophilic surface. Quartz crystal microbalance-dissipation (QCM-D) experiments showed that an active AFP was tightly adsorbed to the silica surface. However, polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap), 2 commercial hydrate kinetic inhibitors that do not eliminate ME, were not as tightly adsorbed. A mutant AFP inhibited tetrahydrofuran clathrate hydrate growth, but not ME. QCM-D analysis showed that adsorption of the mutant AFP was more similar to PVCap than the active AFP. It was concluded that although there is no evidence for memory in ice reformation, the crystallization of ice and hydrates, and the elimination of the more rapid recrystallization of hydrates, can be mediated by the same proteins. The properties of adsorbed layers can be effectively monitored by QCM-D. These study results provided useful information about the inhibition mechanism of heterogeneous nucleation of clathrate hydrate. The technique facilitates the screening of potential low dose hydrate inhibitors and residues in AFPs that are involved in silica adsorption. 24 refs., 1 tab., 4 figs.

  4. Fluorescence microscopy studies of the hyperactive antifreeze protein from an insect

    Science.gov (United States)

    Pertaya, N.; di Prinzio, C. L.; Wilen, L.; Thomson, E.; Wettlaufer, J. S.; Marshall, C. B.; Davies, P. L.; Braslavsky, I.

    2006-03-01

    Antifreeze proteins (AFPs) protect animals from freezing by binding to extracellular ice and inhibiting its growth. Since the initial discovery of AFPs in fish, non-homologous types have been found in insects, plants, bacteria, fungi, and vertebrates. Different AFP types have diverse structures and varied activities. For example, AFPs produced by insects are much more active in inhibiting ice crystal growth compared to most AFPs found in fish or plants. By putting a fluorescent tag on an insect AFP we were able to visualize AFP binding to ice, to determine the ice crystal surfaces to which the AFP adheres, and to follow the kinetics of AFP binding to ice. We expect this approach will contribute to a better understanding of the mechanism of AFP activity and in particular the hyperactivity of insect AFPs.

  5. The Surface of Ice in the presence of Antifreeze Proteins studied by Atomic Force Microscopy

    Science.gov (United States)

    Zepeda, Salvador; Orme, Christine; Yeh, Yin

    2002-03-01

    The surface of ice has been a topic of interest for centuries. In particular, the surface structure and properties have been explored with the advent of new surface techniques. Several groups have convincingly shown a surface transition layer to exist between the solid-vapor interface as well as the solid-liquid interface. In addition, the characteristics of this region may be directly correlated with growth morphologies of ice. Certain peptide molecules have the ability to significantly alter the growth morphology of an ice crystal. Do these molecules simply disrupt this transition region? Or do they anchor themselves deep into it reaching the bulk-ice phase? And is there a similar mechanism by which they function? We use AFM to study the morphological changes to the true ice surface due to the presence antifreeze proteins. We will discuss the implications of our results on the longstanding debate to the above questions.

  6. Evolution of Type II Antifreeze Protein Genes in Teleost Fish: A Complex Scenario Involving Lateral Gene Transfers and Episodic Directional Selection

    OpenAIRE

    Ulf Sorhannus

    2012-01-01

    I examined hypotheses about lateral transfer of type II antifreeze protein (AFP) genes among “distantly” related teleost fish. The effects of episodic directional selection on amino acid evolution were also investigated. The strict consensus results showed that the type II AFP and type II antifreeze-like protein genes were transferred from Osmerus mordax to Clupea harengus, from the ancestral lineage of the Brachyopsis rostratus—Hemitripterus americanus clade to the ancestor of the Hypomesus ...

  7. Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature.

    Science.gov (United States)

    Wen, Xin; Wang, Sen; Duman, John G; Arifin, Josh Fnu; Juwita, Vonny; Goddard, William A; Rios, Alejandra; Liu, Fan; Kim, Soo-Kyung; Abrol, Ravinder; DeVries, Arthur L; Henling, Lawrence M

    2016-06-14

    The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding insects in winter, and yet, paradoxically are found in some freeze-tolerant insects. Here, we report a previously unidentified role for AFPs in effectively inhibiting trehalose precipitation in the hemolymph (or blood) of overwintering beetle larvae. We determine the trehalose level (29.6 ± 0.6 mg/mL) in the larval hemolymph of a beetle, Dendroides canadensis, and demonstrate that the hemolymph AFPs are crucial for inhibiting trehalose crystallization, whereas the presence of trehalose also enhances the antifreeze activity of AFPs. To dissect the molecular mechanism, we examine the molecular recognition between AFP and trehalose crystal interfaces using molecular dynamics simulations. The theory corroborates the experiments and shows preferential strong binding of the AFP to the fast growing surfaces of the sugar crystal. This newly uncovered role for AFPs may help explain the long-speculated role of AFPs in freeze-tolerant species. We propose that the presence of high levels of molecules important for survival but prone to precipitation in poikilotherms (their body temperature can vary considerably) needs a companion mechanism to prevent the precipitation and here present, to our knowledge, the first example. Such a combination of trehalose and AFPs also provides a novel approach for cold protection and for trehalose crystallization inhibition in industrial applications. PMID:27226297

  8. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax.

    Science.gov (United States)

    Friis, Dennis S; Johnsen, Johannes L; Kristiansen, Erlend; Westh, Peter; Ramløv, Hans

    2014-06-01

    The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm , of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.

  9. Influence of Block Copolymerization on the Antifreeze Protein Mimetic Ice Recrystallization Inhibition Activity of Poly(vinyl alcohol).

    Science.gov (United States)

    Congdon, Thomas R; Notman, Rebecca; Gibson, Matthew I

    2016-09-12

    Antifreeze (glyco) proteins are produced by many cold-acclimatized species to enable them to survive subzero temperatures. These proteins have multiple macroscopic effects on ice crystal growth which makes them appealing for low-temperature applications-from cellular cryopreservation to food storage. Poly(vinyl alcohol) has remarkable ice recrystallization inhibition activity, but its mode of action is uncertain as is the extent at which it can be incorporated into other high-order structures. Here the synthesis and characterization of well-defined block copolymers containing poly(vinyl alcohol) and poly(vinylpyrrolidone) by RAFT/MADIX polymerization is reported, as new antifreeze protein mimetics. The effect of adding a large second hydrophilic block is studied across a range of compositions, and it is found to be a passive component in ice recrystallization inhibition assays, enabling retention of all activity. In the extreme case, a block copolymer with only 10% poly(vinyl alcohol) was found to retain all activity, where statistical copolymers of PVA lose all activity with very minor changes to composition. These findings present a new method to increase the complexity of antifreeze protein mimetic materials, while retaining activity, and also to help understand the underlying mechanisms of action.

  10. Expression of a Carrot 36 kD Antifreeze Protein Gene Improves Cold Stress Tolerance in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Antifreeze proteins (AFPs) enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants. In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S:AFP and Prd29A:AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress.However, the use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition (2℃). The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.

  11. Antifreeze (glyco)protein mimetic behavior of poly(vinyl alcohol): detailed structure ice recrystallization inhibition activity study.

    Science.gov (United States)

    Congdon, Thomas; Notman, Rebecca; Gibson, Matthew I

    2013-05-13

    This manuscript reports a detailed study on the ability of poly(vinyl alcohol) to act as a biomimetic surrogate for antifreeze(glyco)proteins, with a focus on the specific property of ice-recrystallization inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biological antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alcohol) (PVA) has been shown to display remarkable ice recrystallization inhibition activity despite its major structural differences to native antifreeze proteins. Here, controlled radical polymerization is used to synthesize well-defined PVA, which has enabled us to obtain the first quantitative structure-activity relationships, to probe the role of molecular weight and comonomers on IRI activity. Crucially, it was found that IRI activity is "switched on" when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains with hydrophilic or hydrophobic units was found to diminish activity. Hydrophobic modifications to the backbone were slightly more tolerated than side chain modifications, which implies an unbroken sequence of hydroxyl units is necessary for activity. These results highlight that, although hydrophobic domains are key components of IRI activity, the random inclusion of addition hydrophobic units does not guarantee an increase in activity and that the actual polymer conformation is important.

  12. Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

    Science.gov (United States)

    Stevens, Corey A; Drori, Ran; Zalis, Shiran; Braslavsky, Ido; Davies, Peter L

    2015-09-16

    By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization. PMID:26267368

  13. Structural Basis for the Inhibition of Gas Hydrates by α-Helical Antifreeze Proteins.

    Science.gov (United States)

    Sun, Tianjun; Davies, Peter L; Walker, Virginia K

    2015-10-20

    Kinetic hydrate inhibitors (KHIs) are used commercially to inhibit gas hydrate formation and growth in pipelines. However, improvement of these polymers has been constrained by the lack of verified molecular models. Since antifreeze proteins (AFPs) act as KHIs, we have used their solved x-ray crystallographic structures in molecular modeling to explore gas hydrate inhibition. The internal clathrate water network of the fish AFP Maxi, which extends to the protein's outer surface, is remarkably similar to the {100} planes of structure type II (sII) gas hydrate. The crystal structure of this water web has facilitated the construction of in silico models for Maxi and type I AFP binding to sII hydrates. Here, we have substantiated our models with experimental evidence of Maxi binding to the tetrahydrofuran sII model hydrate. Both in silico and experimental evidence support the absorbance-inhibition mechanism proposed for KHI binding to gas hydrates. Based on the Maxi crystal structure we suggest that the inhibitor adsorbs to the gas hydrate lattice through the same anchored clathrate water mechanism used to bind ice. These results will facilitate the rational design of a next generation of effective green KHIs for the petroleum industry to ensure safe and efficient hydrocarbon flow. PMID:26488661

  14. Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

    Science.gov (United States)

    Stevens, Corey A; Drori, Ran; Zalis, Shiran; Braslavsky, Ido; Davies, Peter L

    2015-09-16

    By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization.

  15. Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

    Science.gov (United States)

    Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-15

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

  16. Antifreeze proteins in the primary urine of larvae of the beetle Dendroides canadensis.

    Science.gov (United States)

    Nickell, Philip K; Sass, Sandra; Verleye, Dawn; Blumenthal, Edward M; Duman, John G

    2013-05-01

    To avoid freezing while overwintering beneath the bark of fallen trees, Dendroides canadensis (Coleoptera: Pyrochroidae) larvae produce a family of antifreeze proteins (DAFPs) that are transcribed in specific tissues and have specific compartmental fates. DAFPs and associated thermal hysteresis activity (THA) have been shown previously in hemolymph and midgut fluid, but the presence of DAFPs has not been explored in primary urine, a potentially important site that can contain endogenous ice-nucleating compounds that could induce freezing. A maximum mean THA of 2.65±0.33°C was observed in primary urine of winter-collected D. canadensis larvae. THA in primary urine increased significantly through autumn, peaked in the winter and decreased through spring to levels of 0.2-0.3°C in summer, in a pattern similar to that of hemolymph and midgut fluid. THA was also found in hindgut fluid and excreted rectal fluid, suggesting that these larvae not only concentrate AFPs in the hindgut, but also excrete AFPs from the rectal cavity. Based on dafp transcripts isolated from Malpighian tubule epithelia, cDNAs were cloned and sequenced, identifying the presence of transcripts encoding 24 DAFP isoforms. Six of these Malpighian tubule DAFPs were known previously, but 18 are new. We also provide functional evidence that DAFPs can inhibit ice nucleators present in insect primary urine. This is potentially critical because D. canadensis larvae die if frozen, and therefore ice formation in any body fluid, including the urine, would be lethal.

  17. Structure and evolutionary origin of Ca(2+-dependent herring type II antifreeze protein.

    Directory of Open Access Journals (Sweden)

    Yang Liu

    Full Text Available In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs. AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP, a Ca(2+-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca(2+-dependent lectins with unique ice-binding features. The 1.7 A crystal structure of hAFP with bound Ca(2+ and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca(2+-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca(2+ through the coordination with a water molecule of the ice lattice. This Ca(2+-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.

  18. Production of a recombinant type 1 antifreeze protein analogue by L. lactis and its applications on frozen meat and frozen dough.

    Science.gov (United States)

    Yeh, Chuan-Mei; Kao, Bi-Yu; Peng, Hsuan-Jung

    2009-07-22

    In this study, a novel recombinant type I antifreeze protein analogue (rAFP) was produced and secreted by Lactococcus lactis, a food-grade microorganism of major commercial importance. Antifreeze proteins are potent cryogenic protection agents for the cryopreservation of food and pharmaceutical materials. A food-grade expression and fermentation system (BSE- and antibiotic-free) for the production and secretion of high levels of rAFP was developed. Lyophilized, crude rAFP produced by L. lactis was tested in a frozen meat and frozen dough processing model. The frozen meat treated with the antifreeze protein showed less drip loss, less protein loss, and a high score on juiciness by sensory evaluation. Frozen dough treated with the rAFP showed better fermentation capacity than untreated frozen dough. Breads baked from frozen dough treated with rAFP acquired the same consumer acceptance as fresh bread. PMID:19545118

  19. Lateral transfer of a lectin-like antifreeze protein gene in fishes.

    Directory of Open Access Journals (Sweden)

    Laurie A Graham

    Full Text Available Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche.

  20. In silico characterization of antifreeze proteins using computational tools and servers

    Indian Academy of Sciences (India)

    K Sivakumar; S Balaji; Gangaradhakrishnan

    2007-09-01

    In this paper, seventeen different fish Antifreeze Proteins (AFPs) retrieved from Swiss-Prot database are analysed and characterized using In silico tools. Primary structure analysis shows that most of the AFPs are hydrophobic in nature due to the high content of non-polar residues. The presence of 11 cysteines in the rainbow smelt fish and sea raven fish AFPs infer that these proteins may form disulphide (SS) bonds, which are regarded as a positive factor for stability. The aliphatic index computed by Ex-Pasy’s ProtParam infers that AFPs may be stable for a wide range of temperature. Secondary structure analysis shows that most of the fish AFPs have predominant α-helical structures and rest of the AFPs have mixed secondary structure. The very high coil structural content of rainbow smelt fish and sea raven fish AFPs are due to the rich content of more flexible glycine and hydrophobic proline amino acids. Proline has a special property of creating kinks in polypetide chains and disrupting ordered secondary structure. SOSUI server predicts one transmembrane region in winter flounder fish and atlantic cod and two transmembrane regions in yellowtail flounder fish AFP. The predicted transmembrane regions were visualized and analysed using helical wheel plots generated by EMBOSS pepwheel tool. The presence of disulphide (SS) bonds in the AFPs Q01758 and P05140 are predicted by CYS_REC tool and also identified from the three-dimensional structure using Rasmol tool. The disulphide bonds identified from the three-dimensional structure using the Rasmol tool might be correct as the evaluation parameters are within the acceptable limits for the modelled 3D structures.

  1. Expression, purification and activity determination of the beetle tenebrio molitor antifreeze protein afp84c in escherichia coli

    International Nuclear Information System (INIS)

    Summary: A cDNA encoding antifreeze protein (AFP84c) was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 252 bp encodes a protein of 84 amino acid residues and was fused to the expression vectors pMAL-c2X and pMAL-p2X. The expression plasmids pMAL-c2X-afp84c and pMAL-p2X-afp84c were constructed and transformed into Escherischia coli strains TBI, respectively. Strategy of optimization of induction conditions were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in pMALTM expression system. The target fusion protein was released from the cytoplasm and periplasm by sonication and cold osmotic shock procedure respectively. Recombinant AFP84c was purified by amylose affinity column. The purified target protein displayed a single band in SDS-PAGE. Expressed AFP84c exhibits to increase low temperature resistance of bacteria. (author)

  2. Modeling the Influence of Antifreeze Proteins on Three-Dimensional Ice Crystal Melt Shapes using a Geometric Approach

    CERN Document Server

    Liu, Jun Jie; Dolev, Maya Bar; Celik, Yeliz; Wettlaufer, J S; Braslavsky, Ido

    2012-01-01

    The melting of pure axisymmetric ice crystals has been described previously by us within the framework of so-called geometric crystal growth. Nonequilibrium ice crystal shapes evolving in the presence of hyperactive antifreeze proteins (hypAFPs) are experimentally observed to assume ellipsoidal geometries ("lemon" or "rice" shapes). To analyze such shapes we harness the underlying symmetry of hexagonal ice Ih and extend two-dimensional geometric models to three-dimensions to reproduce the experimental dissolution process. The geometrical model developed will be useful as a quantitative test of the mechanisms of interaction between hypAFPs and ice.

  3. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Johnsen, Johannes Lørup; Kristiansen, Erlend;

    2014-01-01

    The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the...... RmAFP1 has only one disulfide bridge. The melting temperature, Tm, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that...... the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and...

  4. Structure of solvation water around the active and inactive regions of a type III antifreeze protein and its mutants of lowered activity

    Science.gov (United States)

    Grabowska, Joanna; Kuffel, Anna; Zielkiewicz, Jan

    2016-08-01

    Water molecules from the solvation shell of the ice-binding surface are considered important for the antifreeze proteins to perform their function properly. Herein, we discuss the problem whether the extent of changes of the mean properties of solvation water can be connected with the antifreeze activity of the protein. To this aim, the structure of solvation water of a type III antifreeze protein from Macrozoarces americanus (eel pout) is investigated. A wild type of the protein is used, along with its three mutants, with antifreeze activities equal to 54% or 10% of the activity of the native form. The solvation water of the ice-binding surface and the rest of the protein are analyzed separately. To characterize the structure of solvation shell, parameters describing radial and angular characteristics of the mutual arrangement of the molecules were employed. They take into account short-distance (first hydration shell) or long-distance (two solvation shells) effects. The obtained results and the comparison with the results obtained previously for a hyperactive antifreeze protein from Choristoneura fumiferana lead to the conclusion that the structure and amino acid composition of the active region of the protein evolved to achieve two goals. The first one is the modification of the properties of the solvation water. The second one is the geometrical adjustment of the protein surface to the specific crystallographic plane of ice. Both of these goals have to be achieved simultaneously in order for the protein to perform its function properly. However, they seem to be independent from one another in a sense that very small antifreeze activity does not imply that properties of water become different from the ones observed for the wild type. The proteins with significantly lower activity still modify the mean properties of solvation water in a right direction, in spite of the fact that the accuracy of the geometrical match with the ice lattice is lost because of the

  5. Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus

    DEFF Research Database (Denmark)

    Wilkens, Casper; Poulsen, Jens-Christian Navarro; Ramløv, Hans;

    2014-01-01

    Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform...

  6. De novo DESIGN AND SYNTHESIS OF AN ICE-BINDING, DENDRIMERIC, POLYPEPTIDE BASED ON INSECT ANTIFREEZE PROTEINS

    Directory of Open Access Journals (Sweden)

    Ricardo Vera Bravo

    2011-12-01

    Full Text Available A new strategy is presented for the designand synthesis of peptides that exhibitice-binding and antifreeze activity. Apennant-type dendrimer polypeptidescaffold combining an α-helical backbonewith four short β-strand branches wassynthesized in solid phase using Fmocchemistry in a divergent approach. The51-residue dendrimer was characterizedby reverse phase high performance liquidchromatography, mass spectrometry andcircular dichroism. Each β-strand branchcontained three overlapping TXT aminoacid repeats, an ice-binding motif foundin the ice-binding face of the sprucebudworm (Choristoneura fumiferanaand beetle (Tenebrio molitor antifreezeproteins. Ice crystals in the presence ofthe polypeptide monomer displayed flat,hexagonal plate morphology, similar tothat produced by weakly active antifreezeproteins. An oxidized dimeric form of thedendrimer polypeptide also produced flathexagonal ice crystals and was capableof inhibiting ice crystal growth upontemperature reduction, a phenomenontermed thermal hysteresis, a definingproperty of antifreeze proteins. Linkageof the pennant-type dendrimer to a trifunctionalcascade-type polypeptideproduced a trimeric macromolecule thatgave flat hexagonal ice crystals withhigher thermal hysteresis activity thanthe dimer or monomer and an ice crystal burst pattern similar to that producedby samples containing insect antifreezeproteins. This macromolecule was alsocapable of inhibiting ice recrystallization.

  7. Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation.

    Directory of Open Access Journals (Sweden)

    Jaewang Lee

    Full Text Available Ovarian tissue (OT cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function.This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs on mouse ovarian tissue cryopreservation and transplantation.Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III and concentration (0.1, 1, 10 mg/mL used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs and repair (DDR, respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control. Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared.In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL

  8. Research Progress in Antifreeze Proteins and Application in Food Industry%抗冻蛋白的研究进展及其在食品工业中的应用

    Institute of Scientific and Technical Information of China (English)

    汪少芸; 赵珺; 吴金鸿; 陈琳

    2011-01-01

    抗冻蛋白是一类具有热滞效应、冰晶形态效应和重结晶抑制效应的蛋白质,因其特殊的结构和功能,抗冻蛋白引起了研究人员的极大兴趣.探讨了近年来抗冻蛋白的研究进展,介绍了目前已知的抗冻蛋白的来源、特性、测定方法、基因结构及在食品工业中的应用.抗冻蛋白对冷冻食品有显著的品质改良功能,是未来冷冻食品工业中极具潜力的抗冻添加剂.%Antifreeze proteins (AFPs) are the thermal hysteresis proteins that have the ability to modify the growth and inhibit the recrystallization of the ice. Antifreeze proteins aroused great interests of many researchers due to its special structure and functions. In this article, the recent advance in antifreeze protein was reviewed, and the types, properties, measurements, gene structures of antifreeze protein, and its applications in food industry were introduced. The application trials indicated that antifreeze protein could significantly improve the qualities of frozen foods, which suggested the potential food additives of antifreeze protein in future frozen food industry.

  9. Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect.

    Science.gov (United States)

    Yu, Shu-Hui; Yang, Pu; Sun, Tao; Qi, Qian; Wang, Xue-Qing; Chen, Xiao-Ming; Feng, Ying; Liu, Bo-Wen

    2016-06-01

    The Chinese white wax scale insect, Ericerus pela, can survive at extremely low temperatures, and some overwintering individuals exhibit supercooling at temperatures below -30°C. To investigate the deep supercooling ability of E. pela, transcriptomic and proteomic analyses were performed to delineate the major gene and protein families responsible for the deep supercooling ability of overwintering females. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that genes involved in the mitogen-activated protein kinase, calcium, and PI3K-Akt signaling pathways and pathways associated with the biosynthesis of soluble sugars, sugar alcohols and free amino acids were dominant. Proteins responsible for low-temperature stress, such as cold acclimation proteins, glycerol biosynthesis-related enzymes and heat shock proteins (HSPs) were identified. However, no antifreeze proteins (AFPs) were identified through sequence similarity search methods. A random forest approach identified 388 putative AFPs in the proteome. The AFP gene ep-afp was expressed in Escherichia coli, and the expressed protein exhibited a thermal hysteresis activity of 0.97°C, suggesting its potential role in the deep supercooling ability of E. pela.

  10. Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect.

    Science.gov (United States)

    Yu, Shu-Hui; Yang, Pu; Sun, Tao; Qi, Qian; Wang, Xue-Qing; Chen, Xiao-Ming; Feng, Ying; Liu, Bo-Wen

    2016-06-01

    The Chinese white wax scale insect, Ericerus pela, can survive at extremely low temperatures, and some overwintering individuals exhibit supercooling at temperatures below -30°C. To investigate the deep supercooling ability of E. pela, transcriptomic and proteomic analyses were performed to delineate the major gene and protein families responsible for the deep supercooling ability of overwintering females. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that genes involved in the mitogen-activated protein kinase, calcium, and PI3K-Akt signaling pathways and pathways associated with the biosynthesis of soluble sugars, sugar alcohols and free amino acids were dominant. Proteins responsible for low-temperature stress, such as cold acclimation proteins, glycerol biosynthesis-related enzymes and heat shock proteins (HSPs) were identified. However, no antifreeze proteins (AFPs) were identified through sequence similarity search methods. A random forest approach identified 388 putative AFPs in the proteome. The AFP gene ep-afp was expressed in Escherichia coli, and the expressed protein exhibited a thermal hysteresis activity of 0.97°C, suggesting its potential role in the deep supercooling ability of E. pela. PMID:26799455

  11. Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.

    Science.gov (United States)

    Middleton, Adam J; Marshall, Christopher B; Faucher, Frédérick; Bar-Dolev, Maya; Braslavsky, Ido; Campbell, Robert L; Walker, Virginia K; Davies, Peter L

    2012-03-01

    The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.

  12. Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.

    Science.gov (United States)

    Kondo, Hidemasa; Hanada, Yuichi; Sugimoto, Hiroshi; Hoshino, Tamotsu; Garnham, Christopher P; Davies, Peter L; Tsuda, Sakae

    2012-06-12

    Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

  13. Molecular and quantum mechanical studies on the monomer recognition of a highly-regular β-helical antifreeze protein

    Institute of Scientific and Technical Information of China (English)

    YANG; Zuoyin; JIA; Zongchao; LIU; Ruozhuang; CHEN; Guangj

    2004-01-01

    The possible interaction models for an antifreeze protein from Tenebrio molitar (TmAFP) have been systematically studied using the methods of molecular mechanics, molecular dynamics and quantum chemistry. It is hoped that these approaches would provide insights into the nature of interaction between protein monomers through sampling a number of interaction possibilities and evaluating their interaction energies between two monomers in the course of recognition. The results derived from the molecular mechanics indicate that monomer's β-sheets would be involved in interaction area and the side chains on two β-faces can match each other at the two-dimensional level. The results from molecular mechanics and ONIOM methods show that the strongest interaction energy could be gained through the formation of H-bonds when the two β-sheets are involved in the interaction model. Furthermore, the calculation of DFT and analysis of van der Waals bond charge density confirm further that recognition between the two TCTs mainly depends on inter-molecular hydroxyls. Therefore, our results demonstrate that during the course of interaction the most favorable association of TmAFPs is via their β-sheets.

  14. Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus.

    Science.gov (United States)

    Nishimiya, Yoshiyuki; Kondo, Hidemasa; Takamichi, Manabu; Sugimoto, Hiroshi; Suzuki, Mamoru; Miura, Ai; Tsuda, Sakae

    2008-10-10

    We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca(2+)-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 A resolution. The lpAFP structure displayed a relatively planar surface, which encompasses two loop regions (Cys86-Lys89 and Asn91-Cys97) and a short beta-strand (Trp109-Leu112) with three unstructured segments (Gly57-Ile58, Ala103-Ala104, and Pro113-His118). Electrostatic calculation of the protein surface showed that the relatively planar surface was divided roughly into a hydrophobic area (composed of the three unstructured segments lacking secondary structure) and a hydrophilic area (composed of the loops and beta-strand). Site-directed mutation of Ile58 with Phe at the center of the hydrophobic area decreased activity significantly, whereas mutation of Leu112 with Phe at an intermediate area between the hydrophobic and hydrophilic areas retained complete activity. In the hydrophilic area, a peptide-swap mutant in the loops retained 60% activity despite simultaneous mutations of eight residues. We conclude that the epicenter of the ice-binding site of lpAFP is the hydrophobic region, which is centered by Ile58, in the relatively planar surface. We built an ice-binding model for lpAFP on the basis of a lattice match of ice and constrained water oxygen atoms surrounding the hydrophobic area in the lpAFP structure. The model in which lpAFP has been docked to a secondary prism (2-1-10) plane, which is different from the one determined for Ca(2+)-independent type II AFP from sea raven (11-21), appears to explain the results of the mutagenesis analysis. PMID:18674542

  15. Saccharide antifreeze compositions

    Science.gov (United States)

    Walters, Kent; Duman, John G; Serianni, Anthony S

    2013-12-10

    The invention provides an antifreeze glycolipid compounds and composition comprising a polysaccharide moiety of Formula I; ##STR00001## wherein D-Manp represents a D-mannopyranose moiety, D-Xylp represents a D-xylopyranose moiety, and n is about 5 to about 70; and one or more lipid moieties covalently linked to the polysaccharide moiety of Formula I or electrostatically associated with the polysaccaride moiety for Formula I. The antifreeze glycolipid compounds and compositions can be used for a variety of industrial, agricultural, medical, and cosmetic applications where recrystallization-inhibition, cyroprotection, or cryopreservation is desired. The antifreeze glycolipid compounds or compositions can be used as, for example, as cryoprotectants for tissue preservation and transplantation, improving the texture of processed frozen food and frozen meats, frostbit protection, crop protection, and green alternatives for land vehicle antifreeze and aircraft de-icing.

  16. 胡萝卜与黄粉虫抗冻融合基因在拟南芥中的表达与抗冻性分析%Expression and Antifreeze Feature Analysis of the Daucus carota and Tentbrio molitor Fusion Antifreeze Protein Gene in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    张振华; 陈介南; 卢孟柱; 章怀云; 刘伯斌

    2012-01-01

    抗冻蛋白(antifreeze protein,AFP)是一类能控制冰晶生长和抑制冰晶之间发生重结晶的蛋白质,能在低温结冰条件下保护生物体不受伤害.抗冻蛋白具有2种明显不同的抗冻活性——热滞(thermal hysteresis,TH)活性和重结晶抑制(recrystallizationinhibition,RI)活性.鱼类和昆虫抗冻蛋白的特征是高TH低RI活性,而植物抗冻蛋白的特征是低TH高RI活性.为了获得高TH、高RI活性的抗冻蛋白(antifreeze protein,AFP),克隆得到胡萝卜抗冻蛋白基因(DcAFP)和黄粉虫抗冻蛋白基因(TmAFP)cDNA 全序列.通过重叠延伸PCR,将2个基因前后串联构建融合基因Dc- TmAFP和Tm -DcAFP,并构建了植物表达载体,通过农杆菌介导转化模式植物拟南芥,成功获得4个基因的转基因拟南芥植株.对转基因拟南芥植株进行抗冻试验处理,并进行了存活率统计和叶片黄化率统计分析.分析结果表明,不同功能的抗冻蛋白对植物的抗冻性有不同的帮助,热滞效应高效的黄粉虫抗冻蛋白对植物降低冰点方面效果大,而在植物遭受较长时间冻害时,抑制重结晶效应高效的胡萝卜抗冻蛋白对植物质外体中流体保持稳定性作用较大,从而提高植物的耐冻能力.%Antifreeze proteins (AFPs) are a family of proteins capable of protecting organisms from damage in freezing or sub-freezing conditions by controlling the growth of ice and inhibiting the recrystallization between ice granules, which are termed thermal hysteresis (TH) activity and recrystallization inhibition (RI) activity respectively. In comparison with the characteristic of high TH and low RI of fish AFPs and insect AFPs, antifreeze proteins of plants was low TH and high RI activity. In order to obtain antifreeze proteins with high TH and high RI activity, the complete cDNA of antifreeze protein genes (DcAFP and TmAFP) were cloned from Daucus carota and Tentbrio molitor. The two fusion antifreeze protein genes, Dc

  17. Identification of antifreeze proteins and their functional residues by support vector machine and genetic algorithms based on n-peptide compositions.

    Directory of Open Access Journals (Sweden)

    Chin-Sheng Yu

    Full Text Available For the first time, multiple sets of n-peptide compositions from antifreeze protein (AFP sequences of various cold-adapted fish and insects were analyzed using support vector machine and genetic algorithms. The identification of AFPs is difficult because they exist as evolutionarily divergent types, and because their sequences and structures are present in limited numbers in currently available databases. Our results reveal that it is feasible to identify the shared sequential features among the various structural types of AFPs. Moreover, we were able to identify residues involved in ice binding without requiring knowledge of the three-dimensional structures of these AFPs. This approach should be useful for genomic and proteomic studies involving cold-adapted organisms.

  18. DSC Study on the Thermal Hysteresis Activity of Plant Antifreeze Proteins%沙冬青抗冻蛋白热滞活性的DSC研究

    Institute of Scientific and Technical Information of China (English)

    周晓蕾; 陈滔滔; 王保怀; 李芝芬; 费云标; 魏令波; 高素琴

    2001-01-01

    Differential scanning calorimetry (DSC) was used to measure thethermal hysteresis activity(THA) of plant antifreeze proteins(AFPs). The results reveal that DSC is a good method to screen and study AFPs. In the sixteen components extracted from Ammopipanthus mongolicus leaves, one(P3S1) was found to have apparent thermal hysteresis activity by DSC. As the amount of ice nuclei in the sample decreased, the THA of P3S1 increased from 0.01 ℃ to 0.65 ℃ . It is notable that the two-peak thermal hysteresis effect was observed. Two endothermic peaks appeared in the melting process of P3S1, while the freezing peak also consisted of two peaks. The peaks appeared antecedently showed larger thermal effect. This phenomenon shows P3S1 has two different kinds of interaction with water and ice crystal. It is probably an important property of a class of AFPs.

  19. Identification and Evaluation of Cryoprotective Peptides from Chicken Collagen: Ice-Growth Inhibition Activity Compared to That of Type I Antifreeze Proteins in Sucrose Model Systems.

    Science.gov (United States)

    Du, Lihui; Betti, Mirko

    2016-06-29

    The ability of chicken collagen peptides to inhibit the growth of ice crystals was evaluated and compared to that of fish antifreeze proteins (AFPs). This ice inhibition activity was assessed using a polarized microscope by measuring ice crystal dimensions in a sucrose model system with and without collagen peptides after seven thermal cycles. The system was stabilized at -25 °C and cycled between -16 and -12 °C. Five candidate peptides with ice inhibition activity were identified using liquid chromatography and tandem mass spectrometry and were then synthesized. Their ice inhibition capacity was compared to that of type I AFPs in a 23% sucrose model system. Specific collagen peptides with certain amino acid sequences reduced the extent of ice growth by approximately 70% at a relatively low concentration (1 mg/mL). These results suggest that specific collagen peptides may act in a noncolligative manner, inhibiting ice crystal growth like type I AFPs, but less efficiently. PMID:27293017

  20. Validation of antifreeze properties of glutathione based on its thermodynamic characteristics and protection of baker's yeast during cryopreservation.

    Science.gov (United States)

    Zhang, Chao; Zhang, Hui; Wang, Li; Yao, Huiyuan

    2007-06-13

    The antifreeze ability of glutathione was evaluated on the basis of its thermodynamic characteristics and protection of baker's yeast during cryopreservation at -30 degrees C. The thermodynamic characteristics and protection of baker's yeast of glutathione were similar to those of known antifreeze proteins, such as carrot antifreeze protein and holly antifreeze protein. These properties included lowering the freezing point at about 0.20 degrees C non-colligatively, decreasing freezable water content, controlling the movement of free water for its strong hydrophilicity, and improving baker's yeast survival during the simulated processing of frozen dough. Therefore, glutathione was viewed to be an antifreeze protein like substance on the basis of its unique thermodynamic characteristics and protection of baker's yeast. The method combining thermodynamic characteristic analysis and protection evaluation is a new and simple way to screen new antifreeze proteins. PMID:17508758

  1. Antifreeze glycopeptide diastereomers

    Directory of Open Access Journals (Sweden)

    Lilly Nagel

    2012-10-01

    Full Text Available Antifreeze glycopeptides (AFGPs are a special class of biological antifreeze agents, which possess the property to inhibit ice growth in the body fluids of arctic and antarctic fish and, thus, enable life under these harsh conditions. AFGPs are composed of 4–55 tripeptide units -Ala-Ala-Thr- glycosylated at the threonine side chains. Despite the structural homology among all the fish species, divergence regarding the composition of the amino acids occurs in peptides from natural sources. Although AFGPs were discovered in the early 1960s, the adsorption mechanism of these macromolecules to the surface of the ice crystals has not yet been fully elucidated. Two AFGP diastereomers containing different amino acid configurations were synthesized to study the influence of amino acid stereochemistry on conformation and antifreeze activity. For this purpose, peptides containing monosaccharide-substituted allo-L- and D-threonine building blocks were assembled by solid-phase peptide synthesis (SPPS. The retro-inverso AFGP analogue contained all amino acids in D-configuration, while the allo-L-diastereomer was composed of L-amino acids, like native AFGPs, with replacement of L-threonine by its allo-L-diastereomer. Both glycopeptides were analyzed regarding their conformational properties, by circular dichroism (CD, and their ability to inhibit ice recrystallization in microphysical experiments.

  2. Preparation of Food-based Antifreeze Peptides and Research on the Ice Crystal Inhibition%食品源抗冻多肽的制备及冰晶抑制作用研究

    Institute of Scientific and Technical Information of China (English)

    洪晶; 汪少芸; 吴金鸿; 饶平凡

    2013-01-01

    Objective:Antifreeze protein is becoming a popular research point because it could inhibit ice crystal growth, reduce damage of cell membranes and maintain products' quality during food during storage and handling. Methods:This paper reports that gelatin peptides of a certain molecular size range with compact-packed structural domain derived from Papain hydrolysis of bovine gelatin are able to inhibit recrystallization of ice crystals in ice cream mix and show natural antifreeze activity. Results:The optimum conditions for producing antifreeze peptides were hydrolysis at pH 7.0 for 30 min at 37℃ and an Papain to gelatin ratio of 1 :10. The gelatin peptides were fractionated on size exclusion (Sephadex C-SO) and ion exchange (sulfopropyl-Sephadex C-2S) columns, and the molecular mass distribution of the antifreeze peptide fractions was determined by matrixassisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The gelatin peptide fractions in the molecular mass range of 700~1 318 u strongly inhibited ice recrystallization in ice cream mix. Conclusion:The highly effective antifreeze peptide on ice crystal inhibition shows specific rules during cold-heat-stage cycles, the key approach is how to control hydrolysis conditions. It probably exists the surface hydropholic-complementary interaction between antifreeze peptide and ice molecules.%目的:因抗冻蛋白具有控制冰晶生长,减少细胞损伤及保持产品原有组织结构、质地和品质的特点和突出意义而成为研究的热点.方法:以食品源的食用明胶为原材料,通过控制木瓜蛋白酶的切割条件,将活性多肤切割为具有特定的肽链长度和结构组成,从而使抗冻活性得以高效实现.结果:酶切多肽抗冻活性的实现受酶/底物比、酶解时间、酶解温度等条件的影响.优化的酶解条件为:pH 7.0,酶/底物配比1:10;酶解时间30 min;酶解温度37℃.通过Sephadex G-50和Sephadex C-25色谱分离

  3. The Roles of Insects Body Cold-resistance Effects on Ice Nucleation Proteins and Insect Antifreeze Proteins%冰核蛋白和昆虫抗冻蛋白在虫体抗寒中的作用

    Institute of Scientific and Technical Information of China (English)

    周世豪; 袁哲明

    2012-01-01

    为了冰核蛋白和昆虫抗冻蛋白共同维持虫体在寒冬中的动态平衡.尝试着从冰核蛋白和昆虫抗冻蛋白的结构和功能出发,阐述两者在虫体中的抗寒作用,有助于日后更为深入的研究.%In order to the ice nucleation proteins (INPs) and the insects antifreeze proteins (AFPs) are two closely related to the body of insects, intervening of antagonism and synergy have maintained the dynamical equilibrium in the body of insects in winter. This paper expounds on two of the body in insect role, further cold more in-depth research.

  4. An open source cryostage and software analysis method for detection of antifreeze activity

    DEFF Research Database (Denmark)

    Lørup Buch, Johannes; Ramløv, H

    2016-01-01

    The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method....... The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing...

  5. 印楝Azadirachta Indica A.Juss的冷驯化与抗冻蛋白的研究%On Cold-Acclimation and Antifreeze Proteins of Azadirachta Indica A.Juss

    Institute of Scientific and Technical Information of China (English)

    王颖; 杨光伟

    2012-01-01

    The micropropagation system has been established through plant tissue culture technology with traditional herbal plant Azadirachta Indica A. Juss as material before cold acclimation process is carried out; in vivo antifreeze proteins of neem have been investigated and analyzed. The main results are as follows: ①After cold acclimation, total amount of neem protein increases and several new proteins produce. However, with the cold treatment for too long a time, amount of the neem cold-induced proteins decrease, and even some of them are degradated. ②The stability of antifreeze proteinfrom neem is related to the time of cold treatment in cold acclimation. The shortest time for antifreeze protein production is two weeks in 5℃ , and the cold resistant limit of neem is in 5℃ for 20 days. When the temperature drops below 0℃ , AFPs are accumulated in the first days of cold treatment(0~ 15 d). However, while the treatment time i.s prolonged, Antifreeze protein are degradated and dismissed on the 30th day. ③ Purified antifreeze protein is obtained, and the relative molecular mass is around 36 KD.%采用木本植物材料——印楝,通过组织培养建立快繁体系,然后对其进行冷驯化处理,并分析检测印楝植物体内抗冻蛋白.主要结果如下:①冷驯化处理后印楝的总蛋白一些表现为量的增加同时会有新的蛋白产生.但脱驯化或处理时间过长时,抗冻蛋白在量的表达上会有逐渐减少或消失的现象.②在对印楝的冷驯化中,发现不同的温度处理后蛋白稳定存在的时间不同.抗冻蛋白出现的最早时期为5℃处理2周左右,印楝能耐受的稳定最低温为5℃,所持续的最长时间约为20 d.在0℃低温处理后,虽然在处理初期(0~15 d)也有抗冻蛋白的产生,但随处理时间的延长,这种差异逐渐减少,在处理30 d时完全消失.③得到了分离纯化的抗冻蛋白,其相对分子质量约为3.6×104.

  6. Inhibition of Gas Hydrate Nucleation and Growth: Efficacy of an Antifreeze Protein from the Longhorn BeetleRhagium mordax

    DEFF Research Database (Denmark)

    Perfeldt, Christine Malmos; Chua, Pei Cheng; Daraboina, Nagu;

    2014-01-01

    showed inhibition of hydrates compared to amino acids (l-valine and l-threonine) and the protein bovine serum albumin (BSA). This indicates that proteins or amino acids do not generally inhibit hydrate formation. The promising performance of RmAFP1 as a new green kinetic hydrate inhibitor could further...

  7. 白菜型冬油菜质外体抗冻蛋白研究%Study on apoplast anti-freeze proteins in winter turnip rape (Brassica rape L.)

    Institute of Scientific and Technical Information of China (English)

    杨刚; 刘林波; 杨建胜; 方园; 张娟; 史鹏辉; 孙万仓; 刘自刚; 曾秀存; 武军艳; 方彦; 李学才; 陈奇

    2016-01-01

    The objective of this paper was to lay the basis for studying cold resistance of winter rapeseed. The anti-freeze activities of apoplast proteins were determined in the ‘Longyou 6’ winter rape leaves and roots under cold vernalization. The apoplast proteins were separated by SDS-PAGE and high expression proteins identified in MALDI-TOF/TOF mass spectrometry under field and pot experiments. The results showed that apoplast protein content of ‘Longyou 6’ leaves increased significantly (P < 0.05) after cold acclimation in an artificial climate chamber, reaching 92.31 µg•g-1(FW) on the fifth day, which represented an increase of 246.12% over CK. Apoplast protein content after 10-15 days of cold acclimation dropped compared with that after 5 days, but was still significantly higher than that of CK (P < 0.05). Apoplast protein content continued to increase with increasing cold acclimation time from 20 to 25 days (P < 0.05). Apoplast protein content decreased significantly with after 10 days of de-acclimation. In the process of cold acclimation, apoplast protein content of ‘Longyou 6’ leaves significantly accumulated. However, it decreased significantly after de-acclimation. Obviously, apoplast proteins of‘Longyou 6’ winter rape belonged to low temperature induced proteins. Anti-freeze activity detection analysis suggested that apoplast proteins had re-crystallization inhibition activity. Mass spectrometry identification revealed a variety of proteins with unclear functions along with β-1-3-glucanase consistent anti-freeze proteins reported in winter rye. The class glucanase detected by mass spectrometry was found to have weaker ice crystal forms due to modification effect with reclamation and anti-freeze activity test. The test suggested that this class glucanase was a low activity anti-freeze protein. Many anti-freeze proteins were synthesized and secreted by winter rape in apoplast of leaves and roots under low temperature stress. The proteins

  8. 抗冻蛋白应用前景及基因工程表达研究进展%Researches Advances in Application of Antifreeze Protein and Its Gene Engineering Expression

    Institute of Scientific and Technical Information of China (English)

    蔡文萍; 马纪

    2012-01-01

    抗冻蛋白(antifreeze protein,AFP)又称为热滞蛋白,是一类抑制冰晶生长的蛋白,它具有三个基本特征:热滞效应(thermal hysteresis activity,THA)、冰晶形态效应和重结晶抑制效应(frecrystallization inhibition,RI),抗冻蛋白是一类广泛存在于鱼类、植物、真菌、昆虫中的蛋白,不同生物的AFPs的化学结构、理化性质、空间构型各不相同,并且同类生物之间的AFPs同源性也不高,不存在相似性序列或结构模式,说明它们可能是在不同的有机体中独立进化而来,没有共同的演化规律.随着研究的深入,抗冻蛋白可广泛应用于医学农学食品工业等领域,起到冷冻保护剂,食品添加剂的作用,前人已经对抗冻蛋白的结构、生化性质和抗冻机制进行了阐述,研究主要对抗冻蛋白应用方面及基因工程的表达作了系统综述,为抗冻蛋白的深入研究奠定基础.%Antifreeze protein (AFP) , also known as thermal hysteresis protein, It is a class of proteinThat inhibiting the growth of ice crystals. It has three basic characteristics; thermal hysteresis effect (THA), the effect of ice crystals form, recrystallization inhibitory effect (RI), To date, AFPs have been widely found in a variety of organisms, such as fish, insects, plants, bacteria and fungi, isolated from a number of fish, plants , bacteria, fungi and arthropods . Different biological AFPs chemical structure, physical and chemical properties, spatial configuration of each are not identical, and similar biological between AFPs homology is not high, there is no similar sequence or structural patterns, indicating that they may be in different organisms evolved independently, there is no common evolution law. Along with the development of research, Antifreeze protein can be widely used in agriculture, medicine food sciences and industry, It played a role in the cryoprotectant and food additives. The biochemical characteristics, as well as antifreeze

  9. Antifreeze life cycle assessment (LCA

    Directory of Open Access Journals (Sweden)

    Kesić Jelena

    2005-01-01

    Full Text Available Antifreeze based on ethylene glycol is a commonly used commercial product The classification of ethylene glycol as a toxic material increased the disposal costs for used antifreeze and life cycle assessment became a necessity. Life Cycle Assessment (LCA considers the identification and quantification of raw materials and energy inputs and waste outputs during the whole life cycle of the analyzed product. The objectives of LCA are the evaluation of impacts on the environment and improvements of processes in order to reduce and/or eliminate waste. LCA is conducted through a mathematical model derived from mass and energy balances of all the processes included in the life cycle. In all energy processes the part of energy that can be transformed into some other kind of energy is called exergy. The concept of exergy considers the quality of different types of energy and the quality of different materials. It is also a connection between energy and mass transformations. The whole life cycle can be described by the value of the total loss of exergy. The physical meaning of this value is the loss of material and energy that can be used. The results of LCA are very useful for the analyzed products and processes and for the determined conditions under which the analysis was conducted. The results of this study indicate that recycling is the most satisfactory solution for the treatment of used antifreeze regarding material and energy consumption but the re-use of antifreeze should not be neglected as a solution.

  10. Antifreeze and cryoprotective activities of ice-binding collagen peptides from pig skin.

    Science.gov (United States)

    Cao, Hui; Zhao, Ying; Zhu, Yu Bing; Xu, Fei; Yu, Jing Song; Yuan, Min

    2016-03-01

    A novel "hyperactive" ice-binding peptide from porcine collagen was prepared by alkaline protease hydrolysis and a series of column chromatography separations, and then its antifreeze and cryoprotective properties were reported. Using differential scanning calorimetry (DSC), the thermal hysteresis (TH) of ice-binding collagen peptides was closely related to their concentration and crystal fraction. Collagen hydrolysates with maximal TH were obtained by hydrolysis at pH 8.0, DH 15.0%, and 5% alkaline protease at 55°C. After purification by column chromatography, the AP-3 ice-binding collagen peptide (GLLGPLGPRGLL) with 1162.8Da molecular weights exhibited the highest TH (5.28°C), which can be classified as "hyperactive". Recrystallisation and melt-resistance of ice cream were improved by AP-3 ice-binding collagen peptide at 0.2% (w/v) in a similar manner to natural antifreeze proteins. Moreover, the addition of AP-3 collagen peptides in ice cream greatly elevated the glass transition temperature (Tg) to -17.64°C. PMID:26471678

  11. Structure and collective dynamics of hydrated anti-freeze protein type III from 180 K to 298 K by X-ray diffraction and inelastic X-ray scattering

    Science.gov (United States)

    Yoshida, Koji; Baron, Alfred Q. R.; Uchiyama, Hiroshi; Tsutsui, Satoshi; Yamaguchi, Toshio

    2016-04-01

    We investigated hydrated antifreeze protein type III (AFP III) powder with a hydration level h (=mass of water/mass of protein) of 0.4 in the temperature range between 180 K and 298 K using X-ray diffraction and inelastic X-ray scattering (IXS). The X-ray diffraction data showed smooth, largely monotonic changes between 180 K and 298 K without freezing water. Meanwhile, the collective dynamics observed by IXS showed a strong change in the sound velocity at 180 K, after being largely temperature independent at higher temperatures (298-220 K). We interpret this change in terms of the dynamic transition previously discussed using other probes including THz IR absorption spectroscopy and incoherent elastic and quasi-elastic neutron scattering. This finding suggests that the dynamic transition of hydrated proteins is observable on the subpicosecond time scale as well as nano- and pico-second scales, both in collective dynamics from IXS and single particle dynamics from neutron scattering. Moreover, it is most likely that the dynamic transition of hydrated AFP III is not directly correlated with its hydration structure.

  12. 脊尾白虾虾糜的制备及其抗冷冻变性工艺%Technology of shrimp surimi preparation from Exopalaemon carinicauda and its anti-freeze denaturation

    Institute of Scientific and Technical Information of China (English)

    曹文红; 赵子科; 田申; 陈良

    2015-01-01

    This paper studied the rinsing technology and anti-freeze denaturation technology of shrimp surimi made from Exopalaemon carinicauda.The effect of different rinsing conditions on gel strength and elasticity of the minced shrimp were determined and estimated.With the weighted value of the gel strength and elasticity as the indexes,the rinsing process technology was optimized with an orthogonal trial.The optimal conditions were:rinsing time 7 min,shrimp meat:water ratio 1∶9,CaCl2 concentration 0.7%.In order to understand the influence of frozen storage on the quality of the rinsed shrimp meat,trehalose,sodium lactate,sorbitol and sucrose were used as cryoprotectants,salt-soluble protein content,Ca2+-ATPase activity,total sulfhydryl content,gel strength and pH of the rinsed shrimp meat were determined and compared during a storage of 8 weeks.The results indicated that all cryoprotectants showed good anti-freeze denaturation activity on rinsed shrimp meat as compared to the control samples.The trehalose added samples had better anti-frozen effect than other cryoprotectants added samples.The results of this research show that shrimp surimi of Exopalaemon carinicauda can remain better qualities after a relatively long time storage.%以脊尾白虾为原料,研究虾糜漂洗工艺及其抗冷冻变性工艺.以凝胶强度和弹性为指标,探索不同漂洗条件对虾糜凝胶性能的影响,以凝胶强度和弹性的加权平均值为指标进行正交试验得出虾糜最佳的漂洗工艺为:漂洗时间7 min,料水比1∶9(g∶ mL),漂洗液CaCl2浓度0.7%.为研究冻藏对虾糜品质的影响,以海藻糖、乳酸钠、山梨糖醇、蔗糖等为抗冻剂,比较了添加不同抗冻剂时8周冻藏期间内虾糜的盐溶蛋白含量、Ca2-AT-Pase、总巯基含量、凝胶强度、pH值的变化规律.结果显示:不同抗冻剂均能较好地抑制脊尾白虾虾糜蛋白质的冷冻变性,海藻糖的抗冻效果优于另外两种抗冻剂.研究表

  13. Cloning and sequencing of antifreeze protein gene inDaucus carota var \\%sativus\\% Hoffm Deutschl%胡萝卜var sativus Hoffm Deutschl抗冻蛋白基因的克隆及测序

    Institute of Scientific and Technical Information of China (English)

    尹明安; 崔鸿文; 樊代明; 郭立

    2001-01-01

    Antifreeze protein gene (afp) in three native carrot cultivars(Daucus carota var \\%sativus\\% Hoffm Deutschl),Wuzhong carrot in Ningxia,H uaxian carrot in Shaanxi and Hanzhong carrot in Shaanxi,was cloned by PCR (polym erase chain reaction).Wuzhong carrots afp was sequenced and its sequence w as compared with that of Daucus carota var \\%autumn\\% King from British.Ther e were 35 different bases between two varieties in 1004 sequenced nucleotides,among which there were 20 nonsense mutations and 15 sense mutations.Based on sense mutations homology was 98.5%.%以宁夏吴忠胡萝卜、陕西华县胡萝卜、陕西汉中胡萝卜3个地方品种为材料,用PCR方法克隆了中国胡萝卜var\\%sativus\\%HoffmDeutschl的抗冻蛋白基因\\%afp\\%,测定了宁夏吴忠胡萝卜\\%afp\\%的核苷酸序列,和英国胡萝卜var\\%autumn\\%King\\%afp\\%序列对比,在所测1004个核苷酸中,有35个碱基不同,其中无义突变20个,有义突变15个。按有义突变计,同源性为\\{98.5%\\}

  14. Synthesis of Cyclic Antifreeze Glycopeptide and Glycopeptoids and Their Ice Recrystallization Inhibition Activity

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Mija; Murugan, Ravichandran N.; Bang, Jeong Kyu; Kim, Hak Jun [Korea Basic Science Institute, Daejeon (Korea, Republic of); Shin, Song Yub [Chousn Univ., Gwangju (Korea, Republic of); Kim, Eunjung; Lee, Jun Hyuck [Korea Polar Research Institute, Incheon (Korea, Republic of)

    2012-11-15

    Until now, few groups reported the antifreeze activity of cyclic glycopeptides; however, the tedious synthetic procedure is not amenable to study the intensive structure activity relationship. A series of N-linked cyclic glycopeptoids and glycopeptide have been prepared to evaluate antifreeze activity as a function of peptide backbone cyclization and methyl stereochemical effect on the rigid Thr position. This study has combined the cyclization protocol with solid phase peptide synthesis and obtained significant quantities of homogeneous cyclic glycopeptide and glycopeptoids. Analysis of antifreeze activity revealed that our cyclic peptide demonstrated RI activity while cyclic glycopeptoids showed no RI activity. These results suggest that the subtle changes in conformation and Thr orientation dramatically influence RI activity of N-linked glycopeptoids.

  15. Preparing and evaluating delivery systems for proteins

    DEFF Research Database (Denmark)

    Jorgensen, L; Moeller, E H; van de Weert, M;

    2006-01-01

    From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping...... and long-term storage of the formulation. Therefore, the development and evaluation of successful and promising drug delivery systems is essential. In the present review, some of the particulate drug delivery systems for parenteral delivery of protein are presented and discussed. The challenge...... for incorporation of protein in particulate delivery systems is exemplified by water-in-oil emulsions....

  16. Preparation of Quenchbodies by protein transamination reaction.

    Science.gov (United States)

    Dong, Jinhua; Jeong, Hee-Jin; Ueda, Hiroshi

    2016-07-01

    Quenchbody (Q-body) is an antibody fragment labeled with fluorescent dye(s), which functions as a biosensor via the antigen-dependent removal of the quenching effect on fluorophores. It is based on the principle that the fluorescence of the dye(s) attached to the antibody N-terminal region is quenched primarily by the tryptophan residues present in the variable regions, and this quenching is released when the antigen binds to the antibody, resulting in increased fluorescence intensity. Hence Q-body is utilized in various immunoassays for the rapid and sensitive detection of analytes. So far, Q-bodies have been prepared by using a cell-free translation system or by combining Escherichia coli expression and post-labeling steps. However, the above methods need antibody gene cloning, and are time-consuming. In this study, we report a novel approach to prepare Q-bodies by protein N-terminal transamination. We used the antigen-binding fragment (Fab) of an antibody against the bone-Gla-protein (BGP), a biomarker for bone diseases, which was expressed in E. coli. The purified Fab was treated with Rapoport's salt to convert the amino group at the N-terminus to a ketone group, which in turn was allowed to react with fluorescent probes that have aminooxy or hydrazide groups, to prepare a Q-body. The Q-body prepared by this method could detect the BGP-C7 antigen at concentrations as low as 10 nM. Since the approach can label the protein N-terminus directly, it could be applied for preparing Q-bodies from natural antibodies and for the rapid screening of high-performance Q-bodies. PMID:26811222

  17. Patrones electroforéticos de proteínas y actividad anticongelante en el apoplasto de la hoja de la especie andina tropical Senecio niveoaureus PROTEIN ELECTROPHORETIC PATTERNS AND ANTIFREEZING ACTIVITY IN THE LEAF APOPLAST OF THE TROPICAL ANDEAN SPECIES Senecio niveoaureus

    Directory of Open Access Journals (Sweden)

    F ÁLVAREZFLÓREZ

    2006-06-01

    Full Text Available Las plantas de alta montaña tienen diferentes adaptaciones para sobrevivir a cambios drásticos de temperatura, especialmente a condiciones de congelamiento. En plantas de invierno, la supervivencia a temperaturas bajas está relacionada con la capacidad de las células para producir proteínas específicas de bajo peso molecular (proteínas anticongelantes y exportarlas al apoplasto. Para establecer si plantas tropicales de alta montaña sobreviven las temperaturas bajas a través del mismo mecanismo, se colectaron hojas de plantas de Senecio niveoaureus durante 24 horas y a dos alturas 3.300 y 3.600 msnm en el Páramo de Palacio, Chingaza, Colombia. Se observaron proteínas apoplásticas de pesos moleculares entre 3512 kDa. Los patrones electroforéticos fueron diferentes dependiendo de la altura y la hora de muestreo, sin embargo, se observaron variaciones en el patrón de bandeo que no pueden ser atribuidas ni a la temperatura ni al gradiente altitudinal únicamente. Se detectó actividad anticongelante en el apoplasto de hojas de S. niveoaureus, siendo este el primer reporte en especies tropicales de alta montaña.Tropical high mountain plants have different adaptations to survive extreme daily temperature fluctuations and specially freezing night conditions. In winter plant species, survival to low temperatures is related to the ability of the cell to produce specific low molecular weight proteins (antifreezing proteins and to export them to the apoplast. In order to see if high mountain tropical plants survive to low temperatures through the same mechanism we collected, during a 24 hourperiod, leaves from Senecio niveoaureus growing at 3,300 and 3,600 m.o.s.l, in the Páramo de Palacio, Chingaza, Colombia. Leaf apoplast proteins had MW between 3512 kDa. Electrophoretic patterns were different depending on the altitude and the time of sampling. However the observed variations could not be linked to changes in temperature or to the

  18. Preparation of protein samples for gel electrophoresis by sequential extraction

    Institute of Scientific and Technical Information of China (English)

    钟伯雄; 翁宏飚; 等

    2002-01-01

    Since preparation and solubilization of protein samples are crucial factors in proteome research,the authors established a sequential extraction technique to prepare protein samples from the body wall of the 5th instar larvae of silkworm.Bombyx mori.Two kinds of protein samples were obtained from the body wall using the method.Between the two types of samples only about 15% proteins were identical;the majority were different,indicating that more species of proteins could be obtained with the sequential extraction method;which will be useful for preparation of protein samples for proteome study.

  19. Bacterial Ice Crystal Controlling Proteins

    OpenAIRE

    Lorv, Janet S. H.; Rose, David R; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. R...

  20. Preparation of Functional Soybean Protein Isolate

    Institute of Scientific and Technical Information of China (English)

    Liu Zhitong; Li Xiaolian; Zhao Guangming

    2000-01-01

    Soybean protein isolate(SPI)is a high purity soybean protein product. Its protein content is over 90% .A popular processing method is alkali dissolution and acid precipitation. This method can produce various functional SPIs by changing the temperature, pH, types of alkali and acid, and by different pretreatment and post transformation treatment. The properties addressed in this paper would open a big market for the appli cation of SPI.

  1. Preparation of Functional Soybean Protein Isolate

    Institute of Scientific and Technical Information of China (English)

    Liuzhitong; LiXiaolian; 等

    2000-01-01

    Soybean protein isolate(SPI) is a high purity soybean protein product.Its protein content is over 90%.A popular processing method is alkali dissolution and acid precipitation.This method can produce various functional SPIs by changing the temperature,pH,types of alkali and acid,and by different pretreatment and post transformation treatment.The properties addressed in this paper would open a big market for the appli cation of SPI.

  2. Prediction of Antifreeze Critical Strength of Infant Age Concrete

    Institute of Scientific and Technical Information of China (English)

    LIU Jun; LIU Runqing

    2008-01-01

    The rule of infant age concrete strength development under low temperature and complex affecting factors is researched. An efficient and reliable mathematical forecast model is set up to predict the infant age concrete antifreeze critical strength under low temperature at construction site. On the basis of the revision of concrete equivalent coefficient under complex influencing factors, least-squares curve-fitting method is applied to approximate the concrete strength under standard curing and the forecast formula of concrete compressive strength could be obtained under natural temperature condition by various effects. When the amounts of donble-doped are 10% fly ashes and 4% silica fumes as cement replacement, the antifreeze critical strength changes form 3.5-4.1MPa under different low temperature curing. The equivalent coefficient correction formula of concrete under low temperature affected by various factors could be obtained. The obtainede quivalent coefficient is suitable for calculating the strength which is between 10% to 40% of standard strength and the curing temperature from 5-20 ℃. The forecast value of concrete antifreeze critical strength under low temperature could be achieved by combining the concrete antifreeze critical strength value with the compressive strength forecast of infant age concrete under low temperature. Then the theory for construction quality control under low temperature is provided.

  3. RAPID TEST METHOD FOR EVALUATION OF ANTIFREEZE ADDITIVE EFFICIENCY

    Directory of Open Access Journals (Sweden)

    S. V. Gushchin

    2015-01-01

    Full Text Available Usage of chemical additives while executing concrete works at negative temperatures is considered as a convenient and economical method. Range of the used antifreeze additives is rather wide. A great number of new additives are advertised but their characteristics have not been practically studied. Evaluation of the antifreeze additive efficiency is unfortunately rather long process and it does not provide comprehensive data on concrete structure formation processes. Due to this development of rapid and comprehensive methodology for construction companies is urgently required.Freezing processes of antifreeze additive aqueous solutions and hardening of cement paste with them have been investigated in the paper. The paper proposes a methodology for determination of freezing point for aqueous solutions of chemical additives of various applications. Identity of  freezing point for a chemical additive aqueous solution and cement paste with an equal concentration of the additive in the paste pore fluid has been determined while taking  calcium nitrate and sodium formate additives as an example. The paper demonstrates the possibility to evaluate efficiency of antifreeze additive action on the basis of kinetics in temperature changes of the cement paste with additives by its consecutive freezing and defrosting.  A methodology for operational evaluation in the field of chemical additive application for concreting items at negative temperatures has been offered in the paper.  The methodology does not require  deficient and expensive test-equipment. It can be applied at ordinary construction companies and it is comprehensible for personnel of low-qualification.  The paper shows the possibility to develop an original methodology for designing concrete structure which is based on operating efficiency determinations  for single and integrated antifreeze additives.

  4. Preparation of Modified Films with Protein from Grouper Fish.

    Science.gov (United States)

    Valdivia-López, M A; Tecante, A; Granados-Navarrete, S; Martínez-García, C

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials. PMID:27597950

  5. Preparation of Modified Films with Protein from Grouper Fish

    Science.gov (United States)

    Tecante, A.; Granados-Navarrete, S.; Martínez-García, C.

    2016-01-01

    A protein concentrate (PC) was obtained from Grouper fish skin and it was used to prepare films with different amounts of sorbitol and glycerol as plasticizers. The best performing films regarding resistance were then modified with various concentrations of CaCl2, CaSO4 (calcium salts), and glucono-δ-lactone (GDL) with the purpose of improving their mechanical and barrier properties. These films were characterized by determining their mechanical properties and permeability to water vapor and oxygen. Formulations with 5% (w/v) protein and 75% sorbitol and 4% (w/v) protein with a mixture of 15% glycerol and 15% sorbitol produced adequate films. Calcium salts and GDL increased the tensile fracture stress but reduced the fracture strain and decreased water vapor permeability compared with control films. The films prepared represent an attractive alternative for being used as food packaging materials. PMID:27597950

  6. An ignored variable: solution preparation temperature in protein crystallization.

    Science.gov (United States)

    Chen, Rui-Qing; Lu, Qin-Qin; Cheng, Qing-Di; Ao, Liang-Bo; Zhang, Chen-Yan; Hou, Hai; Liu, Yong-Ming; Li, Da-Wei; Yin, Da-Chuan

    2015-01-01

    Protein crystallization is affected by many parameters, among which certain parameters have not been well controlled. The temperature at which the protein and precipitant solutions are mixed (i.e., the ambient temperature during mixing) is such a parameter that is typically not well controlled and is often ignored. In this paper, we show that this temperature can influence protein crystallization. The experimental results showed that both higher and lower mixing temperatures can enhance the success of crystallization, which follows a parabolic curve with an increasing ambient temperature. This work illustrates that the crystallization solution preparation temperature is also an important parameter for protein crystallization. Uncontrolled or poorly controlled room temperature may yield poor reproducibility in protein crystallization.

  7. An automatic refolding apparatus for preparative-scale protein production.

    Directory of Open Access Journals (Sweden)

    Yanye Feng

    flexible strategy may provide a powerful tool for preparative scale protein production.

  8. Effect of Anti-freezing Admixtures on Alkali-silica Reaction in Mortars

    Institute of Scientific and Technical Information of China (English)

    LIU Junzhe; LI Yushun; LV Lihua

    2005-01-01

    The influence of anti-freezing admixture on the alkali aggregate reaction in mortar was analyzed with accelerated methods. It is confirmed that the addition of sodium salt ingredients of anti-freezing admixture accelerates the alkali silica reaction to some extent, whereas calcium salt ingredient of anti-freezing admixture reduces the expansion of alkali silica reaction caused by high alkali cement. It is found that the addition of the fly ash considerably suppresses the expansion of alkali silica reaction induced by the anti-freezing admixtures.

  9. Bacterial ice crystal controlling proteins.

    Science.gov (United States)

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  10. AccaDueO - Solar heating system without antifreeze; AccaDueO - Solaranlage ohne Frostschutzmittel - Schlussbericht

    Energy Technology Data Exchange (ETDEWEB)

    Engeler, L.; Salerno, B.

    2003-12-15

    This illustrated final report for the Swiss Federal Office of Energy (SFOE) describes a solar collector system that uses a heat transfer fluid without antifreeze additives. The so-called 'drain-back' system supplies heat for heating and hot water preparation in a three-family house in Waldenburg, Switzerland, together with a wood-fired boiler. The results of measurements made on the collectors and the storage tank are presented in tabular and graphical form and discussed. The opinions of experts, inhabitants and the general public are noted.

  11. Lipid oxidation in omega-3 emulsions prepared with milk proteins

    DEFF Research Database (Denmark)

    Horn, Anna Frisenfeldt; Nielsen, Nina Skall; Andersen, Ulf;

    An increasing body of evidence supports the health beneficial effects of omega-3 polyunsaturated fatty acids. Therefore, incorporation of marine oils into foods has also gained an increasing interest. However, the highly unsaturated lipids present in marine oils are prone to lipid oxidation....... The properties of the emulsifier used and the structure at the interface are therefore expected to be of great importance for oxidation in emulsions. This presentation will include results from mainly three different studies of lipid oxidation in omega-3 emulsions prepared with milk proteins and protein...

  12. Preparation and characterization of polyclonal antibodies against ARL-1 protein

    Institute of Scientific and Technical Information of China (English)

    Jun-Fei Jin; Liu-Di Yuan; Li Liu; Zhu-Jiang Zhao; Wei Xie

    2003-01-01

    AIM: To prepare and characterize polyclonal antibodies against aldose reductase-like (ARL-1) protein.METHODS: ARL-L gene was inserted into the E.coli expression vector pGEX-4T-1(His)6C and vector pQE-30. Recombinant ARL1 proteins named ARL-(His)6 and ARL-GST were expressed.They were purified by affinity chromatography. Sera from domestic rabbits immunized with ARL-(His)6 were purified by CNBr-activated sepharose 4B coupled ARL-GST. Polyclonal antibodies were detected by Western blotting.RESULTS: Recombinant proteins of ARL-(His)6 with molecular weight of 35.7 KD and ARL-GST with molecular weight of 60.8 KD were highly expressed. The expression levels of ARL-GST and ARL-(His)6 were 15.1% and 27.7 %among total bacteria proteins, respectively. They were soluble, predominantly in supernatant. After purification by non-denatured way, SDS-PAGE showed one band. In the course of polyclonal antibodies purification, only one elution peak could be seen. Western blotting showed positive signals in the two purified proteins and the bacteria transformed with pGEX-4T-1(His)6 C-ARL and pQE-30-ARL individually.CONCLUSION: Polyclonal antibodies are purified and highly specific against ARL-1 protein. ARL-GST and ARL-(His)6 are highly expressed and purified.

  13. Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection.

    Science.gov (United States)

    Abeyrathne, Priyanka D; Chami, Mohamed; Pantelic, Radosav S; Goldie, Kenneth N; Stahlberg, Henning

    2010-01-01

    Electron crystallography is a powerful technique for the structure determination of membrane proteins as well as soluble proteins. Sample preparation for 2D membrane protein crystals is a crucial step, as proteins have to be prepared for electron microscopy at close to native conditions. In this review, we discuss the factors of sample preparation that are key to elucidating the atomic structure of membrane proteins using electron crystallography.

  14. Antifreeze Activity of Xylomannan from the Mycelium and Fruit Body of Flammulina velutipes.

    Science.gov (United States)

    Kawahara, Hidehisa; Matsuda, Yoshiyuki; Sakaguchi, Takuya; Arai, Naoki; Koide, Yoshihide

    2016-01-01

    An identified class of antifreeze, a xylomannan-based thermal hysteresis (TH)-producing glycolipid, has been discovered from diverse taxa, including plants, insects, and amphibians. We isolated xylomannan from the mycelium and fruit body of the basidiomycete Flammulina velutipes using successive hot extraction with water, 2% and 25% aqueous KOH, and gel filtration chromatography. The xylomannan from the fruit body had a recrystallization inhibiting (RI) activity (RI=0.44) at 0.5 mg/mL. The dried weight yield of the fruit body (7.7×10(-2)%, w/w) was higher than that of the mycelium. Although the purified xylomannan from both soures were composed of mannose and xylose in a 2 : 1 molar ratio, the molecular weight of the xylomannan from the mycelium and fruit body was 320,000 and 240,000, respectively. The RI activity of mycelial xylomannan was higher than that from the fruit body (RI=0.57) at 45 µg/mL. Although this RI activity was able to remain constant after exposure to various conditions, we confirmed that the decrease of RI activity was stimulated by the decrease of molecular weight that was caused by heating during the alkaline condition. The survival rate of the CHO cells at -20℃ for two days increased to 97% due to the addition of 20 µg/mL of purified xylomannan. This was the first report to indicate that xylomannan from the mycelium of Flammulina velutipes had a high level of ice recrystallization inhibiting activity like antifreeze proteins from plants and had rhe potential to become a new material for cell storage. PMID:27667520

  15. Production of protein hydrolysates from fish byproduct prepared by enzymatic hydrolysis

    OpenAIRE

    Murna Muzaifa; Novi Safriani; Fahrizal Zakaria

    2012-01-01

    The objective of this research was to study the production of fish protein hydrolysate (FPH) from fish by-product prepared by enzymatichydrolysis. Fish by-product were prepared using Alcalase and Flavourzyme enzyme and properties of FPH were analyzed. The resultsshowed that FPH prepared using Alcalase enzyme had greater amount of protein (82.66%) than FPH prepared using Flavourzyme enzyme(73.51%). Solubility, emulsifying and foaming properties of FPH prepared using Alcalase were also better t...

  16. SynProt: A Database for Proteins of Detergent-Resistant Synaptic Protein Preparations

    Science.gov (United States)

    Pielot, Rainer; Smalla, Karl-Heinz; Müller, Anke; Landgraf, Peter; Lehmann, Anne-Christin; Eisenschmidt, Elke; Haus, Utz-Uwe; Weismantel, Robert; Gundelfinger, Eckart D.; Dieterich, Daniela C.

    2012-01-01

    Chemical synapses are highly specialized cell–cell contacts for communication between neurons in the CNS characterized by complex and dynamic protein networks at both synaptic membranes. The cytomatrix at the active zone (CAZ) organizes the apparatus for the regulated release of transmitters from the presynapse. At the postsynaptic side, the postsynaptic density constitutes the machinery for detection, integration, and transduction of the transmitter signal. Both pre- and postsynaptic protein networks represent the molecular substrates for synaptic plasticity. Their function can be altered both by regulating their composition and by post-translational modification of their components. For a comprehensive understanding of synaptic networks the entire ensemble of synaptic proteins has to be considered. To support this, we established a comprehensive database for synaptic junction proteins (SynProt database) primarily based on proteomics data obtained from biochemical preparations of detergent-resistant synaptic junctions. The database currently contains 2,788 non-redundant entries of rat, mouse, and some human proteins, which mainly have been manually extracted from 12 proteomic studies and annotated for synaptic subcellular localization. Each dataset is completed with manually added information including protein classifiers as well as automatically retrieved and updated information from public databases (UniProt and PubMed). We intend that the database will be used to support modeling of synaptic protein networks and rational experimental design. PMID:22737123

  17. Antifreeze Peptides and Glycopeptides, and Their Derivatives: Potential Uses in Biotechnology

    Directory of Open Access Journals (Sweden)

    Hyun-Cheol Kim

    2013-06-01

    Full Text Available Antifreeze proteins (AFPs and glycoproteins (AFGPs, collectively called AF(GPs, constitute a diverse class of proteins found in various Arctic and Antarctic fish, as well as in amphibians, plants, and insects. These compounds possess the ability to inhibit the formation of ice and are therefore essential to the survival of many marine teleost fishes that routinely encounter sub-zero temperatures. Owing to this property, AF(GPs have potential applications in many areas such as storage of cells or tissues at low temperature, ice slurries for refrigeration systems, and food storage. In contrast to AFGPs, which are composed of repeated tripeptide units (Ala-Ala-Thrn with minor sequence variations, AFPs possess very different primary, secondary, and tertiary structures. The isolation and purification of AFGPs is laborious, costly, and often results in mixtures, making characterization difficult. Recent structural investigations into the mechanism by which linear and cyclic AFGPs inhibit ice crystallization have led to significant progress toward the synthesis and assessment of several synthetic mimics of AFGPs. This review article will summarize synthetic AFGP mimics as well as current challenges in designing compounds capable of mimicking AFGPs. It will also cover our recent efforts in exploring whether peptoid mimics can serve as structural and functional mimics of native AFGPs.

  18. Inhibition of Condensation Frosting by Arrays of Hygroscopic Antifreeze Drops.

    Science.gov (United States)

    Sun, Xiaoda; Damle, Viraj G; Uppal, Aastha; Linder, Rubin; Chandrashekar, Sriram; Mohan, Ajay R; Rykaczewski, Konrad

    2015-12-29

    The formation of frost and ice can have negative impacts on travel and a variety of industrial processes and is typically addressed by dispensing antifreeze substances such as salts and glycols. Despite the popularity of this anti-icing approach, some of the intricate underlying physical mechanisms are just being unraveled. For example, recent studies have shown that in addition to suppressing ice formation within its own volume, an individual salt saturated water microdroplet forms a region of inhibited condensation and condensation frosting (RIC) in its surrounding area. This occurs because salt saturated water, like most antifreeze substances, is hygroscopic and has water vapor pressure at its surface lower than water saturation pressure at the substrate. Here, we demonstrate that for macroscopic drops of propylene glycol and salt saturated water, the absolute RIC size can remain essentially unchanged for several hours. Utilizing this observation, we demonstrate that frost formation can be completely inhibited in-between microscopic and macroscopic arrays of propylene glycol and salt saturated water drops with spacing (S) smaller than twice the radius of the RIC (δ). Furthermore, by characterizing condensation frosting dynamics around various hygroscopic drop arrays, we demonstrate that they can delay complete frosting over of the samples 1.6 to 10 times longer than films of the liquids with equivalent volume. The significant delay in onset of ice nucleation achieved by dispensing propylene glycol in drops rather than in films is likely due to uniform dilution of the drops driven by thermocapillary flow. This transport mode is absent in the films, leading to faster dilution, and with that facilitated homogeneous nucleation, near the liquid-air interface. PMID:26651017

  19. Solution conformation of C-linked antifreeze glycoprotein analogues and modulation of ice recrystallization.

    Science.gov (United States)

    Tam, Roger Y; Rowley, Christopher N; Petrov, Ivan; Zhang, Tianyi; Afagh, Nicholas A; Woo, Tom K; Ben, Robert N

    2009-11-01

    Antifreeze glycoproteins (AFGPs) are a unique class of proteins that are found in many organisms inhabiting subzero environments and ensure their survival by preventing ice growth in vivo. During the last several years, our laboratory has synthesized functional C-linked AFGP analogues (3 and 5) that possess custom-tailored antifreeze activity suitable for medical, commercial, and industrial applications. These compounds are potent inhibitors of ice recrystallization and do not exhibit thermal hysteresis. The current study explores how changes in the length of the amide-containing side chain between the carbohydrate moiety and the polypeptide backbone in 5 influences ice recrystallization inhibition (IRI) activity. Analogue 5 (n = 3, where n is the number of carbons in the side chain) was a potent inhibitor of ice recrystallization, while 4, 6, and 7 (n = 4, 2, and 1, respectively) exhibited no IRI activity. The solution conformation of the polypeptide backbone in C-linked AFGP analogues 4-7 was examined using circular dichroism (CD) spectroscopy. The results suggested that all of the analogues exhibit a random coil conformation in solution and that the dramatic increase in IRI activity observed with 5 is not due to a change in long-range solution conformation. Variable-temperature (1)H NMR studies on truncated analogues 26-28 failed to elucidate the presence of persistent intramolecular bonds between the amide in the side chain and the peptide backbone. Molecular dynamics simulations performed on these analogues also failed to show persistent intramolecular hydrogen bonds. However, the simulations did indicate that the side chain of IRI-active analogue 26 (n = 3) adopts a unique short-range solution conformation in which it is folded back onto the peptide backbone, orienting the more hydrophilic face of the carbohydrate moiety away from the bulk solvent. In contrast, the solution conformation of IRI-inactive analogues 25, 27, and 28 had fully extended side chains

  20. A novel preparation of milk protein/polyethylene terephthalate fabric

    Science.gov (United States)

    Zhou, J. F.; Zheng, D. D.; Zhong, L.; Zhang, F. X.; Zhang, G. X.

    2016-07-01

    In this work, -NH2 groups were introduced to polyethylene terephthalate (PET) fibers by nitration and reduction method, and then milk protein was grafted on the nitrated and reduced PET (NR PET) fibers by sucrose glycidyl ether crosslinking agent. FTIR suggested the milk protein was successfully grafted on PET fiber surface. SEM images showed a layer of substance covered on the PET fiber surface. DSC demonstrated an excellent thermal stability of milk protein/PET fiber. The moisture regain was improved by milk protein/PET fiber. Moreover, the crease recovery angle and stiffness were retained by the milk protein/PET fabric.

  1. Production of protein hydrolysates from fish byproduct prepared by enzymatic hydrolysis

    Directory of Open Access Journals (Sweden)

    Murna Muzaifa

    2012-03-01

    Full Text Available The objective of this research was to study the production of fish protein hydrolysate (FPH from fish by-product prepared by enzymatichydrolysis. Fish by-product were prepared using Alcalase and Flavourzyme enzyme and properties of FPH were analyzed. The resultsshowed that FPH prepared using Alcalase enzyme had greater amount of protein (82.66% than FPH prepared using Flavourzyme enzyme(73.51%. Solubility, emulsifying and foaming properties of FPH prepared using Alcalase were also better than those prepared usingFlavourzyme enzyme. The FPH derived from fish by-product using enzyme may potentially serve as a good source of protein. It could be usedas an emulsifier and as a foaming agent.

  2. Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking

    NARCIS (Netherlands)

    Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan

    2015-01-01

    A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80. °C for 15. min. During heating of w/o emulsions containing 10% (w/v) WPI protein

  3. Preparation of monoclonal antibodies against radiation-induced protein

    International Nuclear Information System (INIS)

    We obtained the 6 monoclonal antibodies against gamma-induced proteins of Deinococcus radiodurans, and these antibodies were designated as Mab-3F, 4B, 4D, 4F, 4G and 12G. Using these antibodies, we investigated the relations between gamma-induced proteins and other stress protein in strain R1, and the induction of proteins were compared among strain R1, resistant mutant (rec1) and radiosensitive mutant (rec30). We found new 6 proteins recognized by these monoclonal antibodies which were induced after gamma-irradiation especially in strain R1 and rec 1, but not induced in strain rec30. We suppose that these proteins participate in repair of DNA damages including double strand breaks caused by gamma-irradiation. One of them was around 46kDa protein band recognized by Mab-12G, and this protein was so induced in a large quantity after irradiation that the protein could detect by gold staining. In addition to this observation, we found some proteins which were induced in R1 and rec 1 by gamma-irradiation and other stress, but not in strain rec30, such as 31kDa protein band recognized by Mab-3F, 4B and 4G, and other 11 proteins which were especially induced in irradiated strain R1. The latter proteins might be reinforcement factor to radioresistance such as GroE and DnaK, or participant in repair of damage by gamma-irradiation in strain R1. (author)

  4. Preparation of flat gold terraces for protein chip developments

    OpenAIRE

    Elie-Caille, Céline; Rauch, Jean-Yves; Rouleau, Alain; Boireau, Wilfrid

    2009-01-01

    A simple method to prepare flat gold terraces on mica for atomic force microscopy biomolecular characterisation is described. The procedure includes preheating of the substrate, metal deposition and an annealing step. All of these steps are at elevated temperatures (300–420°C). This approach allows one to prepare large flat gold terraces (200– 500 nm), which constitute ideal substrates for visualisation and characterisation of a self-assembly monolayer of biomolecules at the nanoscale. The au...

  5. Sample Preparation for Mass Spectrometry Analysis of Protein-Protein Interactions in Cancer Cell Lines and Tissues.

    Science.gov (United States)

    Beigbeder, Alice; Vélot, Lauriane; James, D Andrew; Bisson, Nicolas

    2016-01-01

    A precisely controlled network of protein-protein interactions constitutes the basis for functional signaling pathways. This equilibrium is more often than not disrupted in cancer cells, by the aberrant expression or activation of oncogenic proteins. Therefore, the analysis of protein interaction networks in cancer cells has become crucial to expand our comprehension of the molecular underpinnings of tumor formation and progression. This protocol describes a sample preparation method for the analysis of signaling complexes by mass spectrometry (MS), following the affinity purification of a protein of interest from a cancer cell line or a solid tumor. In particular, we provide a spin tip-based protease digestion procedure that offers a more rapid and controlled alternative to other gel-based and gel-free methods. This sample preparation protocol represents a useful strategy to identify protein interactions and to gain insight into the molecular mechanisms that contribute to a given cancer phenotype. PMID:27581032

  6. Antifreeze polymeric additives for fuels; Aditivos polimericos anticongelantes para combustiveis

    Energy Technology Data Exchange (ETDEWEB)

    Muniz, Aline S.; Carvalho, Agne Roani de; Sakae, George Hideki; Oliveira, Angelo R.S.; Cesar-Oliveira, Maria Aparecida F. [Universidade Federal do Parana - UFPR - Departamento de Quimica - LABPOL-Laboratorio de Polimeros Sinteticos, Centro Politecnico, Curitiba, PR (Brazil)], e-mails: mafco@ufpr.br, alinemuniz@ufpr.br

    2011-07-01

    Owing to the current interest in the reduction of environmental pollution, several researchers are seeking renewable sources of energy which can at least partially replace combustibles derived from petroleum. Diesel oil is the combustible that most seriously pollutes the environment and is thus the biodiesel that is being considered as a fuel which can be replaced by a renewable combustible; this can possibly be used in diesel engines without any modifications. However, certain problems have to be overcome with regard to the temperature at which the biodiesel should be stored and used, since there is a tendency for biodiesel to solidify at low temperatures. This suggests that there is a need for the use of anti-freeze additives. This work behind the main focus additives with only 25 ppm, were able to reduce the pour point of fuel, achieving significant results, for example, the additive M14A18 lowered the pour point (PP) of B20 to -20 degree C, showing that the use of increasing amounts of biodiesel to diesel can aggregate. The main focus of work behind the development of additives that with only 25 ppm, were able to reduce the pour point of fuel, producing significant results such as those obtained with the use of additive M14A18 which lowered the pour point of the B20 to -20 degree C, showing the possibility of using increasing amounts of biodiesel added to diesel. (author)

  7. Nonequilibrium antifreeze peptides and the recrystallization of ice.

    Science.gov (United States)

    Knight, C A; Wen, D; Laursen, R A

    1995-02-01

    Evidence is presented that the nonequilibrium antifreeze peptide (AFP) from winter flounder has a special ability to inhibit recrystallization in ice only when an appreciable amount of liquid is present, as is the case when the system contains salts and the temperature is not too low. In this circumstance the AFP binds to the ice surface at the ice-solution interfaces in grain boundaries, preventing migration of the solution and effectively immobilizing the boundaries. In the absence of liquid, recrystallization inhibition appears to be a common property of many peptides. This is consistent with the view that the special effects of AFPs require a structural fit onto ice, and therefore require the AFP molecules to have the mobility to achieve that fit. Since the concentration of salt required to induce the special recrystallization inhibition effects of AFPs is lower (recrystallization. The proposition that mobility is needed for AFP molecules to produce their special influence upon ice growth argues against any special effects of AFPs in devitrification.

  8. Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough.

    Science.gov (United States)

    Panadero, Joaquin; Randez-Gil, Francisca; Prieto, Jose Antonio

    2005-12-28

    The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0 degrees C. In this study a recombinant antifreeze peptide GS-5 was expressed from the polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions. PMID:16366681

  9. DSCG binding protein and process for preparing same

    Energy Technology Data Exchange (ETDEWEB)

    Pecht, I.; Mazurek, N.

    1987-07-28

    An essentially pure protein is described consisting essentially of the protein, (CBP), present in nature in membranes of basophile cells and in mast cells, having a molecular weight of about 60,000 +- 2,000 determined by SDS polyacrylamide electrophoresis an isoelectric point of about 3.9 and an amino acid composition of about 4 units of asparagine, 3 units of threonine and serine, 3 units glycine, 2 units alanine, 2 units proline, 1 unit cysteine, 2 units valine, 1 unit methionine, 1 unit isoleucine, 2 units leucine, 1 unit tyrosine, 1 unit phenylalanine, 2 units histamine, 2 units lysine and 1 unit arginine. The protein is able to build calcium and having a calcium dependent affinity to the disodium salt of 1,2 bis(-2 carboxychromon-5-yloxy)-2-hydroxy propane (DSCG).

  10. Preparation of bromine-77 labeled proteins using bromoperoxidase

    Energy Technology Data Exchange (ETDEWEB)

    Lambert, F.; Slegers, G. (Ghent Univ. (Belgium). Lab. of Radiopharmacy)

    1994-01-01

    The halogenating enzyme bromoperoxidase, isolated from the brown algae Ascophyllum nodosum, was used to catalyze the radiobromination of an anti-hPLAP monoclonal antibody. Radiobromination of bovine serum albumin was also performed and used as control method. The proteins are labeled with bromine-77, produced by the [sup 75]As ([alpha], 2n) [sup 77]Br reaction. The essential reaction parameters, such as concentration of protein and hydrogen peroxide, the amount of bromoperoxidase, the pH of the reaction mixture, the reaction time and temperature were optimized. The immunoreactivity of the radiolabeled monoclonal antibody was checked with a ''Sandwich'' ELISA. (author).

  11. Preparation of racombinant proteins for use in tissue cultures

    OpenAIRE

    MIČULKOVÁ, Klára

    2012-01-01

    cDNA derived from exons 2 and 3 of the Ser2 (Sericin 2) gene of Bombyx mori and encoding 121 amino acids was expressed in Escherichia coli as a fusion protein with hexahistidine to allow purification by affinity chromatogramy on Ni-resine column. Several expression systems were used and the codon usage was optimized but the yield of recombinant protein remained low. Commercial sericin extract was therefore used in assay with human embryonic stem cells (hESC) ? the extract in some cases reduce...

  12. Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

    Science.gov (United States)

    Peterson, Megan J.; Snyder, W. Kalani; Westerman, Shelley; McFarland, Benjamin J.

    2011-01-01

    We describe how to produce and purify proteins from "Escherichia coli" inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This 7-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies,…

  13. Preparation and Observation of Fresh-frozen Sections of the Green Fluorescent Protein Transgenic Mouse Head

    International Nuclear Information System (INIS)

    Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein (GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections

  14. Effect of Antifreeze Peptide Pretreatment on Ice Crystal Size, Drip Loss, Texture, and Volatile Compounds of Frozen Carrots.

    Science.gov (United States)

    Kong, Charles H Z; Hamid, Nazimah; Liu, Tingting; Sarojini, Vijayalekshmi

    2016-06-01

    Ice crystal formation is of primary concern to the frozen food industry. In this study, the effects of antifreeze peptides (AFPs) on ice crystal formation were assessed in carrot during freezing and thawing. Three synthetic analogues based on naturally occurring antifreeze peptides were used in this study. The AFPs exhibited modification of ice crystal morphology, confirming their antifreeze activity in vitro. The ability of the synthetic AFPs to minimize drip loss and preserve color, structure, texture, and volatiles of frozen carrot was evaluated using the techniques of SEM, GC-MS, and texture analysis. The results prove the potential of these AFPs to preserve the above characteristics in frozen carrot samples. PMID:27138051

  15. Smelt was the likely beneficiary of an antifreeze gene laterally transferred between fishes

    Directory of Open Access Journals (Sweden)

    Graham Laurie A

    2012-09-01

    Full Text Available Abstract Background Type II antifreeze protein (AFP from the rainbow smelt, Osmerus mordax, is a calcium-dependent C-type lectin homolog, similar to the AFPs from herring and sea raven. While C-type lectins are ubiquitous, type II AFPs are only found in a few species in three widely separated branches of teleost fishes. Furthermore, several other non-homologous AFPs are found in intervening species. We have previously postulated that this sporadic distribution has resulted from lateral gene transfer. The alternative hypothesis, that the AFP evolved from a lectin present in a shared ancestor and that this gene was lost in most species, is not favored because both the exon and intron sequences are highly conserved. Results Here we have sequenced and annotated a 160 kb smelt BAC clone containing a centrally-located AFP gene along with 14 other genes. Quantitative PCR indicates that there is but a single copy of this gene within the smelt genome, which is atypical for fish AFP genes. The corresponding syntenic region has been identified and searched in a number of other species and found to be devoid of lectin or AFP sequences. Unlike the introns of the AFP gene, the intronic sequences of the flanking genes are not conserved between species. As well, the rate and pattern of mutation in the AFP gene are radically different from those seen in other smelt and herring genes. Conclusions These results provide stand-alone support for an example of lateral gene transfer between vertebrate species. They should further inform the debate about genetically modified organisms by showing that gene transfer between ‘higher’ eukaryotes can occur naturally. Analysis of the syntenic regions from several fishes strongly suggests that the smelt acquired the AFP gene from the herring.

  16. Efficient preparation and analysis of membrane and membrane protein systems.

    Science.gov (United States)

    Javanainen, Matti; Martinez-Seara, Hector

    2016-10-01

    Molecular dynamics (MD) simulations have become a highly important technique to consider lipid membrane systems, and quite often they provide considerable added value to laboratory experiments. Rapid development of both software and hardware has enabled the increase of time and size scales reachable by MD simulations to match those attainable by several accurate experimental techniques. However, until recently, the quality and maturity of software tools available for building membrane models for simulations as well as analyzing the results of these simulations have seriously lagged behind. Here, we discuss the recent developments of such tools from the end-users' point of view. In particular, we review the software that can be employed to build lipid bilayers and other related structures with or without embedded membrane proteins to be employed in MD simulations. Additionally, we provide a brief critical insight into force fields and MD packages commonly used for membrane and membrane protein simulations. Finally, we list analysis tools that can be used to study the properties of membrane and membrane protein systems. In all these points we comment on the respective compatibility of the covered tools. We also share our opinion on the current state of the available software. We briefly discuss the most commonly employed tools and platforms on which new software can be built. We conclude the review by providing a few ideas and guidelines on how the development of tools can be further boosted to catch up with the rapid pace at which the field of membrane simulation progresses. This includes improving the compatibility between software tools and promoting the openness of the codes on which these applications rely. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26947184

  17. Optimising functional properties during preparation of cowpea protein concentrate.

    Science.gov (United States)

    Mune Mune, Martin Alain; Minka, Samuel René; Mbome, Israël Lape

    2014-07-01

    Response surface methodology (RSM) was used for modelisation and optimisation of protein extraction parameters in order to obtain a protein concentrate with high functional properties. A central composite rotatable design of experiments was used to investigate the effects of two factors, namely pH and NaCl concentration, on six responses: water solubility index (WSI), water absorption capacity (WAC), oil holding capacity (OHC), emulsifying activity (EA), emulsifying stability (ES) and foam ability (FA). The results of analysis of variance (ANOVA) and correlation showed that the second-order polynomial model was appropriate to fit experimental data. The optimum condition was: pH 8.43 and NaCl concentration 0.25M, and under this condition WSI was ⩾17.20%, WAC⩾383.62%, OHC⩾1.75g/g, EA⩾0.15, ES⩾19.76min and FA⩾66.30%. The suitability of the model employed was confirmed by the agreement between the experimental and predicted values for functional properties.

  18. A novel preparation technique of red (sparkling wine for protein analysis

    Directory of Open Access Journals (Sweden)

    Elisabeth I. Vogt

    2016-06-01

    Full Text Available Despite their low concentration, proteins can influence several key enological parameters such as foam stability or haze formation in (sparkling wine. Most studies focus on white (sparkling wine since the higher content of phenolic compounds in red wines impairs proteomic research. The aim of the study was the development of a method for the preparation of red (sparkling wine proteins for proteomic analysis. Three methods of sample preparation were assessed on silver stained SDS-PAGE gels and with MALDI-TOF MS. Our new method was highly suitable for the preparation of proteins for the aforementioned applications. The results showed a substantial increase in signal intensity with a simultaneous decrease in background noise. The preparation protocol consists of (i dialysis and freeze drying of the sample, (ii removal of phenolic compounds by water-saturated phenol and (iii protein precipitation by addition of ammonium acetate. Employment of this method followed by SDS-PAGE analysis allowed for silver stained gels with diminished background or streaking and clearly resolved protein bands. Analysis of spectra obtained from samples prepared according to the proposed protocol showed increased intensity and signal-to-noise ratio in MALDI-TOF MS. Furthermore it was demonstrated that this method can be applied to various kinds of grape products.

  19. Preparation of iron bound succinylated milk protein concentrate and evaluation of its stability.

    Science.gov (United States)

    Shilpashree, B G; Arora, Sumit; Sharma, Vivek; Bajaj, Rajesh Kumar; Tomar, S K

    2016-04-01

    Major problems associated with the fortification of soluble iron salts include chemical reactivity and incompatibility with other components. Milk protein concentrate (MPC) are able to bind significant amount of iron due to the presence of both casein and whey protein. MPC in its native state possess very poor solubility, therefore, succinylated derivatives of MPC (succ. MPC) were also used for the preparation of protein-iron complex. Preparation of the complex involved centrifugation (to remove insoluble iron), ultrafiltration (to remove unbound iron) and lyophilisation (to attain in dry form). Iron binding ability of MPC enhanced significantly (P<0.05) upon succinylation. Stability of bound iron from both varieties of complexes was monitored under different conditions encountered during processing. Higher stability (P<0.05) of bound iron was observed in succ. MPC-iron complex than native protein complex. This method could be adopted for the production of stable iron enriched protein, an organic iron source.

  20. [Fractional and amino acid composition of krill proteins and the potential for obtaining protein preparations].

    Science.gov (United States)

    Orlova, T A; Churina, E E; Kuranova, L K

    1985-01-01

    Studies of the fractional composition of krill proteins demonstrated that the content of protein fractions changes depending on the time of krill catch. The highest amount of water-soluble proteins is contained by krill caught in December (64%), of salt-soluble by krill caught in June (12%), base-soluble by krill caught in May, September and February (34%). Krill protein contains from 50 to 60% of water- and salt-soluble fractions. Analysis of the amino acid composition of krill proteins showed that it does not differ essentially from that of adequate food proteins.

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

    Science.gov (United States)

    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

  2. Observation on the modifying activity of the protein from Elytrzgia repens rhizome for ice crystal

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In winter, spring and summer, the rhizome of wild Elytrzgia repens of Heilongjiang Province was selected to extract the soluble which whole protein and the apoplastic protein, and analyzed by SDS-PAGE. The result indicated that there were two specific polypeptides in two types protein from winter; their relative molecular weight were identified as 52 ku and 26 ku by analyzing software; the apoplastic protein from winter had the ability of modifing the growth of ice crystal which appeared hexagonal in shape observed with the phase-contrast photomicroscope. So the apoplastic protein from winter has the antifreeze characters and the 52 ku protein is more likely the antifreeze protein.

  3. Preparation of 15N labelled protein sample by gene engineering technology

    International Nuclear Information System (INIS)

    Using the advanced multi-dimension heteronuclear pulses and isotope labelled protein technique, nuclear magnetic resonance spectroscopy has become an important tool in analysis of the solution conformation of protein. On the basis of the high level expression of a protein-trichosanthin in recombinant E.coli using DNA, 15N was used to label the protein, the 15N labelled trichosanthin was obtained by affinity chromatography on Ni-NTA agarose. Terminating pregnant effect in mice showed that this recombinant protein had the same activity as natural trichosanthin. A 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum was obtained from an AM-500 NMR spectrometer, demonstrating that this method is suitable in preparing labelled protein sample for NMR

  4. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    Science.gov (United States)

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  5. Radioactive labeling of proteins in cultured postimplantation mouse embryos. I. Influence of the embryo preparation method

    International Nuclear Information System (INIS)

    Conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations followed by fluorography. The aim was to obtain highly radioactive proteins under conditions as physiological as possible. Embryos at Days 10, 11, and 12 of gestation were prepared in different ways and incubated for 4 h in Tyrode's solution containing [3H]amino acids (mixture) at a concentration of 27 microCi/ml medium. The preparations were: (a) yolk sac opened, placenta and blood circulation intact; (b) yolk sac and amnion opened, placenta and blood circulation intact (Day 10 embryos only); (c) placenta, yolk sac, and amnion removed (embryo naked); (d) naked embryos cut randomly into pieces (Day 10 embryos only). After incubation whole embryos or certain parts (tail, liver, rest body) were investigated by determining the radioactivity taken up by the protein. The results are given in dpm per mg protein per embryo. Radioactivity of proteins was about 3 times higher in naked embryos than in embryos left in their yolk sacs. This was true for all three stages investigated. However, the degree of radioactivity in the various parts of naked embryos differed by a factor of 15, whereas radioactivity was evenly distributed in embryos incubated in their yolk sacs. Therefore, embryos prepared according to the first method (see above) fulfilled the conditions required at the best

  6. Solution structures, dynamics, and ice growth inhibitory activity of peptide fragments derived from an antarctic yeast protein.

    Directory of Open Access Journals (Sweden)

    Syed Hussinien H Shah

    Full Text Available Exotic functions of antifreeze proteins (AFP and antifreeze glycopeptides (AFGP have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.

  7. 复合防冻剂的防冻机理及施工要求%On the Antifreeze Mechanism and the Construction Requirements of Antifreeze Compound

    Institute of Scientific and Technical Information of China (English)

    王超

    2011-01-01

    Concrete is the most widely used material in construction,.The durability of concrete which is strongly influenced by its frost resistance has raised great attention.Therefore,improving and developing concrete of good frost resistance has very significant economic and social benefits.Using concrete with antifreeze compound can achieve convenient construction,can conserve energy,can save concrete cost and improve the quality of concrete constructed in winter.It can not only reduce the liquid freezing point of concrete,but can promote freezing and save water as well.Therefore,understanding the antifreeze mechanism and the construction requirements of antifreeze compound can achieve better technical economic benefit.%在建筑工程中,混凝土是使用最广泛的一种材料,混凝土的耐久性受到人们的普遍关注,其中冻害性是影响混凝土耐久性的一个最重要的因素。改善并开发抗冻性能良好的混凝土具有非常重大的经济效益与社会效益。而采用掺复合防冻剂混凝土具有施工简便、节能、节约混凝土冬施费用,提高混凝土冬施质量等优点,它不仅能够降低混凝土中液相冰点,同时还具有促凝、早强和减水作用。

  8. Preparation of a Rechargeable Battery Using Waste Protein from the Fish Scales

    International Nuclear Information System (INIS)

    The electrochemical redox reactions of the oxytocin and fish scale protein which are mainly collagen were exploited for the preparation of a rechargeable protein battery named as fish scale battery. This battery was found to depend upon the concentration of oxidizing and reducing agents, voltage of the charger and the time for charging. Some of these parameters were optimized using a single cell of this battery and some others were optimized by using five cell battery. The five cell protein battery gives a maximum and stable voltage of 8500 millivolt. The way of charging and theoretical aspects of the battery is also discussed in this communication. (author)

  9. Preparation and characterisation of protein hydrolysates from Indian defatted rice bran meal.

    Science.gov (United States)

    Bandyopadhyay, Kakali; Misra, Gautam; Ghosh, Santinath

    2008-01-01

    Rice bran meal is a very good source of protein along with other micronutrients. Rice bran meal has been utilized to produce protein isolates and respective protein hydrolysates for potential application in various food products. De-oiled rice bran meal, available from Indian rice bran oil extraction plants, was initially screened by passing through an 80-mesh sieve (yield about 70%). A fraction (yield-30%) rich in fibre and silica was initially discarded from the meal. The protein content of the through fraction increased from 20.8% to 24.1% whereas silica content reduced from 3.1% to 0.4%. Rice bran protein isolate (RPI) was prepared by alkaline extraction followed by acidic precipitation at isoelectric point. This protein isolate was hydrolysed by papain at pH 8.0 and at 37 degrees C for 10, 20, 30, 45 and 60 minutes. The peptides produced by partial hydrolysis had been evaluated by determining protein solubility, emulsion activity index (EAI), emulsion stability index (ESI), foam capacity and foam stability (FS). All protein hydrolysates showed better functional properties than the original protein isolate. These improved functional properties of rice bran protein hydrolysates would make it useful for various application especially in food, pharmaceutical and related industries. PMID:18075222

  10. Human protein C: new preparations. Effective replacement therapy for some clotting disorders.

    Science.gov (United States)

    2003-02-01

    (1) Depending on its severity, congenital protein C deficiency can cause a variety of problems, such as increasing the frequency of venous thrombosis in high risk situations; recurrent venous thrombosis; skin necrosis at the start of treatment with a vitamin K antagonist; and severe thrombotic events in neonates. For many years the only available replacement treatment consisted of fresh frozen plasma which, among other adverse effects, carries a risk of hypervolemia. (2) Two human protein C concentrates prepared from donated blood have been given marketing authorisation in Europe for intravenous replacement therapy (Ceprotin from Baxter, and Protexel from LFB). (3) Their clinical files contain only retrospective case series (22 children with severe deficiency treated with Ceprotin; and 10 patients of various ages and with different degrees of severity treated with Protexel). The two preparations have not been compared with each other. (4) In patients with severe protein C deficiency, including neonates, replacement therapy with human protein C is effective, especially for treating cutaneous thrombosis and preventing thrombosis in high risk situations. (5) In patients with moderate deficiency, a short-course of human protein C prophylaxis reduces the frequency of thrombosis in high risk situations. (6) In long-term prophylaxis, human protein C replacement therapy, added to ongoing (but inadequately effective) vitamin K antagonist therapy, seems to reduce the risk of recurrent venous thrombosis even though it has some constraints. (7) The adverse effects of the two preparations are poorly documented. Allergic reactions and bleeding have been reported. Human protein C is a blood product, and therefore carries a risk of infection. (8) Ceprotin offers a small advantage, being available in two dose strengths: for a given dose the volume injected is halved. (9) In practice, Ceprotin and Protexel are the reference drugs for replacement therapy of constitutional protein C

  11. Evaluation of anti-freeze viscosity modifier for potential external tank applications

    Science.gov (United States)

    Lynn, R. O. L.

    1981-01-01

    Viscosity modifiers and gelling agents were evaluated in combination with ethylene glycol and dimethyl sulfoxide water eutectics. Pectin and agarose are found to gel these eutectics effectively in low concentration, but the anti-freeze protection afforded by these compositions is found to be marginal in simulations of the intended applications. Oxygen vent shutters and vertical metallic surfaces were simulated, with water supplied as a spray, dropwise, and by condensation from the air.

  12. Role of ice nucleation and antifreeze activities in pathogenesis and growth of snow molds.

    Science.gov (United States)

    Snider, C S; Hsiang, T; Zhao, G; Griffith, M

    2000-04-01

    ABSTRACT We examined the ability of snow molds to grow at temperatures from -5 to 30 degrees C and to influence the growth of ice through assays for ice nucleation and antifreeze activities. Isolates of Coprinus psychromorbidus (low temperature basidiomycete variant), Microdochium nivale, Typhula phacorrhiza, T. ishikariensis, T. incarnata, and T. canadensis all grew at -5 degrees C, whereas Sclerotinia borealis and S. homoeocarpa did not grow at temperatures below 4 degrees C. The highest threshold ice nucleation temperature was -7 degrees C. Because snow molds are most damaging to their hosts at temperatures above this, our results imply that the pathogenesis of these fungi is not dependent on ice nucleation activity to cause freeze-wounding of host plants. All snow molds that grew at subzero temperatures also exhibited antifreeze activity in the growth medium and in the soluble and insoluble hyphal fractions, with the exception of M. nivale and one isolate of T. canadensis. The lack of high ice nucleation activity combined with the presence of antifreeze activity in all fungal fractions indicates that snow molds can moderate their environment to inhibit or modify intra- and extracellular ice formation, which helps explain their ability to grow at subzero temperatures under snow cover.

  13. Ultrasonic atomization for spray drying: a versatile technique for the preparation of protein loaded biodegradable microspheres.

    Science.gov (United States)

    Bittner, B; Kissel, T

    1999-01-01

    Bovine serum albumin (BDA) loaded microspheres with a spherical shape and smooth surface structure were successfully prepared from poly(lactide-co-glycolide) using an ultrasonic nozzle installed in a Niro laboratory spray dryer. Process and formulation parameters were investigated with respect to their influence on microsphere characteristics, such as particle size, loading capacity, and release properties. Preparation of microspheres in yields of more than 50% was achieved using an ultrasonic atomizer connected to a stream of carrier air. Microsphere characteristics could be modified by changing several technological parameters. An increased polymer concentration of the feed generated larger particles with a significantly reduced initial release of the protein. Moreover, microspheres with a smooth surface structure were obtained from the organic polymer solution with the highest viscosity. Microparticles with a low BSA loading showed a large central cavity surrounded by a thin polymer layer in scanning electron microspheres. A high protein loading led to an enlargement of the shell layer, or even to dense particles without any cavities. A continuous in vitro release pattern of BSA was obtained from the particles with low protein loading. Glass transition temperatures (Tg) of the microspheres before and after lyophilization did not differ from those of the BSA loaded particles prepared by spray drying with a rotary atomizer. Analysis of the polymer by gel permeation chromatography indicated that ultrasonication had no effect on polymer molecular weight. Molecular weight and polydispersity of the pure polymer, placebo microspheres prepared by spray drying, and placebo microspheres prepared using the ultrasonic nozzle were in the same range. In conclusion, ultrasonic atomization represents a versatile and reliable technique for the production of protein loaded biodegradable microspheres without inducing a degradation of the polymer matrix. Particle characteristics

  14. Improved characterization of EV preparations based on protein to lipid ratio and lipid properties.

    Directory of Open Access Journals (Sweden)

    Xabier Osteikoetxea

    Full Text Available In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody and ganglioside GM1 (cholera toxin subunit B. We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition, may prove useful for quality control of extracellular vesicle related basic and clinical studies.

  15. [Preparation and detection of anti-influenza A virus polymerase basic protein 1 polyclonal antibody].

    Science.gov (United States)

    Qin, Yujie; Zhang, Tinghong; Ye, Xin

    2016-01-01

    Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1. PMID:27363203

  16. Preliminary study on preparation of E.coli cell-free system for protein expression

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In the new era of "Omics",the traditional techniques of protein expression in vivo can not come up with the exponential increase of genetic information.The cellfree protein synthesis system provides a new strategy of protein expression with advantages of rapid,convenient and high-throughput expression.The preparation of cell extracts,the optimization of substrate concentrations and the energy regeneration system are the key factors for the successful construction of cell-free protein expression system.In this work,the cell extract was prepared from RNase I- defective strain E.coli A19.The cell growth phase,the pressure for cell disruption and the storage condition of cell extracts were optimized.Meanwhile,the optimal substrate concentrations and the energy regeneration system were selected.Under the optimized conditions,the green fluorescent protein (GFP) reporter gene was expressed in the E.coli cell-free system with high expression level (Ca.154 μg/mL) which was 29 times higher than the expression level before optimization.

  17. Infant food from quality protein maize and chickpea: optimization for preparing and nutritional properties.

    Science.gov (United States)

    Alarcón-Valdez, C; Milán-Carrillo, J; Cárdenas-Valenzuela, O G; Mora-Escobedo, R; Bello-Pérez, L A; Reyes-Moreno, C

    2005-06-01

    The present study had two objectives: to determine the best combination of nixtamalized maize flour (NMF) from quality protein maize and extruded chickpea flour (ECF) for producing an infant food, and to evaluate the nutritional properties of the optimized NMF/ECF mixture and the infant food. Response surface methodology (RSM) was applied to determine the best combination of NMF/ECF; the experimental design (Lattice simplex) generated 11 assays. Mixtures from each assay were evaluated for true protein and available lysine. Each one of 11 mixtures was used for preparing 11 infant foods that were sensory evaluated for acceptability. A common optimum value for the three response variables was obtained utilizing the desirability method. The best combination of NMF/ECF for producing an infant food was NMF = 26.7%/ECF = 73.3%; this optimized mixture had a global desirability of 0.87; it contained 19.72% dry matter (DM) proteins, 6.10% (DM) lipids, 71.45% (DM) carbohydrates, and 2.83% (DM) minerals; its essential amino acids profile covered the amino acids requirements for children 10-12 years old. The infant food prepared from optimized mixture had an in vitro protein digestibility of 87.9%, and a calculated protein efficiency ratio of 1.86. Infant food could be used to support the growth of infants in developing countries.

  18. PREPARATION AND CHARACTERIZATION OF SOY PROTEIN ISOLATE (SPI)/MONTMORILLONITE (MMT) BIONANOCOMPOSITES

    Institute of Scientific and Technical Information of China (English)

    Lixue Xiang; Chang-yu Tang; Jing Can; Chao-yu Wang; Ke Wang; Qin Zhang; Qiang Fu; Shu-gao Zhao

    2009-01-01

    The bionanocomposites of soy protein isolate (SPI)/montmorillonite (MMT) have been prepared successfully via simple melt mixing, in which MMT was used as nanofiller and glycerol was used as plasticizer. Their structures and properties were characterized with X-ray diffraction (XRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), thermogravimetric analysis and tensile testing. XRD, TEM and SEM results indicated that the MMT layers could be easily intercalated by the SPI matrix even by simple melt processing. The exfoliated MMT layers were randomly dispersed in the protein matrix as MMT content was low (less than 5 wt%), an incomplete exfoliation was evident from SEM results, and some primary particles were observed as the MMT content was high (from 5 wt% to 9 wt%). A significant improvement of the mechanical strength and thermal stability of SPI/MMT nanocomposites has been achieved. Our work suggests that simple melt processing is an efficient way to prepare SPI/MMT nanocomposites with exfoliated structure.

  19. [Preparation of procion brilliant blue-doped silica nanorods and their recognition properties for proteins].

    Science.gov (United States)

    Deng, Qiliang; Chen, Yang; Wu, Jianhua; Li, Yanli; Liu, Chunjing; Song, Weijing

    2011-09-01

    Protein enrichment and separation is one of the pivotal preliminary steps of proteomics studies, which is important to medical diagnosis and treatment. In this study, procion brilliant blue-doped silica nanorod was prepared via self-assembly sol-gel technology without any additional template. Procion brilliant blue was covalently linked to 3-aminopropyltriethyloxy silane in ethanol. Tetraethylorthosilane (TEOS) was then added into the mixture, subsequently hydrolyzed and co-condensed for 3 h under stirring. The resulted nanorods were isolated by centrifugation, re-dispersed in deionized water, and centrifuged again. This wash process was repeated three times. Finally, the nanorods were dried under vacuum. Procion brilliant blue acted simultaneously as a self-assembly template during the preparation process, and subsequently as recognition probe for proteins. Scanning electron microscopy (SEM) image showed that the nanotubes were 2-16 microm in length and 200-500 nm in diameter. The obtained nanorods were further characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSA), separately. All these results indicated that procion brilliant blue were successfully doped into silica nanorods. The recognition property of nanorods for bovine serum albumin (BSA) was investigated under static condition. The resulted nanorods showed high binding capacity (57.6 mg/g) for BSA and fast adsorption equilibrium (within 60 min). The nanorods were also evaluated with four typical proteins, hemoglobin, trypsin, lysozyme and pepsin, with different relative molecular masses and isoelectric points. The results indicated that the prepared nanorods exhibited the highest binding capacity for trypsin (87.5 mg/g) and the least binding for hemoglobin (Hb, 3.0 mg/g). This easy preparation protocol and excellent recognition property make the prepared materials a bright future in proteomics research. PMID:22233075

  20. PREPARATION AND CHARACTERIZATION OF SOY PROTEIN ISOLATE(SPI)/MONTMORILLONITE(MMT) BIONANOCOMPOSITES

    Institute of Scientific and Technical Information of China (English)

    傅强

    2009-01-01

    The bionanocomposites of soy protein isolate(SPI)/montmorillonite(MMT) have been prepared successfully via simple melt mixing,in which MMT was used as nanofiller and glycerol was used as plasticizer.Their structures and properties were characterized with X-ray diffraction(XRD),differential scanning calorimetry(DSC),scanning electron microscopy(SEM),thermogravimetric analysis and tensile testing.XRD、TEM and SEM results indicated that the MMT layers could be easily intercalated by the SPI matrix even by si...

  1. Preparation of Soybean Protein Concentrate with Mixed Solvents of Hexane-Aqueous Alcohol

    Institute of Scientific and Technical Information of China (English)

    ZhangWeinong; LiuDachuan

    2002-01-01

    Preparation of soybean protein concentrate with the mixed solvents of hexane-aqueous alcohol was studied in this paper.The optimum technology parameters were obtained by orthogonal tests.The results of experiments showed that the qualities of the product were good not only on taste of the product were good not only on tasted and color,but also on high solubility-NSI value was 48.80%.

  2. In-vitro antioxidant and antibacterial properties of fermentatively and enzymatically prepared chicken liver protein hydrolysates.

    Science.gov (United States)

    Chakka, Ashok Kumar; Elias, Mercy; Jini, R; Sakhare, P Z; Bhaskar, N

    2015-12-01

    Protein hydrolysates were prepared from chicken liver using fermentation and enzymatic hydrolysis. The lactic acid bacteria Pediococcus acidilactici NCIM5368 was employed in the fermentation process and a commercial protease (Alcalase® 2.5) was used in enzymatic hydrolysis. Chicken liver hydrolysates prepared by fermentation (FCLH) and enzymatic hydrolysis (ECLH) revealed appreciable amounts of protein [55.85 and 61.34 %; on dry weight basis, respectively]. Fermentation and enzymatic hydrolysis resulted in 14.3 and 26.12 % of degree of hydrolysis. Total antioxidant activity, reducing power, scavenging of superoxide, 2- diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid (ABTS) radicals were determined for both FCLH & ECLH. FCLH & ECLH showed total antioxidant activity of 0.99 and 1.13 μg AAE mg(-1) proteins, respectively; while, they scavenged 96.14 and 92.76 % of DPPH radicals respectively. FCLH showed higher ABTS radical scavenging activity (32.16 %) than ECLH (19.29 %). Superoxide anion scavenging activity of FCLH & ECLH were found to be 95.02 & 88.94 %, respectively. Residues obtained after both treatments also exhibited antioxidant activities. FCLH reported highest antagonistic activity against Listeria monocytogenes (30 mm); while, ECLH showed antibacterial activity only against Micrococcus luteus (12 mm). Both hydrolysates have the potential to be a protein rich ingredient for use in formulated foods and possible help in reduction of oxidative stress. PMID:26604378

  3. Preparation of Soy Protein Fruit Juice%大豆蛋白果汁的研制

    Institute of Scientific and Technical Information of China (English)

    郭睿; 杨晓泉; 杨熙

    2012-01-01

    The preparation method of high-protein fruit juice was studied. When added soy protein, the acidic beverages like fruit juice would have the phenomenon of precipitation. Use Glucono-Delta-Lactone (GDL) to induce soy protein to form the gelatin, then add this protein into the juice. Study the effect of different concentrations of GDL and protein on the stability of products, and illustrate the mechanism of this method on stabilizing the protein in fruit juice. The result showed that reducing the potential and Z-Average of protein was the main reason of stabilizing the protein in the fruit juice, and moderate concentration of GDL and protein, as well as homogenization pressure and number of times would play a significant improvement on the stability of the products.%研究了高蛋白果汁的制备方法。在果汁等酸性饮料中添加大豆蛋白,会导致产品中蛋白质的沉淀。利用葡萄糖酸内酯(GDL)诱导大豆蛋白形成凝胶,将蛋白加入到果汁中,研究了不同GDL诱导量和不同蛋白浓度对产品稳定性的影响,并阐明了此方法可以稳定果汁中蛋白的机理。研究表明,降低蛋白的荷电量和粒度是在果汁中稳定蛋白的主要原因,并且适度的GDL和蛋白的浓度,以及均质压力和次数,都会对产品的稳定性起到明显的改善作用。

  4. Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking.

    Science.gov (United States)

    Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan

    2015-03-01

    A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80°C for 15 min. During heating of w/o emulsions containing 10% (w/v) WPI proteins in the water phase, the emulsions displayed turbid-transparent-turbid phase transitions, which is ascribed to the change in the size of the protein-containing water droplets caused by thermal cross-linking between denatured protein molecules. The transparent stage indicated the formation of WPI NPs. WPI NPs of different sizes were obtained by varying the mixing speed. WPI NPs of 200-500 nm were selected to prepare o/w Pickering emulsions because of their good stability against coalescence. By Confocal Laser Scanning Microscopy, it was observed that WPI NPs were closely packed and distributed at the surface of the emulsion droplets. By measuring water contact angles of WPI NPs films, it was found that under most conditions WPI NPs present good partial wetting properties, but that at the isoelectric point (pI) and high ionic strength the particles become more hydrophobic, resulting in less stable Pickering emulsion. Thus, at pH above and below the pI of WPI NPs and low to moderate ionic strengths (1-10 mM), and with a WPI NPs concentration of 2% (w/v), a stable Pickering emulsion can be obtained. The results may provide useful information for applications of WPI NPs in environmentally friendly and food grade applications, notably in food, pharmaceutical and cosmetic products.

  5. Coagulant properties of Moringa oleifera protein preparations: application to humic acid removal.

    Science.gov (United States)

    Santos, Andréa F S; Paiva, Patrícia M G; Teixeira, José A C; Brito, António G; Coelho, Luana C B B; Nogueira, Regina

    2012-01-01

    This work aimed to characterize the coagulant properties of protein preparations from Moringa oleifera seeds in the removal of humic acids from water. Three distinct preparations were assayed, namely extract (seeds homogenized with 0.15 M NaCl), fraction (extract precipitated with 60% w/v ammonium sulphate) and cMoL (protein purified with guar gel column chromatography). The extract showed the highest coagulant activity in a protein concentration between 1 mg/L and 180 mg/L at pH 7.0. The zeta potential of the extract (-10 mV to -15 mV) was less negative than that of the humic acid (-41 mV to -42 mV) in a pH range between 5.0 and 8.0; thus, the mechanism that might be involved in this coagulation activity is adsorption and neutralization of charges. Reduction of total organic carbon (TOC) and dissolved organic carbon (DOC) was observed in water samples containing 9 mg/L carbon as humic acid when treated with 1 mg/L of the extract. A decrease in colour and in the aromatic content of the treated water was also observed. These results suggested that the extract from M. oleifera seeds in a low concentration (1 mg/L) can be an interesting natural alternative for removing humic acid from water in developing countries. The extract dose determined in the present study does not impart odour or colour to the treated water.

  6. Spray-drying performance of a bench-top spray dryer for protein aerosol powder preparation.

    Science.gov (United States)

    Maa, Y F; Nguyen, P A; Sit, K; Hsu, C C

    1998-11-01

    The objective of this work was to improve a bench-top spray dryer's efficiency in both production recovery and throughput for preparing protein aerosol powders. A Büchi mini-spray dryer was used to prepare the powders of recombinant humanized anti-IgE antibody. The resulting powder's physical properties such as particle size, residual moisture, and morphology, along with its recovery and production rate was the basis of this development work. Mass balance suggests that approximately 10-20% of powder was lost in the exhaust air, consisting primarily of particles less than 2 micrometer. Also, significant loss (20-30%) occurred in the cyclone. Attempts were made to improve product recovery in the receiving vessel using dual-cyclone configurations, different cyclone designs, cyclones with anti-static treatment, and different receiver designs. System modifications such as replacing the original bag-filter unit with a vacuum system effectively reduced drying air flow resistance, allowing the protein to be dried at a lower inlet air temperature and the production scale to be increased. We concluded that the modified spray-drying system is advantageous over the original bench-top spray dryer. This improvement will be beneficial to early-stage research and development involving high-valued protein powders. PMID:10099432

  7. Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis.

    Science.gov (United States)

    Hodgman, C Eric; Jewett, Michael C

    2013-10-01

    Cell-free protein synthesis (CFPS) has emerged as a powerful platform technology to help satisfy the growing demand for simple, affordable, and efficient protein production. In this article, we describe a novel CFPS platform derived from the popular bio-manufacturing organism Saccharomyces cerevisiae. By developing a streamlined crude extract preparation protocol and optimizing the CFPS reaction conditions we were able to achieve active firefly luciferase synthesis yields of 7.7 ± 0.5 µg mL(-1) with batch reactions lasting up to 2 h. This duration of synthesis is the longest ever reported for a yeast CFPS batch reaction. Furthermore, by removing extraneous processing steps and eliminating expensive reagents from the cell-free reaction, we have increased relative product yield (µg protein synthesized per $ reagent cost) over an alternative commonly used method up to 2000-fold from ∼2 × 10(-4) to ∼4 × 10(-1)  µg $(-1) , which now puts the yeast CPFS platform on par with other eukaryotic CFPS platforms commercially available. Our results set the stage for developing a yeast CFPS platform that provides for high-yielding and cost-effective expression of a variety of protein therapeutics and protein libraries.

  8. Preparation of photoreactive phospholipid polymer nanoparticles to immobilize and release protein by photoirradiation.

    Science.gov (United States)

    Chen, Weixin; Inoue, Yuuki; Ishihara, Kazuhiko

    2015-11-01

    Photoreactive and cytocompatible polymer nanoparticles for immobilizing and releasing proteins were prepared. A water-soluble and amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-4-(4-(1-methacryloyloxyethyl)-2-methoxy-5-nitrophenoxy) butyric acid (PL)) (PMB-PL) was synthesized. The PMB-PL underwent a cleavage reaction at the PL unit with photoirradiation at a wavelength of 365 nm. Additionally, the PMB-PL took polymer aggregate in aqueous medium and was used to modify the surface of biodegradable poly(L-lactic acid) (PLA) nanoparticle as an emulsifier. The morphology of the PMB-PL/PLA nanoparticle was spherical and approximately 130 nm in diameter. The carboxylic acid group in the PL unit could immobilize proteins by covalent bonding. The bound proteins were released by a photoinduced cleavage reaction. Within 60s, up to 90% of the immobilized proteins was released by photoirradiation. From these results and with an understanding of the fundamental properties of MPC polymers, we concluded that PMB-PL/PLA nanoparticles have the potential to be used as smart carriers to deliver proteins to biological systems, such as the inside of living cells.

  9. STUDY ON PROPERTIES OF SKID RESISTANCE ON FREEZING PAVEMENTS AND QUANTITATIVE EVALUATION METHOD OF ANTIFREEZING EFFECTS

    Science.gov (United States)

    Tanaka, Shunsuke; Takeichi, Kiyoshi; Masuyama, Yukiei; Takahashi, Naoto

    Snow and ice control in winter roads trends to be controlled by the skid friction coefficients in North America and North European countries at present, but the measurements are not necessarily easy. We studied on a simplified measurement method based on the relationship between skid friction coefficients and the bare pavement ratio (BPR) in the laboratory tests and field tests. The factors of BPR, surface textures and antifreezing materials which affect the skid friction coefficient are reviewed by a multiple linear regression analysis and a spectrum analysis, considering different freezing surfaces. These studies indicate that conclusions induced by laboratory tests could be applied to roads in service.

  10. Preparation of inulin-type fructooligosaccharides using fast protein liquid chromatography coupled with refractive index detection.

    Science.gov (United States)

    Li, J; Cheong, K L; Zhao, J; Hu, D J; Chen, X Q; Qiao, C F; Zhang, Q W; Chen, Y W; Li, S P

    2013-09-20

    A fast protein liquid chromatography coupled with refractive index detection (FPLC-RID) method was firstly developed for preparation and purification of fructooligosaccharides with different degree of polymerization from burdock, Arctium lappa. After extraction with 60% ethanol and decolorization with MCI gel CHP20P, total fructooligosaccharides were purified on Bio-Gel P-2 column eluted with water at the flow rate of 0.3 ml/min, which was the optimized conditions. The obtained fructooligosaccharides with degree of polymerization of 3-9 were identified based on their methylation analysis, MS and NMR data. This method has the advantages of high automation, good recovery and easy performance, which could be used for preparation of FOS from other sources, as well as other targeted compounds without UV absorbance.

  11. Preparation of inulin-type fructooligosaccharides using fast protein liquid chromatography coupled with refractive index detection.

    Science.gov (United States)

    Li, J; Cheong, K L; Zhao, J; Hu, D J; Chen, X Q; Qiao, C F; Zhang, Q W; Chen, Y W; Li, S P

    2013-09-20

    A fast protein liquid chromatography coupled with refractive index detection (FPLC-RID) method was firstly developed for preparation and purification of fructooligosaccharides with different degree of polymerization from burdock, Arctium lappa. After extraction with 60% ethanol and decolorization with MCI gel CHP20P, total fructooligosaccharides were purified on Bio-Gel P-2 column eluted with water at the flow rate of 0.3 ml/min, which was the optimized conditions. The obtained fructooligosaccharides with degree of polymerization of 3-9 were identified based on their methylation analysis, MS and NMR data. This method has the advantages of high automation, good recovery and easy performance, which could be used for preparation of FOS from other sources, as well as other targeted compounds without UV absorbance. PMID:23962565

  12. Amino acid incorporation into the protein of mitochondrial preparations from cerebral cortex and spinal cord.

    Science.gov (United States)

    Bachelard, H S

    1966-07-01

    1. Washed guinea-pig cerebral-cortex mitochondria incorporate [(14)C]leucine into their protein at a rate comparable with the rates reported for liver or heart mitochondria only if the mitochondria are separated from myelin and nerve endings by density-gradient centrifugation. 2. The non-mitochondrial components (myelin and nerve endings) of brain mitochondrial preparations incorporated [(14)C]leucine at a negligible rate. 3. The mitochondria do not require an exogenous supply of energy or a full supply of amino acids to support the process. 4. The incorporation rate was linear up to 2hr. aerobic incubation at 30 degrees and was inhibited by chloramphenicol, only slightly by actinomycin D and not by penicillin or pretreatment with ribonuclease. The observed incorporation is considered to be unlikely to be due to contaminating cytoplasmic ribosomes or bacteria. 5. The process was also studied in mitochondrial preparations from rabbit cerebral cortex and spinal cord.

  13. A novel method for preparation of HAMLET-like protein complexes.

    Science.gov (United States)

    Permyakov, Sergei E; Knyazeva, Ekaterina L; Leonteva, Marina V; Fadeev, Roman S; Chekanov, Aleksei V; Zhadan, Andrei P; Håkansson, Anders P; Akatov, Vladimir S; Permyakov, Eugene A

    2011-09-01

    Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA - OA samples. In this study we present a novel method for preparation of α-LA - OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA-OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca(2+) transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA - OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties.

  14. Preparation of mesoporous silica thin films by photocalcination method and their adsorption abilities for various proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Katsuya, E-mail: katsuya-kato@aist.go.jp [National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560 (Japan); Nakamura, Hitomi [National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560 (Japan); Yamauchi, Yoshihiro; Nakanishi, Kazuma; Tomita, Masahiro [Department of Chemistry for Materials, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8570 (Japan)

    2014-07-01

    Mesoporous silica (MPS) thin film biosensor platforms were established. MPS thin films were prepared from tetraethoxysilane (TEOS) via using sol–gel and spin-coating methods using a poly-(ethylene oxide)-block-poly-(propylene oxide)-block-poly-(ethylene oxide) triblock polymer, such as P123 ((EO){sub 20}(PO){sub 70}(EO){sub 20}) or F127 ((EO){sub 106}(PO){sub 70}(EO){sub 106}), as the structure-directing agent. The MPS thin film prepared using P123 as the mesoporous template and treated via vacuum ultraviolet (VUV) irradiation to remove the triblock copolymer had a more uniform pore array than that of the corresponding film prepared via thermal treatment. Protein adsorption and enzyme-linked immunosorbent assay (ELISA) on the synthesized MPS thin films were also investigated. VUV-irradiated MPS thin films adsorbed a smaller quantity of protein A than the thermally treated films; however, the human immunoglobulin G (IgG) binding efficiency was higher on the former. In addition, protein A–IgG specific binding on MPS thin films was achieved without using a blocking reagent; i.e., nonspecific adsorption was inhibited by the uniform pore arrays of the films. Furthermore, VUV-irradiated MPS thin films exhibited high sensitivity for ELISA testing, and cytochrome c adsorbed on the MPS thin films exhibited high catalytic activity and recyclability. These results suggest that MPS thin films are attractive platforms for the development of novel biosensors. - Highlights: • VUV-treated MPS thin films with removed polymer had uniform pore. • VUV-treated MPS thin films exhibited high sensitivity by ELISA. • Cytochrome c showed the catalytic activity and recyclability on synthesized films.

  15. Protein expression and preparation of polydonal antibody of AD-004 and study on its expression in the adrenal and testis

    Institute of Scientific and Technical Information of China (English)

    乔洁

    2006-01-01

    Objective To prepare rabbit antibody against mouse AD-004 by AD-004 expressed in the prokaryotic expression system and to identify its distribution in the testis and adrenal. Methods The full-length cDNA of mouse AD-004 was cloned into PET28 plasmid, and the protein was induced in E. coli BL21 bacteria by adding IPTG and then purified by Ni2+ -NTA column. The purified protein was used as an immunogene to prepare polyclonal

  16. PREPARATION AND CHARACTERIZATION OF SOLUBLE EGGSHELL MEMBRANE PROTEIN/CHITOSAN BLEND FILMS

    Institute of Scientific and Technical Information of China (English)

    Qing-lei Qi; Qiang Li; Jian-wei Lu; Zhao-xia Guo; Jian Yu

    2009-01-01

    Biopolymer chitosan was used to modify the mechanical properties of soluble eggshell membrane protein (SEP) films. The SEP/chitosan blend films were prepared by solution casting from 10% aqueous acetic acid. Tensile strength and elongation at break of the blend films increased with increasing amount of chitosan. Microphase separation was observed by field emission scanning electron microscopy, although interaction between the two components was revealed by FTIR. The biocompatibility of SEP/chitosan blend flints containing 10%-50% of chitosan, as demonstrated by cell culture of NIH3T3, was much better than that of pure chitosan.

  17. Water-dispersed bone morphogenetic protein nanospheres prepared by co-precipitation method

    Institute of Scientific and Technical Information of China (English)

    江兵兵; 高长有; 胡玲; 沈家骢

    2004-01-01

    A modified complex coacervation-co-precipitation method was used to prepare bone morphogenetic protein(BMP)-loaded nanospheres. Three natural polymers were used as packing materials to obtain nanoscale delivery device for BMP,in the presence of phosphatidylcholine functioning as stabilizer. Positively charged polysaccharide, N,N-diethylaminoethyl dex-tran (DEAE-dextran) tended to form stable, uniform and smaller size particles carrying BMP. Negatively charged bovine serumalbumin (BSA) induced precipitation of the produced BMP particles due to its weak interaction with BMP molecules, although itproduced nanosized BMP spheres. While collagen, a weakly positively charged protein shaped larger particles due to the stronginteraction among themselves. A mechanism of co-precipitation process was also deduced to depict the formation of stablenanospheres.

  18. Water-dispersed bone morphogenetic protein nanospheres prepared by co-precipitation method

    Institute of Scientific and Technical Information of China (English)

    江兵兵; 高长有; 胡玲; 沈家骢

    2004-01-01

    A modified complex coacervation-co-precipitation method was used to prepare bone morphogenetic protein (BMP)-loaded nanospheres. Three natural polymers were used as packing materials to obtain nanoscale delivery device for BMP,in the presence of phosphatidylcholine functioning as stabilizer. Positively charged polysaccharide, N,N-diethylaminoethyl dex-tran (DEAE-dextran) tended to form stable, uniform and smaller size particles carrying BMP. Negatively charged bovine serum albumin (BSA) induced precipitation of the produced BMP particles due to its weak interaction with BMP molecules, although it produced nanosized BMP spheres. While collagen, a weakly positively charged protein shaped larger particles due to the strong interaction among themselves. A mechanism of co-precipitation process was also deduced to depict the formation of stable nanospheres.

  19. A new process for preparation of soybean protein concentrate with hexane-aqueous ethanol mixed solvents.

    Science.gov (United States)

    Zhang, Wei-Nong; Liu, Da-Chuan

    2005-01-01

    A new process for the preparation of soybean protein concentrate (SPC) by directly extracting full-fat soy flour with a mixture of hexane and aqueous ethanol was established. Compared with conventional methods, it has some advantages, such as saving energy and reducing protein denaturation caused by heat action during solvent recovery, because this process saves one step of solvent recovery. The effects of aqueous ethanol concentration and the mixure ratio (hexane to ethanol) on the degree of protein denaturation and product quality were investigated, on the basis of which the orthogonal tests were performed. The optimum technical parameters were obtained by analyzing the results of the orthogonal tests with statistical methods. We found that SPC can be obtained by extracting full-fat soy flour under the following conditions: mixture ratio hexane: 90% ethanol, 9:1, v/v; extraction temperature, 45 degrees C; ratio of solid to solvents, (1:2 w/v); and 5 repeated extractions (15 min each time). The results of quality analysis showed that solubility of the product was improved significantly [nitrogen solubility index (NSI) 46.6%] compared with that for ethanol washing of protein concentrate (NSI 8.7%). PMID:16152943

  20. Prokaryotic expression of soluble Arabidopsis protein AtERF1 and preparation of its polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    ZHANG Yu

    2013-08-01

    Full Text Available AtERF1 encodes a member of the ERF subfamily B-3 of ERF/AP2 transcription factor family.It has been demonstrated almost every member of the B3 subgroup of AP2/ERF genes is involved in defense responses in Arabidopsis.Codon usage within a gene is a critical determinant of achievable protein expression level in E.coli. Gene optimization,therefore,is an effective method for synthetic genes with the aim of enhancing soluble expression,particular in heterologous hosts.In this paper,the AtERF1 protein of Arabidopsis thaliana was expressed in Escherichia coli using its optimized DNA sequence for E.coli. and yielded high level of soluble AtERF-1 protein in recombinant E.coli. The AtERF1 protein was used as an antigen to immune rabbits and obtains high titer antibodies.The immunological specificity of the polyclonal antibodies to AtERF1 was confirmed by Western blot assay.The prepared antibody in this work would facilitate the further functional investigation of AtERF1 in biochemical levels in Arabidopsis anther development.

  1. Preparation and recognition of surface molecularly imprinted core-shell microbeads for protein in aqueous solutions

    International Nuclear Information System (INIS)

    In this paper, a surface molecular imprinting technique was reported for preparing core-shell microbeads of protein imprinting, and bovine hemoglobin or bovine serum albumin were used as model proteins for studying the imprinted core-shell microbeads. 3-Aminophenylboronic acid (APBA) was polymerized onto the surface of polystyrene microbead in the presence of the protein templates to create protein-imprinted core-shell microbeads. The various samples were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) methods. The effect of pH on rebinding of the template hemoglobin, the specific binding and selective recognition were studied for the imprinted microbeads. The results show that the bovine hemoglobin-imprinted core-shell microbeads were successfully created. The shell was a sort of imprinted thin films with porous structure and larger surface areas. The imprinted microbeads have good selectivity for templates and high stability. Due to the recognition sites locating at or closing to the surface, these imprinted microbeads have good property of mass-transport. Unfortunately, the imprint technology was not successfully applied to imprinting bovine serum albumin (BSA).

  2. Foams prepared from whey protein isolate and egg white protein: 2. Changes associated with angel food cake functionality.

    Science.gov (United States)

    Berry, Tristan K; Yang, Xin; Foegeding, E Allen

    2009-06-01

    The effects of sucrose on the physical properties and thermal stability of foams prepared from 10% (w/v) protein solutions of whey protein isolate (WPI), egg white protein (EWP), and their combinations (WPI/EWP) were investigated in wet foams and angel food cakes. Incorporation of 12.8 (w/v) sucrose increased EWP foam stability (drainage 1/2 life) but had little effect on the stability of WPI and WPI/EWP foams. Increased stability was not due to viscosity alone. Sucrose increased interfacial elasticity (E ') of EWP and decreased E' of WPI and WPI/EWP combinations, suggesting that altered interfacial properties increased stability in EWP foams. Although 25% WPI/75% EWP cakes had similar volumes as EWP cakes, cakes containing WPI had larger air cells. Changes during heating showed that EWP foams had network formation starting at 45 degrees C, which was not observed in WPI and WPI/EWP foams. Moreover, in batters, which are foams with additional sugar and flour, a stable foam network was observed from 25 to 85 degrees C for batters made from EWP foams. Batters containing WPI or WPI/EWP mixtures showed signs of destabilization starting at 25 degrees C. These results show that sucrose greatly improved the stability of wet EWP foams and that EWP foams form network structures that remain stable during heating. In contrast, sucrose had minimal effects on stability of WPI and WPI/EWP wet foams, and batters containing these foams showed destabilization prior to heating. Therefore, destabilization processes occurring in the wet foams and during baking account for differences in angel food cake quality. PMID:19646042

  3. Preparation of polyacrylamide based monolith with immobilized pH gradient and its application for protein analysis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Monolithic materials were prepared in capillaries by in situ polymerization of acrylamide, glycidyl methacrylate and N,N′-memylenebisacrylamid in the presence of trinary porogens, including 1,4-butanediol, dodecanol and dimethyl sulphoxide. With Ampholine immobilized on the monolith by chemical bonding according to their pIs, the monolithic immobilized pH gradient (M-IPG) was prepared, and applied to the separation of four standard proteins. Compared with polyacrylate based M-IPG, the hydrophilicity of the new material was improved. It could not only avoid the adsorption of proteins, but also make the synthesized procedure simple, which showed great potential in the analysis of proteins.

  4. Preparation of polyacrylamide based monolith with immobilized pH gradient and its application for protein analysis

    Institute of Scientific and Technical Information of China (English)

    ZHU GuiJie; ZHANG WeiBing; ZHANG LiHua; LIANG Zhen; ZHANG YuKui

    2007-01-01

    Monolithic materials were prepared in capillaries by in situ polymerization of acrylamide, glycidyl methacrylate and N,N'-memylenebisacrylamid in the presence of trinary porogens, including 1,4-butanediol, dodecanol and dimethyl sulphoxide. With Ampholine immobilized on the monolith by chemical bonding according to their pIs, the monolithic immobilized pH gradient (M-IPG) was prepared, and applied to the separation of four standard proteins. Compared with polyacrylate based M-IPG, the hydrophilicity of the new material was improved. It could not only avoid the adsorption of proteins, but also make the synthesized procedure simple, which showed great potential in the analysis of proteins.

  5. AMP-activated protein kinase phosphorylation in brain is dependent on method of sacrifice and tissue preparation

    OpenAIRE

    Scharf, Matthew T.; Mackiewicz, Miroslaw; Naidoo, Nirinjini; O'Callaghan, James P.; Pack, Allan I.

    2007-01-01

    AMP-activated protein kinase is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α-AMP-activated protein kinase phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMP-activated protein kinase phosphorylation in the mouse brain, we compared different methods of sacrifice and tissue preparation. We found that fre...

  6. Preparation of Monoclonal Antibodies Against Prion Proteins With Full-length Hamster PrP

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPSc. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.

  7. New insights into respirable protein powder preparation using a nano spray dryer.

    Science.gov (United States)

    Bürki, K; Jeon, I; Arpagaus, C; Betz, G

    2011-04-15

    In this study the Nano Spray Dryer B-90 (BÜCHI Labortechnik AG, Flawil, Switzerland) was evaluated with regard to the drying of proteins and the preparation of respirable powders in the size range of 1-5 μm. β-galactosidase was chosen as a model protein and trehalose was added as a stabilizer. The influence of inlet temperature, hole size of the spray cap membrane and ethanol concentration in the spray solution was studied using a 3³ full factorial design. The investigated responses were enzyme activity, particle size, span, yield and shelf life. Furthermore, the particle morphology was examined. The inlet temperature as well as the interaction of inlet temperature and spray cap size significantly influenced the enzyme activity. Full activity was retained with the optimized process. The particle size was affected by the hole size of the spray cap membrane and the ethanol content. The smallest cap led to a monodisperse particle size distribution and the greatest yield of particles of respirable size. Higher product recovery was achieved with lower inlet temperatures, higher ethanol contents and smaller cap sizes. Particle morphology differed depending on the cap size. The protein exhibited higher storage stability when spray dried without ethanol and when a larger spray cap size was used. PMID:21335078

  8. Expression Characterization and Preparation of Human Amyloid Precursor Protein in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    XU Guang-wei; WANG Jia-peng; HUANG Xue-mei; ZHANG Ying-jiu

    2009-01-01

    To analyze whether expressed amyloid precursor protein(APP) existed in hydrophilic(cytoplasmid) or hy-drophobic(lipid bilayer) environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coli. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hy-drophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni2. agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coil cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.

  9. Numerical prediction of micro-channel LD heat sink operated with antifreeze based on CFD method

    Science.gov (United States)

    Liu, Gang; Liu, Yang; Wang, Chao; Wang, Wentao; Wang, Gang; Tang, Xiaojun

    2014-12-01

    To theoretically study the feasibility of antifreeze coolants applied as cooling fluids for high power LD heat sink, detailed Computational Fluid Dynamics (CFD) analysis of liquid cooled micro-channels heat sinks is presented. The performance operated with antifreeze coolant (ethylene glycol aqueous solution) compared with pure water are numerical calculated for the heat sinks with the same micro-channels structures. The maximum thermal resistance, total pressure loss (flow resistance), thermal resistance vs. flow-rate, and pressure loss vs. flow-rate etc. characteristics are numerical calculated. The results indicate that the type and temperature of coolants plays an important role on the performance of heat sinks. The whole thermal resistance and pressure loss of heat sinks increase significantly with antifreeze coolants compared with pure water mainly due to its relatively lower thermal conductivity and higher fluid viscosity. The thermal resistance and pressure loss are functions of the flow rate and operation temperature. Increasing of the coolant flow rate can reduce the thermal resistance of heat sinks; meanwhile increase the pressure loss significantly. The thermal resistance tends to a limit with increasing flow rate, while the pressure loss tends to increase exponentially with increasing flow rate. Low operation temperature chiefly increases the pressure loss rather than thermal resistance due to the remarkable increasing of fluid viscosity. The actual working point of the cooling circulation system can be determined on the basis of the pressure drop vs. flow rate curve for the micro-channel heat sink and that for the circulation system. In the same system, if the type or/and temperature of the coolant is changed, the working point is accordingly influenced, that is, working flow rate and pressure is changed simultaneously, due to which the heat sink performance is influenced. According to the numerical simulation results, if ethylene glycol aqueous

  10. Honeycomb-patterned films of polystyrene/poly(ethylene glycol): Preparation, surface aggregation and protein adsorption

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Highly ordered honeycomb-patterned polystyrene (PS)/poly(ethylene glycol) (PEG) films were prepared by a water-assisted method using an improved setup, which facilitated the formation of films with higher regularity, better reproducibility, and larger area of honeycomb structures. Surface aggregation of hydrophilic PEG and adsorption of bovine serum albumin (BSA) on the honeycomb-patterned films were investigated. Field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) were used to observe the surface morphologies of the films before and after being rinsed with water. As confirmed by the FESEM images and the AFM phase images, PEG was enriched in the pores and could be gradually removed by water. The adsorption of fluorescence-labeled BSA on the films was studied in visual form using laser scanning confocal microscopy. Results clearly demonstrated that the protein-resistant PEG was selectively enriched in the pores. This water-assisted method may be a latent tool to prepare honeycomb-patterned biofunctional surfaces.

  11. Identification of the major T-cell antigens present in the Brucella melitensis B115 protein preparation, Brucellergene OCB.

    Science.gov (United States)

    Denoel, P A; Vo, T K; Weynants, V E; Tibor, A; Gilson, D; Zygmunt, M S; Limet, J N; Letesson, J J

    1997-09-01

    Brucellergene is a commercial allergen prepared from Brucella melitensis strain B115 and containing at least 20 cytoplasmic proteins. These proteins were separated by SDS-PAGE. The unstained gel was divided into 18 fractions and proteins were eluted from the gel fractions. The capacity of the separated proteins to elicit delayed-type hypersensitivity (DTH) in infected guinea-pigs or to induce the production of interferon-gamma (IFN-gamma) by blood cells from infected cattle was evaluated. The biological activity of the corresponding protein fractions blotted on to nitrocellulose was measured in a lymphocyte blastogenesis assay. Among the 18 fractions tested, two-spanning the mol. wt ranges 17-22 (fraction 8) and 35-42-kDa (fraction 17)-showed the maximum biological activity in the three tests. These fractions contain two antigens, the Brucella bacterioferritin (BFR) and P39 proteins. Both proteins are good candidates for the detection of cellular immunity to Brucella. PMID:9291893

  12. A pull-down method with a biotinylated bait protein prepared by cell-free translation using a puromycin linker.

    Science.gov (United States)

    Mochizuki, Yuki; Kohno, Fumiaki; Nishigaki, Koichi; Nemoto, Naoto

    2013-03-01

    In this paper, we demonstrate a novel pull-down method that dramatically reduces the cost and preparation time of a bait protein by cell-free translation with a puromycin linker. With the C-terminus of the bait protein linked to biotin through a puromycin molecule after the translation reaction and subsequent mRNA degradation by RNase, the prey protein was easily pulled down by streptavidin-coated magnetic beads in a test tube. Three fluorescent prey protein types were tested and confirmed by gel electrophoresis to be pulled down easily and rapidly, depending on their affinity.

  13. Preparation of silica coated cobalt ferrite magnetic nanoparticles for the purification of histidine-tagged proteins

    Science.gov (United States)

    Aygar, Gülfem; Kaya, Murat; Özkan, Necati; Kocabıyık, Semra; Volkan, Mürvet

    2015-12-01

    Surface modified cobalt ferrite (CoFe2O4) nanoparticles containing Ni-NTA affinity group were synthesized and used for the separation of histidine tag proteins from the complex matrices through the use of imidazole side chains of histidine molecules. Firstly, CoFe2O4 nanoparticles with a narrow size distribution were prepared in an aqueous solution using the controlled co-precipitation method. In order to obtain small CoFe2O4 agglomerates, oleic acid and sodium chloride were used as dispersants. The CoFe2O4 particles were coated with silica and subsequently the surface of these silica coated particles (SiO2-CoFe2O4) was modified by amine (NH2) groups in order to add further functional groups on the silica shell. Then, carboxyl (-COOH) functional groups were added to the SiO2-CoFe2O4 magnetic nanoparticles through the NH2 groups. After that Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA) was attached to carboxyl ends of the structure. Finally, the surface modified nanoparticles were labeled with nickel (Ni) (II) ions. Furthermore, the modified SiO2-CoFe2O4 magnetic nanoparticles were utilized as a new system that allows purification of the N-terminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3.

  14. Raman spectroscopy of antifreeze glycoproteins and their interaction with various substrates

    Science.gov (United States)

    Cui, Y.; Turner, G.; Alexander, V.; Smith, I.; Sease, A.; Guo, M.; Burger, A.; Morgan, S.; Yeh, Yin

    2004-11-01

    Micro-Raman spectra of a mixture of antifreeze glycoproteins (AFGP) 6, 7 and 8 have been measured in the range of 100 - 4500 cm-1 with He-Ne laser excitation. The spectra were obtained for both bulk AFGP and films of AFGP deposited on various substrates. New vibrational peaks have been observed for films which are not present in the spectra of the bulk samples. The results will be presented and mechanisms of interaction between the AFGP molecule and substrates will be proposed. The assignment of new peaks and the effects of the water presence will also be discussed. Research supported by the NSF Center for Biophotonics, managed by U.C. Davis, CA No. PHY 0120999, NSF Research Experiences for Undergraduates DMR-0139180 and by the MBRS program through NIH/NIGMS grant 1S06-GM62813-01.

  15. Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coli BL21,the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG),and it was analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyclonal antibody,wi...

  16. Design and Preparation of Nano-Lignin Peroxidase (NanoLiP) by Protein Block Copolymerization Approach

    OpenAIRE

    Turgay Tay; Ender Köse; Rüstem Keçili; Rıdvan Say

    2016-01-01

    This study describes the preparation of nanoprotein particles having lignin peroxidase (LiP) using a photosensitive microemulsion polymerization technique. The protein-based nano block polymer was synthesized by cross-linking of ligninase enzyme with ruthenium-based aminoacid monomers. This type polymerization process brought stability in different reaction conditions, reusability and functionality to the protein-based nano block polymer system when compared the traditional methods. After cha...

  17. Home-prepared soymilk: Potential to alleviate protein-energy malnutrition in low-income rural communities in South Africa?

    OpenAIRE

    Gabriel N. Medoua; Abdulkadir A. Egal; Sara S. Duvenage; Wilna H Oldewage-Theron

    2013-01-01

    Research findings reported pronounced protein and some energy shortfalls for school-aged children and female caregivers in rural communities in Qwa-Qwa, South Africa. The household gardening project was expanded to include soy cultivation. Subsequently, a process was developed for home-preparation of soymilk to support macronutrient consumption. The limited explorative experimental approach included chemical analysis for total protein (Kjeldahl digestion, spectrophotometric determination), to...

  18. Effect of green tea or rosemary extract on protein oxidation in Bologna type sausages prepared from oxidatively stressed pork.

    Science.gov (United States)

    Jongberg, Sisse; Tørngren, Mari Ann; Gunvig, Annemarie; Skibsted, Leif H; Lund, Marianne N

    2013-03-01

    Bologna type sausages were prepared from oxidatively stressed pork (UV-irradiation, 48 h, 5 °C) using a traditional recipe (control) or the same recipe but added green tea extract (500 ppm total phenolic compounds) or rosemary extract (400 ppm total phenolic compounds). Green tea and rosemary extracts protected against formation of TBARS and protein carbonyls. On the contrary, increased thiol loss and a distinct loss of myosin heavy chain and actin due to polymerization by reducible bonds as determined by SDS-page were found by addition of green tea extract. The enhanced protein polymerization was ascribed to the reaction between quinone compounds from the plant extracts and protein thiol groups to yield phenol-mediated protein polymerization. Analysis by ESR spectroscopy revealed increased radical intensities in sausages added plant extracts, which was ascribed to originate from protein-bound phenoxyl radicals, which may protect against other oxidatively induced protein modifications. PMID:23273462

  19. Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.

    Science.gov (United States)

    Ishchenko, Andrii; Cherezov, Vadim; Liu, Wei

    2016-09-20

    Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include

  20. Preparation of salted meat products, e.g. cured bacon - by injecting liquid comprising meat proteins hydrolysed with enzymes

    DEFF Research Database (Denmark)

    1997-01-01

    Preparation of salted meat products comprises the following:(1) meat is chopped into fine pieces and mixed with water to form a slurry; (2) enzymes hydrolyse proteins in the meat; (3) adding a culture to the resulting medium, which comprises short peptide chains or amino acids; (4) forming...

  1. Expression and purification of the nucleocapsid protein of Schmallenberg virus, and preparation and characterization of a monoclonal antibody against this protein.

    Science.gov (United States)

    Zhang, Yongning; Wu, Shaoqiang; Wang, Jianchang; Wernike, Kerstin; Lv, Jizhou; Feng, Chunyan; Zhang, Jihong; Wang, Caixia; Deng, Junhua; Yuan, Xiangfen; Lin, Xiangmei

    2013-11-01

    Schmallenberg virus (SBV) is a novel orthobunyavirus that primarily infects ruminants such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV has been shown to be an ideal target antigen for serological detection. To prepare a monoclonal antibody (mAb) against the N protein, the full-length coding sequence of the SBV N gene was cloned into pET-28a-c(+) and pMAL-c5X vectors to generate two recombinant plasmids, which were expressed in Escherichia coli BL21 as histidine (His)-tagged (His-SBV-N) and maltose-binding protein (MBP)-tagged (MBP-SBV-N) fusion proteins, respectively. After affinity purification of His-SBV-N with Ni-NTA agarose and MBP-SBV-N with amylose resin, His-SBV-N was used to immunize BALB/c mice, while MBP-SBV-N was utilized to screen for mAb-secreting hybridomas. Six hybridoma cell lines stably secreting mAbs against N were obtained. Clone 2C8 was selected for further study because of its rapid growth characteristics in vitro and good reactivity with recombinant SBV N proteins in enzyme-linked immunosorbent assays. The epitope recognized by 2C8 is located at amino acids 51-76 of the SBV N protein. Western blot analyses showed that 2C8 reacts with both recombinant SBV N proteins and SBV isolates. It is also cross-reactive with the N proteins of genetically related Shamonda, Douglas and Akabane viruses, but not with the Rift Valley fever virus N protein. The successful preparation of recombinant N proteins and mAbs provides valuable materials that can be used in the serological diagnosis of SBV. PMID:23988909

  2. Preparation and some functional properties of rice bran protein concentrate at different degree of hydrolysis using bromelain and alkaline extraction.

    Science.gov (United States)

    Apinunjarupong, Suthep; Lapnirun, Surawoot; Theerakulkait, Chockchai

    2009-01-01

    Rice bran protein was extracted by using defatted rice bran and water at 1:6 (w/w) and 6% of bromelain at pH 9.0, 50 degrees C, 500 rpm for 15 and 30 mins. The degree of hydrolysis (DH) of rice bran protein extract (RBPE) was 19 and 36.5%, respectively, and their nitrogen solubility was higher than the controls. Rice bran protein concentrate (RBPC) was prepared by spray drying. Emulsion activity of RBPC produced from 19% DH RBPE was increased while emulsion stability index was not significantly different from the control. Foam capacity and rehydration ability of RBPC were greater than the control. PMID:19291580

  3. Design and Preparation of Nano-Lignin Peroxidase (NanoLiP by Protein Block Copolymerization Approach

    Directory of Open Access Journals (Sweden)

    Turgay Tay

    2016-06-01

    Full Text Available This study describes the preparation of nanoprotein particles having lignin peroxidase (LiP using a photosensitive microemulsion polymerization technique. The protein-based nano block polymer was synthesized by cross-linking of ligninase enzyme with ruthenium-based aminoacid monomers. This type polymerization process brought stability in different reaction conditions, reusability and functionality to the protein-based nano block polymer system when compared the traditional methods. After characterization of the prepared LiP copolymer nanoparticles, enzymatic activity studies of the nanoenzymes were carried out using tetramethylbenzidine (TMB as the substrate. The parameters such as pH, temperature and initial enzyme concentration that affect the activity, were investigated by using prepared nanoLip particles and compared to free LiP. The reusability of the nano-LiP particles was also investigated and the obtained results showed that the nano-LiP particles exhibited admirable potential as a reusable catalyst.

  4. Preparation and properties of fast temperature-responsive soy protein/PNIPAAm IPN hydrogels

    Directory of Open Access Journals (Sweden)

    Liu Yong

    2014-01-01

    Full Text Available The interpenetrating polymer network of fast temperature-responsive hydrogels based on soy protein and poly(N-isopropylacrylamide were successfully prepared using the sodium bicarbonate (NaHCO3 solutions as the reaction medium. The structure and properties of the hydrogels were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, differential scanning calorimetry and thermal gravimetric analysis. The swelling and deswelling kinetics were also investigated in detail. The results have shown that the proposed hydrogels had high porous structure, good miscibility and thermal stability, and fast temperature responsivity. The presence of NaHCO3 had little effect on the volume phase transition temperature (VPTT of the hydrogels, and the VPTTs were at about 32°C. Compared with the traditional hydrogels, the proposed hydrogels had much faster swelling and deswelling rate. The swelling mechanism of the hydrogels was the non-Fickian diffusion. This fast temperature-responsive hydrogels may have potential applications in the field of biomedical materials.

  5. Mechanism of antifreeze proteins action, based on Hierarchic theory of water and new ''clusterphilic'' interaction

    CERN Document Server

    Kaivarainen, A

    2001-01-01

    A basically new Hierarchic theory, general for solids and liquids (Kaivarainen, 2001, 2000, 1995, 1992), has been briefly described and illustrated by computer simulations on examples of water and ice. Full description of theory and its numerous applications are presented in series of articles at the arXiv of Los-Alamos (see http://arXiv.org/abs/physics/0102086). New clusterphilic interactions, intermediate between hydrophilic and hydrphobic, are introduced. They can be subdivided into: intramolecular - when water cluster is localized in the ''open'' states of big interdomain or intersubunit cavities and intermolecular clusterphilic interactions. Intermolecular clusterphilic interactions can be induced by very different macromolecules. The latter displays themselves in bordering of water cluster by macromolecules and forming so-called ''clustrons''. Clusterphilic interactions can play an important role in self-organization of biosystems, especially multiglobular allosteric enzymes, microtubules and the actin ...

  6. In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins

    Directory of Open Access Journals (Sweden)

    Buhrman Jason S

    2012-09-01

    Full Text Available Abstract Background Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins. Results Glutathione was conjugated to low molecular weight poly(ethylene glycol diacrylate (PEGDA via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH homogenates, we were able to purify a glutathione S-transferase (GST green fluorescent protein (GFP fusion protein (GST-GFP from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads. Conclusions GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.

  7. Preparation of recombinant human bone morphogenetic protein-2 loaded dextran-based microspheres and their characteristics

    Institute of Scientific and Technical Information of China (English)

    Fa-ming CHEN; Zhi-fen WU; Qin-tao WANG; Hong WU; Yong-jie ZHANG; Xin NIE; Yan JIN

    2005-01-01

    Aim: To prepare new pharmaceutical forms with sustained delivery properties of recombinant human bone morphogenetic protein-2 (rhBMP2) for tissue engineering and guided tissue regeneration (GTR) use. Methods: rhBMP2-1oaded dextranbased hydrogel microspheres (rhBMP2-MPs), which aimed to keep rhBMP2 bioactivity and to achieve long-term sustained release of rhBMP2, were prepared by double-phase emulsified condensation polymerization. The physical, chemical performances and biological characteristics of those microspheres were studied both in vitro and in vivo. Results: The microspheres' average diameter was 30.33±4.32 μm with 75.4% ranging from 20 μm to 40 μm and the drug loading and encapsulation efficiency were 7.82% and 82.25%, respectively. The rhBMP2-releasing profiles in vitro showed that rhBMP2 release could be maintained more than 10 d. The rhBMP2-MPs, with good swelling and biodegradation behavior,could be kept for 6 months at below 4 ℃ without significant characteristic change or bioactivity loss. Cytology studies showed that rhBMP2-MPs could promote the proliferation of periodontal ligament cells (PDLCs) approximately 10 d, while the bioactivity of concentrated rhBMP2 solution could keep no more than 3 d.Scanning electron microscope showed that rhBMP2-MPs could be enchased into the porous structure of calcium phosphate ceremic (CPC) and the eugonic growth of PDLCs in CPC/rhBMP2-MPs scaffolds. Animal experiments indicated that using CPC/rhBMP2-MPs scaffolds could gain more periodontal tissue regeneration than using rhBMP2 compound firsthand with CPC (CPC/rhBMP2). Conclusion:By encapsulating rhBMP2 into dextran-based microspheres, a small quantity of rhBMP2 could achieve equivalent effects to the concentrated rhBMP2 solution and at the same time, could prolong rhBMP2 retention both in vitro and in vivo.

  8. Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius

    Directory of Open Access Journals (Sweden)

    Zheng Xiaojuan

    2009-10-01

    Full Text Available Abstract Background Two-dimensional gel electrophoresis (2-DE is a powerful method to study protein expression and function in living organisms and diseases. This technique, however, has not been applied to avian bursa of Fabricius (BF, a central immune organ. Here, optimized 2-DE sample preparation methodologies were constructed for the chicken BF tissue. Using the optimized protocol, we performed further 2-DE analysis on a soluble protein extract from the BF of chickens infected with virulent avibirnavirus. To demonstrate the quality of the extracted proteins, several differentially expressed protein spots selected were cut from 2-DE gels and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS. Results An extraction buffer containing 7 M urea, 2 M thiourea, 2% (w/v 3-[(3-cholamidopropyl-dimethylammonio]-1-propanesulfonate (CHAPS, 50 mM dithiothreitol (DTT, 0.2% Bio-Lyte 3/10, 1 mM phenylmethylsulfonyl fluoride (PMSF, 20 U/ml Deoxyribonuclease I (DNase I, and 0.25 mg/ml Ribonuclease A (RNase A, combined with sonication and vortex, yielded the best 2-DE data. Relative to non-frozen immobilized pH gradient (IPG strips, frozen IPG strips did not result in significant changes in the 2-DE patterns after isoelectric focusing (IEF. When the optimized protocol was used to analyze the spleen and thymus, as well as avibirnavirus-infected bursa, high quality 2-DE protein expression profiles were obtained. 2-DE maps of BF of chickens infected with virulent avibirnavirus were visibly different and many differentially expressed proteins were found. Conclusion These results showed that method C, in concert extraction buffer IV, was the most favorable for preparing samples for IEF and subsequent protein separation and yielded the best quality 2-DE patterns. The optimized protocol is a useful sample preparation method for comparative proteomics analysis of chicken BF tissues.

  9. Protein-loaded microspheres prepared by sequential adsorption of dextran sulphate and protamine on melamine formaldehyde core.

    Science.gov (United States)

    Balabushevich, Nadezda G; Larionova, Natalia I

    2009-11-01

    Polyelectrolyte multilayer microspheres were fabricated by layer-by-layer self-assembly of a dextran sulphate and a protamine on melamine formaldehyde cores, followed by the partial decomposition of the core. Effects of pH on the encapsulation of proteins and enzymes with different physico-chemical properties (insulin, aprotinin, peroxidase, glucose oxidase (GOD), catalase (Cat)) in the prepared microspheres were then studied. This method of protein encapsulation demonstrated a high loading capacity and efficiency. The protein incorporation and release was regulated by the pH of the solution. Encapsulated enzymes retained a high specific activity depending on the amount of protein incorporated. Bienzyme system GOD/Cat immobilized in the microspheres was suitable for the glucose content assay.

  10. Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

    Science.gov (United States)

    Chow-Shi-Yée, Mélanie; Briard, Jennie G; Grondin, Mélanie; Averill-Bates, Diana A; Ben, Robert N; Ouellet, François

    2016-05-01

    Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells.

  11. Content of amino acids and the quality of protein in Brussels sprouts, both raw and prepared for consumption

    Energy Technology Data Exchange (ETDEWEB)

    Lisiewska, Zofia; Slupski, Jacek; Skoczen-Slupska, Radoslawa; Kmiecik, Waldemar [Department of Raw Materials and Processing of Fruit and Vegetables, Agricultural University of Krakow, Balicka 122, 30-149 Krakow (Poland)

    2009-03-15

    The aim of the investigation was to evaluate the content of amino acids and the quality of protein in Brussels sprouts. The investigation included the raw material, cooked sample and two types of frozen product stored at -20 C for 12 months and then prepared for consumption. The frozen products investigated were obtained using the traditional method (blanching before freezing) and the modified method (cooking before freezing, then defrosting and heating in microwave oven after refrigerated storage) of the ready-to-eat type. Brussels sprouts, both fresh and prepared for consumption, were a good source of protein and amino acids. Proline and glutamic acid were dominating; leucine and tyrosine with phenylalanine were limiting amino acids. The product obtained by modified method contained 16% less amino acids in 16 g N than the raw material and 14% less than the raw material after cooking, and also 10% lower than that of the traditionally obtained product. (author)

  12. Home-prepared soymilk: Potential to alleviate protein-energy malnutrition in low-income rural communities in South Africa?

    Directory of Open Access Journals (Sweden)

    Gabriel N. Medoua

    2013-10-01

    Full Text Available Research findings reported pronounced protein and some energy shortfalls for school-aged children and female caregivers in rural communities in Qwa-Qwa, South Africa. The household gardening project was expanded to include soy cultivation. Subsequently, a process was developed for home-preparation of soymilk to support macronutrient consumption. The limited explorative experimental approach included chemical analysis for total protein (Kjeldahl digestion, spectrophotometric determination, total carbohydrate (Anthone method and total lipid content (extraction, Gravimetric method, separation. Total energy content was calculated. All results were benchmarked against equivalents. Duplicate analysis of samples, respectively prepared from 1:2 (n= 6 and 1:4 (n = 4 volume ratios of rehydrated minced soybeans : water for cooking of soy mash, indicated statistically-significant differences for reported nutrients (p ≤ 0.05. Comparison between sourced commercial soymilk products for drinking indicated no statistical differences (p > 0.05. Although statistically-significant shortfalls were indicated for nearly all such values for home-prepared soymilk (1:4 ratio against industrial ‘SoyCow’ soymilk and values reported in the South African database for standardised nutrient composition of food (p ≤ 0.05, a much-needed contribution will be made to protein (and energy intake through consumption of the product. More efficient extraction (possibly double mincing of rehydrated soybeans and more efficient pressing of cooked soy mash should be explored, followed by an intervention study to evaluate the impact of daily consumption of home-prepared soymilk on the nutritional status of children in low-income communities. The development of recipes to promote the inclusion of undissolved fibre from the soymilk extraction process (okara in dishes prepared at household level, such as bread, is recommended.

  13. Effect of antifreeze glycoprotein 8 supplementation during vitrification on the developmental competence of bovine oocytes.

    Science.gov (United States)

    Liang, Shuang; Yuan, Bao; Kwon, Jeong-Woo; Ahn, Mija; Cui, Xiang-Shun; Bang, Jeong Kyu; Kim, Nam-Hyung

    2016-07-15

    The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury. PMID:26948296

  14. Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin

    OpenAIRE

    Mackin, Robert B.

    2014-01-01

    The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly ...

  15. Protein adsorption on gradient surfaces on polyethylene prepared in a shielded gas plasma

    NARCIS (Netherlands)

    Spijker, Hendrikje; Bos, Roelof; van Oeveren, Willem; de Vries, Jacob; Busscher, Hendrik

    1999-01-01

    In this study, a new and simple method is described to prepare wettability gradients on polymers by means of glow discharge in a partly shielded argon plasma. The surface characteristics of thus prepared gradients on low density polyethylene were determined by contact angle measurements and electron

  16. Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio [Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias s/n, 15782 Santiago de Compostela (Spain); Bermejo-Barrera, Pilar [Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias s/n, 15782 Santiago de Compostela (Spain)], E-mail: pbermejo@usc.es; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad [Department of Analytical Chemistry, Faculty of Sciences, University of A Coruna, Campus da Zapateira s/n, 15071 A Coruna (Spain); Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario [Department of Analytical Chemistry, Faculty of Sciences, University of A Coruna, Campus da Zapateira s/n, 15071 A Coruna (Spain); University Institute of Environment, University of A Coruna, Pazo de Longora, Lians, 15179 Oleiros (Spain)

    2007-11-05

    The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices.

  17. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    Science.gov (United States)

    Yuan, Huihui; Qian, Bin; Zhang, Wei; Lan, Minbo

    2016-02-01

    An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU-PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU-PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU-PVP (6.0 h) film reduced greatly to 0.08 μg/cm2, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  18. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans.

    Science.gov (United States)

    Gao, Xiong-Zhuo; Li, Lan-Fen; Su, Xiao-Dong; Zhao, XiaoJun; Liang, Yu-He

    2007-10-01

    The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni2+-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 A resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 A, beta = 98.82 degrees.

  19. Preparation of gluten-free bread using a meso-structured whey protein particle system

    NARCIS (Netherlands)

    Riemsdijk, van L.E.; Goot, van der A.J.; Hamer, R.J.; Boom, R.M.

    2011-01-01

    This article presents a novel method for making gluten-free bread using mesoscopically structured whey protein. The use of the meso-structured protein is based on the hypothesis that the gluten structure present in a developed wheat dough features a particle structure on a mesoscopic length scale (1

  20. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    International Nuclear Information System (INIS)

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni2+-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°

  1. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiong-Zhuo [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Li, Lan-Fen; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Zhao, XiaoJun, E-mail: zhaoxj@scu.edu.cn [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); Liang, Yu-He, E-mail: zhaoxj@scu.edu.cn [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China)

    2007-10-01

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°.

  2. Inhibition of influenza virus protein synthesis by a plant preparation from Geranium sanguineum L

    International Nuclear Information System (INIS)

    A polyphenolyc complex (PC) with antiviral properties has been isolated from the Bulgarian medicinal plant Geranium sanguineum L A study was undertaken to investigate the effect of PC on virus-specific protein synthesis in influenza virus-infected cells. The expression of viral glycoproteins on the surface of chick embryo fibroblasts infected with virus A/FPV, strain Rostock (H7N1) was suppressed. Virus protein synthesis was selectively inhibited as shown by SDS polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins and proteins immunoprecipitated with monoclonal antibodies. The inhibitory effect was dose-dependent and better pronounced when PC was applied after virus infection. Two variants of influenza virus FPV/Rostock with reduced drug susceptibility were selected. PC affected to a lesser extent the synthesis of viral proteins in cells infected with the variant as compared to the sensitive parental virus. (author)

  3. Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

    Science.gov (United States)

    Gong, Haibiao; Holcomb, Ilona; Ooi, Aik; Wang, Xiaohui; Majonis, Daniel; Unger, Marc A; Ramakrishnan, Ramesh

    2016-01-20

    The diversity of nucleic acid sequences enables genomics studies in a highly multiplexed format. Since multiplex protein detection is still a challenge, it would be useful to use genomics tools for this purpose. This can be accomplished by conjugating specific oligonucleotides to antibodies. Upon binding of the oligonucleotide-conjugated antibodies to their targets, the protein levels can be converted to oligonucleotide levels. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method's application in oligonucleotide extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and subsequently linked covalently with an azide-modified oligonucleotide. In the functional test, the reaction conditions and purification processes were optimized to achieve maximum yield and best performance. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain unique six-base complementary regions at their 3' prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. Following preamplification, the DNA template is detected by qPCR. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM, and CASP3, had dynamic ranges of 5-6 logs. The antibody binders were also used in a multiplexed format in OER assays, and the binders successfully detected their protein targets in cell lysates, and in single cells in combination with the C1 system. This click reaction-based antibody conjugation procedure is cost-effective, needs minimal hands-on time, and

  4. A Biocompatible and Biodegradable Protein Hydrogel with Green and Red Autofluorescence: Preparation, Characterization and In Vivo Biodegradation Tracking and Modeling

    Science.gov (United States)

    Ma, Xiaoyu; Sun, Xiangcheng; Hargrove, Derek; Chen, Jun; Song, Donghui; Dong, Qiuchen; Lu, Xiuling; Fan, Tai-Hsi; Fu, Youjun; Lei, Yu

    2016-01-01

    Because of its good biocompatibility and biodegradability, albumins such as bovine serum albumin (BSA) and human serum albumin (HSA) have found a wide range of biomedical applications. Herein, we report that glutaraldehyde cross-linked BSA (or HSA) forms a novel fluorescent biological hydrogel, exhibiting new green and red autofluorescence in vitro and in vivo without the use of any additional fluorescent labels. UV-vis spectra studies, in conjunction with the fluorescence spectra studies including emission, excitation and synchronous scans, indicated that three classes of fluorescent compounds are presumably formed during the gelation process. SEM, FTIR and mechanical tests were further employed to investigate the morphology, the specific chemical structures and the mechanical strength of the as-prepared autofluorescent hydrogel, respectively. Its biocompatibility and biodegradability were also demonstrated through extensive in vitro and in vivo studies. More interestingly, the strong red autofluorescence of the as-prepared hydrogel allows for conveniently and non-invasively tracking and modeling its in vivo degradation based on the time-dependent fluorescent images of mice. A mathematical model was proposed and was in good agreement with the experimental results. The developed facile strategy to prepare novel biocompatible and biodegradable autofluorescent protein hydrogels could significantly expand the scope of protein hydrogels in biomedical applications.

  5. imFASP: An integrated approach combining in-situ filter-aided sample pretreatment with microwave-assisted protein digestion for fast and efficient proteome sample preparation.

    Science.gov (United States)

    Zhao, Qun; Fang, Fei; Wu, Ci; Wu, Qi; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-03-17

    An integrated sample preparation method, termed "imFASP", which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50-500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples. PMID:26920773

  6. The in vitro antioxidant properties of alcalase hydrolysate prepared from silkie fowl (Gallus gallus) blood protein.

    Science.gov (United States)

    Cheng, Fu-Yuan; Lai, I-Chun; Lin, Liang-Chuan; Sakata, Ryoichi

    2016-07-01

    Two types of proteins including blood plasma protein and blood cell protein were isolated from silkie fowl (Gallus gallus) blood and hydrolyzed using alcalase for 0, 2, 4 and 6 h. The blood plasma protein hydrolysate (BPH) and blood cell protein hydrolysate (BCH) were analyzed for pH value, peptide content and antioxidative properties. The significantly higher peptide contents were observed in BPH than that of BCH, which showed that blood plasma protein was more suitable to hydrolysis by alcalase than blood cell protein. Both BPH and BCH showed strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and Fe(2+) chelating ability. BPH at 4 h of hydrolysis (BPH4) demonstrated significantly higher antioxidant capacity than those treated by alcalase in most of the assays. The BPH4 was separated using ultra-filtration and assessment of the fractions and indicated that low molecular weight of peptides (< 3 kDa) possessed greater DPPH scavenging activity, Fe(2+) chelating ability and inhibitory activity of lipid peroxidation. These results show that BPH has the potential to be ingredients in the food industry as a replacement of synthetic antioxidants. PMID:26556592

  7. “Fuzzy oil drop” model applied to individual small proteins built of 70 amino acids

    OpenAIRE

    Prymula, Katarzyna; Sałapa, Kinga; Roterman, Irena

    2010-01-01

    Abstract The proteins composed of short polypeptides (about 70 amino acid residues) representing the following functional groups (according to PDB notation): growth hormones, serine protease inhibitors, antifreeze proteins, chaperones and proteins of unknown function, were selected for structural and functional analysis. Classification based on the distribution of hydrophobicity in terms of deficiency/excess as the measure of structural and functional specificity is presented. The ...

  8. Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles

    Science.gov (United States)

    Weidner, A.; Gräfe, C.; von der Lühe, M.; Remmer, H.; Clement, J. H.; Eberbeck, D.; Ludwig, F.; Müller, R.; Schacher, F. H.; Dutz, S.

    2015-07-01

    Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40 °C. For incubation temperatures above 50 °C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on corona

  9. Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles.

    Science.gov (United States)

    Weidner, A; Gräfe, C; von der Lühe, M; Remmer, H; Clement, J H; Eberbeck, D; Ludwig, F; Müller, R; Schacher, F H; Dutz, S

    2015-12-01

    Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40 °C. For incubation temperatures above 50 °C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on

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

    Science.gov (United States)

    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

  11. Protein Hydrolysis from Catfish Prepared by Papain Enzyme and Antioxidant Activity of Hydrolyzate

    OpenAIRE

    Ace Baehaki1); Shanti Dwita Lestari; Achmad Rizky Romadhoni

    2015-01-01

    The objective of this research was to make a protein hydrolysates from catfish (Pangasius pangasius) enzymatically using papain enzyme and analyzed the antioxidant activity of protein hydrolysates produced. The research used the method completely randomized design with two replications the treatment were the difference concentration of the papain enzyme (0%, 1%, 2%, 3%, 4%, 5%, and 6%). The parameters of research were antioxidative activity using DPPH (2,2-difenil-1–pikrilhidra...

  12. Effects of preparation methods on protein and amino acid contents of various eggs available in Malay- sian local markets

    Directory of Open Access Journals (Sweden)

    Maznah Ismail

    2013-03-01

    Full Text Available Background. The effect of preparation methods (raw, half-boiled and hard-boiled on protein and amino acid contents, as well as the protein quality (amino acid score of regular, kampung and nutrient enriched Malaysian eggs was investigated. Methods. The protein content was determined using a semi-micro Kjeldahl method whereas the amino acid composition was determined using HPLC. Results. The protein content of raw regular, kampung and nutrient enriched eggs were 49.9 ±0.2%, 55.8 ±0.2% and 56.5 ±0.5%, respectively. The protein content of hard-boiled eggs of regular, kampung and nutrient enriched eggs was 56.8 ±0.1%, 54.7 ±0.1%, and 53.7 ±0.5%, while that for half-boiled eggs of regular, kampung and nutrient enriched eggs was 54.7 ±0.6%, 53.4 ±0.4%, and 55.1 ±0.7%, respectively. There were signifi cant differences (p < 0.05 in protein and amino acid contents of half-boiled, hard-boiled as compared with raw samples, and valine was found as the limiting amino acid. It was found that there were signifi cant differences (p < 0.05 of total amino score in regular, kampung and nutrient enriched eggs after heat treatments.Furthermore, hard-boiling (100°C for 10 minutes and half-boiling (100°C for 5 minutes affects the total amino score, which in turn alter the protein quality of the egg.

  13. Preparation of Soybean Protein Concentrate with Mixed Solvents of Hexane-Aqueous Alcohol

    Institute of Scientific and Technical Information of China (English)

    Zhang Weinong; Liu Dachuan

    2002-01-01

    Preparation of soybean proteinconcentrate with the mixed solvents of hexane-aqueous alcohol was studied in this paper Theoptimum technology parameters were obtainedby orthogonal tests. The results of experimentsshowed that the qualities of the product weregood not only on taste and color, but also onhigh solubility-NSI value was 48.80%.

  14. Preparation of Barley Storage Protein, Hordein, for Analytical Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    DEFF Research Database (Denmark)

    Doll, Hans; Andersen, Bente

    1981-01-01

    The extraction, reduction, and alkylation of barley hordein for routine electrophoresis in sodium dodecyl sulfate-polyacrylamide gels were studied to set up a simple preparation procedure giving well-resolved bands in the electrophoresis gel. Hordein was extracted from single crushed seeds or flour...... in a buffer without propan-2-ol but containing sodium dodecyl sulfate....

  15. Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin.

    Science.gov (United States)

    Mackin, Robert B

    2014-01-01

    The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.

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

    Science.gov (United States)

    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

  17. Preparation of the Human Cytomegalovirus Nuclear Egress Complex and Associated Proteins.

    Science.gov (United States)

    Sharma, Mayuri; Kamil, Jeremy P; Coen, Donald M

    2016-01-01

    Herpesviruses, like most DNA viruses, replicate their genomes in the host cell nucleus. Their DNA is then packaged and assembled into viral nucleocapsids, which, in most cases, are too large to pass through the nuclear pore complex. Instead, herpesviruses use a complex multistep pathway, termed nuclear egress, to exit the nucleus. Key players in this process include two conserved viral proteins that form the nuclear egress complex (NEC). In human cytomegalovirus, these NEC proteins are UL50, embedded in the inner nuclear membrane, and its nucleoplasmic partner UL53. Both are essential for viral nuclear egress. However, other viral components as well as host nuclear envelope proteins may also participate in nuclear egress. Identifying these viral and cellular factors may provide important insight into the herpesvirus lifecycle and its relationship to the underlying, yet still-mysterious, host nuclear egress pathway. We developed an immunoprecipitation-based protocol, described herein, to identify protein-protein interactions involving the NEC from the nuclear fraction of infected cells that express an epitope-tagged version of NEC subunit UL53.

  18. Cyclic enterobacterial common antigen: Potential contaminant of bacterially expressed protein preparations

    International Nuclear Information System (INIS)

    We have previously reported the identification of the cyclic enterobacterial common antigen (ECACYC) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol.185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N-acetyl signals of ECACYC have been observed in 15N-1H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECACYC in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate

  19. High resolution preparation of monocyte-derived macrophages (MDM protein fractions for clinical proteomics

    Directory of Open Access Journals (Sweden)

    Olivieri Oliviero

    2009-02-01

    Full Text Available Abstract Background Macrophages are involved in a number of key physiological processes and complex responses such as inflammatory, immunological, infectious diseases and iron homeostasis. These cells are specialised for iron storage and recycling from senescent erythrocytes so they play a central role in the fine tuning of iron balancing and distribution. The comprehension of the many physiological responses of macrophages implies the study of the related molecular events. To this regard, proteomic analysis, is one of the most powerful tools for the elucidation of the molecular mechanisms, in terms of changes in protein expression levels. Results Our aim was to optimize a protocol for protein fractionation and high resolution mapping using human macrophages for clinical studies. We exploited a fractionation protocol based on the neutral detergent Triton X-114. The 2D maps of the fractions obtained showed high resolution and a good level of purity. Western immunoblotting and mass spectrometry (MS/MS analysis indicated no fraction cross contamination. On 2D-PAGE mini gels (7 × 8 cm we could count more than five hundred protein spots, substantially increasing the resolution and the number of detectable proteins for the macrophage proteome. The fractions were also evaluated, with preliminary experiments, using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS. Conclusion This relatively simple method allows deep investigation into macrophages proteomics producing discrete and accurate protein fractions, especially membrane-associated and integral proteins. The adapted protocol seems highly suitable for further studies of clinical proteomics, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions.

  20. Preparation and application of SARS-associated coronavirus spike protein antibodies

    International Nuclear Information System (INIS)

    Four hybridoma cell lines secreting monoclonal antibody against SARS-associated coronavirus spike protein were obtained and at same time the polyclonal antibodies were also got by immunizing sheep, goats and rabbits with SARS-associated coronavirus spike protein respectively. Immunoradiometric assay (IRMA) and enzyme-linked immunosorbent assay (ELISA) were established using above Abs for detecting SARS-associated coronavirus, and best assay results were observed when McAb CS4C11 was used as labeled Ab and PcAb (sheep) as capture Ab. (author)

  1. Effect of Fillers Prepared from Enzymatically Modified Proteins on Mechanical Properties of Leather

    Science.gov (United States)

    In an environment where petroleum feedstuffs are becoming increasingly too expensive for a good cost-effective return, utilization of renewable resources makes economic sense, particularly when these substrates are waste proteins. We have thus proposed the application of enzymatically modified wast...

  2. Encapsulation of flaxseed oil using a benchtop spray dryer for legume protein-maltodextrin microcapsule preparation.

    Science.gov (United States)

    Can Karaca, Asli; Low, Nicholas; Nickerson, Michael

    2013-05-29

    Flaxseed oil was microencapsulated employing a wall material matrix of either chickpea (CPI) or lentil protein isolate (LPI) and maltodextrin using a benchtop spray dryer. Effects of emulsion formulation (oil, protein and maltodextrin levels) and protein source (CPI vs LPI) on the physicochemical characteristics, oxidative stability, and release properties of the resulting capsules were investigated. Microcapsule formulations containing higher oil levels (20% oil, 20% protein, 60% maltodextrin) were found to have higher surface oil and lower encapsulation efficiencies. Overall, LPI-maltodextrin capsules gave higher flaxseed oil encapsulation efficiencies (∼88.0%) relative to CPI-maltodextrin matrices (∼86.3%). However, both designs were found to provide encapsulated flaxseed oil protection against oxidation over a 25 d room temperature storage study relative to free oil. Overall, ∼37.6% of encapsulated flaxseed oil was released after 2 h under simulated gastric fluid, followed by the release of an additional ∼46.6% over a 3 h period under simulated intestinal fluid conditions.

  3. Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

    DEFF Research Database (Denmark)

    Spetzler, J C; Westphal, V; Winther, Jakob R.;

    1998-01-01

    the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were...

  4. Integrated Automation of High-Throughput Screening and Reverse Phase Protein Array Sample Preparation

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus;

    multiplexing readouts, but this has a natural limitation. High-content screening via image acquisition and analysis allows multiplexing of few parameters, but is connected to substantial time consumption and complex logistics. We report on integration of Reverse Phase Protein Arrays (RPPA)-based readouts...

  5. Encapsulation of flaxseed oil using a benchtop spray dryer for legume protein-maltodextrin microcapsule preparation.

    Science.gov (United States)

    Can Karaca, Asli; Low, Nicholas; Nickerson, Michael

    2013-05-29

    Flaxseed oil was microencapsulated employing a wall material matrix of either chickpea (CPI) or lentil protein isolate (LPI) and maltodextrin using a benchtop spray dryer. Effects of emulsion formulation (oil, protein and maltodextrin levels) and protein source (CPI vs LPI) on the physicochemical characteristics, oxidative stability, and release properties of the resulting capsules were investigated. Microcapsule formulations containing higher oil levels (20% oil, 20% protein, 60% maltodextrin) were found to have higher surface oil and lower encapsulation efficiencies. Overall, LPI-maltodextrin capsules gave higher flaxseed oil encapsulation efficiencies (∼88.0%) relative to CPI-maltodextrin matrices (∼86.3%). However, both designs were found to provide encapsulated flaxseed oil protection against oxidation over a 25 d room temperature storage study relative to free oil. Overall, ∼37.6% of encapsulated flaxseed oil was released after 2 h under simulated gastric fluid, followed by the release of an additional ∼46.6% over a 3 h period under simulated intestinal fluid conditions. PMID:23663097

  6. Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry

    DEFF Research Database (Denmark)

    Balslev, Y; Hansen, Gert Helge

    1989-01-01

    was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase-isomaltase. The labelling efficiency of RPGG was compared to that of protein A-gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling...

  7. Molecularly imprinted polymers prepared using protein-conjugated cleavable monomers followed by site-specific post-imprinting introduction of fluorescent reporter molecules.

    Science.gov (United States)

    Suga, Yusuke; Sunayama, Hirobumi; Ooya, Tooru; Takeuchi, Toshifumi

    2013-10-01

    Molecularly imprinted polymers were prepared using a protein-conjugated disulfide cleavable monomer. After removing the protein by disulfide reduction, a thiol-reactive fluorophore was introduced into the thiol residue located only inside the imprinted cavity, resulting in specific transduction of the binding events into fluorescence spectral change.

  8. Preparation of vesicular stomatitis virus pseudotype with Chikungunya virus envelope protein.

    Science.gov (United States)

    Tong, W; Yin, X-X; Lee, B-J; Li, Y-G

    2015-06-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes Chikungunya fever (CHIKF) in millions of people mainly in developing countries. CHIKF is characterized by high fever, fatigue, headache, nausea, vomiting, rash, myalgia and severe arthralgia. To date, there is no specific treatment and no licensed vaccine against CHIKV infection. In this study, we developed a safe, efficient and easy neutralization assay of CHIKV based on vesicular stomatitis virus (VSV) pseudotype with CHIKV envelope protein and the green fluorescent protein (GFP) or luciferase as reporter gene, which could be used under a reduced safety level. The VSV pseudotype can be applied to the epidemic survey by measuring the expression of GFP or luciferase activity in infected cells. This system can also be used to study the mechanisms of virus entry.

  9. Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro

    OpenAIRE

    Lylo V. V.; Ruban T. P.; Macewicz L. L.; Kornelyuk A. I.; Chernykh S. I.; Lukash L. L.

    2014-01-01

    Aim. To study the effect of EMAP II, IFN-α2b and its medicinal preparations on the amount of O6-methylguanine-DNA methyltransferase (MGMT) protein in human cells in vitro. Methods. The human cells of 4BL and Hep-2 lines were treated with the purified recombinant proteins EMAP II, IFN-α2b and its commercial me dicinal preparations. Changes in the MGMT gene expression were studied at a protein level by Western blot analysis. Results. Treatment of Hep-2 and 4BL cells with EMAP II at the concentr...

  10. Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein

    OpenAIRE

    Liyue Wang; Kao Zhang; Hongyu Lin; Wenyan Li; Jiexia Wen; Jianlou Zhang; Yonghong Zhang; Xiujin Li; Fei Zhong

    2014-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene wa...

  11. Preparation of Mesoporous Nano-Hydroxyapatite Using a Surfactant Template Method for Protein Delivery

    Institute of Scientific and Technical Information of China (English)

    Xiaodong Wu; Xiaofeng Song; Dongsong Li; Jianguo Liu; Peibiao Zhang; Xuesi Chen

    2012-01-01

    Mesoporous nano-hydroxyapatite (n-HA) has gained more and more attention as drug storage and release hosts.The aim of this study is to observe the effect of the ratio of surfactant to the theoretical yield of HA on the mesoporous n-HA,then to reveal the effect of the mesoporous nanostrueture on protein delivery.The mesoporous n-HA was synthesized using the wet precipitation in the presence of cetyltrimethylammonium bromide (CTAB) at ambient temperature and normal atmospheric pressure.The morphology,size,crystalline phase,chemical composition and textural characteristics of the product were well characterized by X-ray Powder Diffraction (XRD),Fourier Transform Infrared Spectroscopy (FTIR),Scanning Electron Microscopy (SEM),Transmission Electron Microscopy (TEM),Dynamic Light Scattering (DLS) and N2 adsorption/desorption,respectively.The protein adsorption/release studies were also carried out by using Bovine Serum Albumin (BSA) as a model protein.The results reveal that the mesoporous n-HA synthesized with CTAB exhibits high pure phase,low crystallinity and the typical characteristics of the mesostructure.The BSA loading increases with the specific surface area and the pore volume of n-HA,and the release rates of BSA are different due to their different pore sizes and pore structures,n-HA synthesized with 0.5% CTAB has the highest BSA loading and the slowest release rate because of its highest surface area and smaller pore size.These mesoporous n-HA materials demonstrate a potential application in the field of protein delivery due to their bioaetive,biocompatible and mesoporous properties.

  12. Preparation of Monoclonal Antibody Against HPT and Its Application to Detecting Marker Protein in Genetically Modified Rice

    Institute of Scientific and Technical Information of China (English)

    LI-CHEN YANG; SU-XIANG ZHANG; GUO-HUA PI; YING-HUA LI; ZHEN ZHU; XIAO-GUANG YANG

    2005-01-01

    Objective To produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice). Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1, D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 1×10-4 to 1×10-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected (80-150ng/mL). But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay. Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.

  13. Preparation of antioxidative corn protein hydrolysates, purification and evaluation of three novel corn antioxidant peptides.

    Science.gov (United States)

    Jin, Du-Xin; Liu, Xiao-Lan; Zheng, Xi-Qun; Wang, Xiao-Jie; He, Jun-Fang

    2016-08-01

    Corn gluten meal is a major co-product of corn wet milling. Corn gluten meal was hydrolyzed with Alcalase, Flavourzyme, Alcalase+Flavourzyme and Flavourzyme+Alcalase. At the substrate concentration of 10%, corn protein hydrolysate catalyzed by Alcalase had a degree of hydrolysis of 17.83%, which was higher than that by Flavourzyme (3.65%). The hydrolysate catalyzed by Alcalase+Flavourzyme exhibited better antioxidant activities and was further purified. Three novel antioxidant peptides were purified by a series of chromatographic techniques. Sequences of the three peptides were identified as Cys-Ser-Gln-Ala-Pro-Leu-Ala, Tyr-Pro-Lys-Leu-Ala-Pro-Asn-Glu and Tyr-Pro-Gln-Leu-Leu-Pro-Asn-Glu, respectively. Among the three peptides, Cys-Ser-Gln-Ala-Pro-Leu-Ala exhibited good reducing power and excellent scavenging capacities for DPPH radical and superoxide anion radical, with IC50 values of 0.116 and 0.39mg/ml, respectively. The results from our study indicate antioxidant potency of corn protein hydrolysates and peptides separated from corn gluten meal and can provide basic understanding for the application of corn protein hydrolysates as natural antioxidants. PMID:26988521

  14. Preparation of ChlL-2 and IBDV VP2 Fusion Protein by Baculovirus Expression System

    Institute of Scientific and Technical Information of China (English)

    Yan Liu; Yongwei Wei; Xiaofeng Wu; Lian Yu

    2005-01-01

    This study aims to produce an effective subunit vaccine against infectious bursal disease virus (IBDV). The genes of chicken interleukin-2 (ChIL-2) and IBDV viral protein 2 (VP2) were amplified and fused by splice overlap extension-polymerase chain reaction (SOE-PCR). The fusion gene was digested by EcoR I/Kpn I and inserted into pBacPAK8 vector, resulting in recombinant transfer plasmid pBacPakVP2-IL2. The recombinant plasmid was transfected into Sf-9 cells accompanied with hybrid nuclear polyhedrosis virus (HyNPV) genome DNA and lipofectin. Plaque-purification indicated that we had got the recombinant Hy-VP2-IL2. Fusion protein VP2-IL2was expressed effectively both in insect cells and bombyx mori. The expression of fusion protein was confirmed by ELISA, SDS-PAGE and Western blotting assay, respectively. This efficient system allows us to meet the need for inexpensive vaccines required by the poultry industry.

  15. Reciprocating free-flow isoelectric focusing device for preparative separation of proteins.

    Science.gov (United States)

    Kong, Fan-Zhi; Yang, Ying; Wang, Yi; Li, Guo-Qing; Li, Shan; Xiao, Hua; Fan, Liu-Yin; Liu, Shao-Rong; Cao, Cheng-Xi

    2015-11-27

    The traditional recycling free-flow isoelectric focusing (RFFIEF) suffered from complex structure, tedious operations and poor extensibility as well as high cost. To address these issues, a novel reciprocating free-flow isoelectric focusing device (ReFFIEF) was developed for proteins or peptides pre-fractionation. In the new device, a reciprocating background flow was for the first time introduced into free flow electrophoresis (FFE) system. The gas cushion injector (GCI) used in the previous continuous free-flow electrophoresis (CFFE) was redesigned for the reciprocating background flow. With the GCI, the reciprocating background flow could be achieved between the GCI, separation chamber and transient self-balance collector (tSBC). In a run, process fluid flowed to and from, forming a stable reciprocating fluid flow in the separation chamber. A pH gradient was created within the separation chamber, and at the same time proteins were focused repeatedly when passing through the chamber under perpendicular electric field. The ReFFIEF procedure was optimized for fractionations of three model proteins, and the optimized method was further used for pre-fractionation of model human serum samples. As compared with the traditional RFFIEF devices developed about 25 years ago, the new ReFFIEF system showed several merits, such as simple design and structure, user-friendly operation and easy to extend as well as low cost.

  16. Antioxidant and antimicrobial activity of lecithin free egg yolk protein preparation hydrolysates obtained with digestive enzymes

    Directory of Open Access Journals (Sweden)

    Aleksandra Zambrowicz

    2012-12-01

    Full Text Available ABSTRACT:Several biological activities have now been associated with egg protein- derived peptides, including antihypertensive, antimicrobial, immunomodulatory, anticancer and antioxidantactivities, highlighting the importance of these biopeptides in human health, and disease prevention and treatment. Special attention has been given to peptides with antioxidant and antimicrobial activities as a new source of natural preservatives in food industry. In this study, the antioxidant properties of the egg-yolk protein by-product (YP hydrolysates were evaluated based on their radical scavenging capacity (DPPH, Fe2+chelating effect and ferric reducing power (FRAP. Furthermore, antimicrobial properties of obtained hydrolysates against Bacillus species were studied. The degrees (DHs of hydrolysis for 4h hydrolysates were: 19.1%, 13.5% and 13.0%, for pepsin, chymotrypsin and trypsin, respectively. Pepsin was the most effective in producing the free amino groups (1410.3 μmolGly/g. The RP-HPLC profiles of the protein hydrolysates showed differences in the hydrophobicity of the generated peptides.Trypsin hydrolysate obtained after 4h reaction demonstrated the strongest DPPH free radical scavenging activity (0.85 µmol Troloxeq/mg. Trypsin and chymotrypsin hydrolysates obtained after 4h reaction exhibited 4 times higher ferric reducing capacity than those treated bypepsin. The hydrolysis products obtained from YP exhibited significant chelating activity. The 4h trypsin hydrolysate exhibited weak antimicrobial activity against B. subtilis B3; B. cereus B512; B. cereus B 3p and B. laterosporum B6.

  17. [Preparation and Identification of High Immunogenic A/PR/8/34 Maternal Strain HA Protein for Influenza Virus Classical Reassortment].

    Science.gov (United States)

    Tang, Jing; Xin, Li; Guo, Junfeng; Zhu, Wenfei; Zhang, Heyuan; Lang, Shaohui; Wang, Dayan; Shu, Yuelong

    2016-03-01

    Preparation of maternal strain A/PR/8/34 HA antiserum for influenza virus classical reassortment. A/PR/8/34 virus was digested by bromelain after inactivation and purification. 5%-20% sucrose continuous density gradient centrifugation method was used to purify HA protein. SIRD method was used to select the target protein. SDS-PAGE method was used to identified HA protein. High Immunogenic A/PR/8/34 HA protein was successfully prepared and HI titer reached 10240. High purity HA antiserum was identified by SIRD method. The key reagent in the classical reassortment of influenza virus was prepared, and the complete set of technical methods were explored, which laid the foundation for the independent research and development of seasonal influenza vaccine strains of China. PMID:27396155

  18. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    Science.gov (United States)

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-01

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

  19. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    Science.gov (United States)

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-01

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth. PMID:24773089

  20. Preparation by enzymolysis and bioactivity of iron complex of fish protein hydrolysate (Fe-FPH)from low value fish

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Preparation of Fe2+ chelate of fish protein hydrolysate (Fe-FPH) obtained from low value fish proteins was introduced and its bioactivity was studied by compound enzymolysis. The optimum conditions for hydrolysate chelating Fe2+ are DH (degree of hydrolysis) at 5%, pH 7.0, 20°C and 15 min chelating time for FM (material not being defatted). Four types of Fe-FPH including CA (deposit after chelating), CB (deposit in 50% of absolute ethanol solution), CC (suspended deposit in 80% of absolute ethanol solution), and CD (bottom deposit in 80% of absolute ethanol solution) were fractionated with absolute ethanol from FM. Structural analysis through infra-red spectrum revealed that Fe2+ was combined strongly with amino-group and carboxyl-group in each chelate and each Fe2+ could form two five-member ring structures. All of the four chelates were shown more significant antioxidative activity and can be used as natural hydrophobic and hydrophilic antioxidant. Among all the chelates, the CB possesses the most effective antioxidative activity at 92% as high as that of a-tocopherol. Among all Fe-FPHs, only CD showed the most effective antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis and can be used as natural antibacterial. It provides a more effective way for utilization of low value fish proteins and key information of Fe-FPH as additive in food industry.

  1. Preparation of protein based surfactants from leather waste fleshings and their reutilization in leather as a water resisting agent

    International Nuclear Information System (INIS)

    Summary: Tanneries generate a huge amount of highly polluting solid and liquid wastes during leather processing at different stages such as fleshings, shavings, tanning, finishing etc. approximately, 250 kg of finished leather product is obtained from 1 ton of raw salted hide while other protein goes into wastes. leather fleshings are about 50-60% of the total solid waste generated in leather processing. three different surfactants have been prepared from soft wax, long chain fatty acid chlorides and leather waste protein isolated from alkaline hydrolysis of fleshings. products are milky in color and have been applied in goat leathers as a replacement of fat liquor and water resisting agent .the resulted crust leathers have been characterized for various physical parameters such as tensile strength, thickness, softness, tear strength, bursting load, water absorption etc, as per their standard test methods. leathers have also been evaluated for grain smoothness, fullness and feeling. leathers have shown satisfactory results as per international requirement specially for water resisting. thus a leather waste protein is converted into a useful product and reutilized in leather making. (author)

  2. Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

    DEFF Research Database (Denmark)

    Houen, G.; Olsen, D.T.; Hansen, P.R.;

    2003-01-01

    protein was conjugated with glutathione, the conjugation ratio determined by acid hydrolysis, and amino acid analysis performed with quantification of carboxymethyl cysteine. Elution of conjugates from the resin by a salt gradient revealed considerable heterogeneity in the degree of derivatization......, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates...

  3. Protein repellent hydrophilic grafts prepared by surface-initiated atom transfer radical polymerization from polypropylene

    DEFF Research Database (Denmark)

    Fristrup, Charlotte Juel; Jankova Atanasova, Katja; Eskimergen, Rüya;

    2012-01-01

    Grafting of poly(ethylene glycol)methacrylate (PEGMA) and N,N-dimethylacrylamide (DMAAm) from UV-initiator modified polypropylene (PP) was performed by Surface-Initiated Atom Transfer Radical Polymerization (SI-ATRP). The modification and hydrophilization of the PP substrates were confirmed...... with Attenuated Total Reflectance (ATR) Fourier Transform Infrared (FTIR) spectroscopy and Water Contact Angle (WCA) measurements. Confocal fluorescence microscopy of modified and unmodified substrates immersed in labelled insulin aspart showed superior repulsion of this protein for the poly(PEGMA) grafts, due...

  4. P53 FUSION PROTEIN EXPRESSION IN PROKARYOTE AND PREPARATION OF MONOCLONAL ANTIBODY TO P53

    Institute of Scientific and Technical Information of China (English)

    Liu Caiyun; Shou Chengchao; Sun Sulian; ZhangLei; Zeng Li

    1998-01-01

    Objective: Conventional immunohistochemistry (IHC) is available to assess P53 mutations, and expensive imported anti-P53 monoclonal antibody has been used in China, it is necessary to study a new monoclonal antibody.Methods: The P53 DNA fragment enconding N-terminal 180 amiao acide was obtained by PCR and was cloned into PGEX-2T plasmid expressing glutathione S-transferase (GST). The P53-GST fusion protein expressed by JM109was used for immunizing BALB/C mice. We have raised one hybridoma strain secreting McAb to human P53(named M126). Results: The IHC analysis of 52paraffin-embedded sections from human breast cancer with M126 and PAB1801 (Zymed Co.) has showed that the positive immunoreactions were 25 cases (48%) and 22cases (42.3%) respectively. The staining of M126 was stronger and preferable to PAB1801. Conclusion: M126can be instead of PAB1801 for studying immunohistochemical analysis on P53 Protein.

  5. The preparation and use of fluorescent-protein conjugates for microvascular research.

    Science.gov (United States)

    McDonagh, P F; Williams, S K

    1984-01-01

    A procedure is described for making large quantities (100 ml) of fluorochrome-labeled albumin. Chromatographic techniques are described for the purification of commercial albumin (BSA) and the purification of albumin from serum. We report experimentally determined optimal conditions for the covalent attachment of fluorescent dyes (rhodamine isothiocyanate (RITC) and fluorescein isothiocyanate (FITC] to albumin. Subsequent removal of all unreacted fluorescent material (UFM) was achieved using charcoal adsorption. We observed no loss of protein following charcoal treatment. The final protein conjugate was analyzed by polyacrylamide gel electrophoresis, gel chromatography, and isoelectric focusing. The conjugates were determined to be free of UFM and homogeneous with respect to molecular weight. However, FITC conjugation lowered the average isoelectric point of albumin by 0.1 to 0.3 pH units. Illustrations of combining fluorescence microscopy with FITC-BSA and RITC-BSA to view microvascular phenomena in skeletal muscle and the heart are given. Knowledge of the biochemical characteristics of the fluorochrome employed is important for proper interpretation of experimental results using this technique.

  6. Depot injectable biodegradable nanoparticles loaded with recombinant human bone morphogenetic protein-2: preparation, characterization, and in vivo evaluation

    Directory of Open Access Journals (Sweden)

    Hassan AH

    2015-07-01

    Full Text Available Ali Habiballah Hassan,1 Khaled Mohamed Hosny,2,3 Zuahir A Murshid,1 Adel Alhadlaq,4 Ahmed Alyamani,5 Ghada Naguib6 1Department of Orthodontics, Faculty of Dentistry, 2Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; 3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt; 4Department of Pediatric Dentistry and Orthodontics, College of Dentistry, King Saud University, Riyadh, 5Department of Oral Surgery, 6Department of Restorative Dentistry, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia Objective: The aim of this study is to utilize the biocompatibility characteristics of biodegradable polymers, viz, poly lactide-co-glycolide (PLGA and polycaprolactone (PCL, to prepare sustained-release injectable nanoparticles (NPs of bone morphogenetic protein-2 (BMP-2 for the repair of alveolar bone defects in rabbits. The influence of formulation parameters on the functional characteristics of the prepared NPs was studied to develop a new noninvasive injectable recombinant human BMP-2 (rhBMP-2 containing grafting material for the repair of alveolar bone clefts.Materials and methods: BMP-2 NPs were prepared using a water-in-oil-in-water double-emulsion solvent evaporation/extraction method. The influence of molar ratio of PLGA to PCL on a suitable particle size, encapsulation efficiency, and sustained drug release was studied. Critical size alveolar defects were created in the maxilla of 24 New Zealand rabbits divided into three groups, one of them treated with 5 µg/kg of rhBMP-2 NP formulations.Results: The results found that NPs formula prepared using blend of PLGA and PCL in 4:2 (w/w ratio showed the best sustained-release pattern with lower initial burst, and showed up to 62.7% yield, 64.5% encapsulation efficiency, 127 nm size, and more than 90% in vitro release. So, this formula was selected for

  7. Isolation and characterisation of sericin antifreeze peptides and molecular dynamics modelling of their ice-binding interaction.

    Science.gov (United States)

    Wu, Jinhong; Rong, Yuzhi; Wang, Zhengwu; Zhou, Yanfu; Wang, Shaoyun; Zhao, Bo

    2015-05-01

    This study aimed to isolate and characterise a novel sericin antifreeze peptide and investigate its ice-binding molecular mechanism. The thermal hysteresis activity of ice-binding sericin peptides (I-SP) was measured and their activity reached as high as 0.94 °C. A P4 fraction, with high hypothermia protective activity and inhibition activity of ice recrystallisation, was obtained from I-SP, and a purified sericin peptide, named SM-AFP, with the sequence of TTSPTNVSTT and a molecular weight of 1009.50 Da was then isolated from the P4 fraction. Treatment of Lactobacillus delbrueckii Subsp. bulgaricus LB340 LYO with 100 μg/ml synthetic SM-AFP led to 1.4-fold increased survival (p Sericin peptides could be developed into beneficial cryoprotectants and used in frozen food processing. PMID:25529728

  8. Effect of egg albumen (protein additive on surimi prepared from lizardfish (Saurida tumbil during frozen storage

    Directory of Open Access Journals (Sweden)

    Solanki Jitesh B.

    2011-07-01

    Full Text Available Lizardfish (Saurida tumbil (Bloch, 1795 is a relatively abundant, low value fish that has widedistribution in India due to its adaptability to different environments. This study is an attempt to explorethe possibilities of better utilization of this species by development of minced-based value addedproducts and the evaluation of shelf life during frozen storage. Lizardfish were mince for the preparationof value added products viz., surimi and surimi with 3% egg albumen. The biochemical, gel strength andsensory parameters were analyzed to study the quality changes and shelf life of these products in frozenstorage at -20oC. The addition of 3% egg albumen exhibited gel enhancing effect by increase in gelstregth 113.56 g.cm, where as same treatment after 120th days of storage, % of total protein was higher12.69 with comapare to without egg albumen surimi.

  9. Preparation and characterization of whey protein film incorporated with TiO2 nanoparticles.

    Science.gov (United States)

    Zhou, J J; Wang, S Y; Gunasekaran, S

    2009-09-01

    Biodegradable titanium dioxide (TiO(2))/whey protein isolate (WPI) blend films were made by casting denatured WPI film solutions incorporated with TiO(2) nanoparticles. X-ray diffraction, UV-vis spectra, and fluorescence spectra of the films showed the successful incorporation of TiO(2) nanoparticles into the WPI matrix and indicated the interactions between TiO(2) and WPI. Mechanical tests revealed the antiplasticizing effect of TiO(2) nanoparticles on the WPI/TiO(2) film. Small amounts (WPI film, but also decrease the moisture barrier properties. The addition of higher amounts (>1 wt%) of TiO(2) improves moisture barrier properties but lowers the tensile properties of the film. Microstructural evaluation confirmed the aggregation and distribution of TiO(2) nanoparticles within the WPI matrix and validated the results of functional properties of the WPI/TiO(2) film.

  10. Preparation and Characterization of Nanocomposites from Whey Protein Concentrate Activated with Lycopene.

    Science.gov (United States)

    Pereira, Rafaela Corrêa; de Deus Souza Carneiro, João; Borges, Soraia Vilela; Assis, Odílio Benedito Garrido; Alvarenga, Gabriela Lara

    2016-03-01

    The production and characterization of nanocomposites based on whey protein concentrate (WPC) and montmorilonite (MMT) incorporated with lycopene as a functional substance is presented and discussed as an alternative biomaterial for potential uses in foodstuff applications. A full factorial design with varying levels of MMT (0% and 2% in w/w) and lycopene (0%, 6%, and 12% in w/w) was used. Color, light transmission, film transparency, moisture, density, solubility, water vapor permeability, and antioxidant activity of the resulting materials were evaluated. Results indicated that lycopene and MMT nanoparticles were successfully included in WPC films using the casting/evaporation method. Inclusion of 2% w/w of MMT in the polymeric matrix significantly improved barrier property against water vapor. Lycopene, besides its good red coloring ability, provided to the films antioxidant activity and UV-vis light protection. These findings open a new perspective for the use of materials for bioactive packaging applications. PMID:26814439

  11. Inactivation of the AIDS-causing retrovirus and other human viruses in antihemophilic plasma protein preparations by pasteurization.

    Science.gov (United States)

    Hilfenhaus, J; Herrmann, A; Mauler, R; Prince, A M

    1986-01-01

    Heat treatment at 60 degrees C for 10 h in solution (pasteurization) was introduced into the manufacturing process of antihemophilic cryoprecipitate (AHC) and factor VIII concentrates (F VIII) to reduce the risk of transmission of hepatitis to hemophiliacs. Since the acquired immunodeficiency syndrome (AIDS) may also be transmitted to hemophiliacs by antihemophilic plasma protein preparations, we have investigated inactivation of the AIDS virus HTLV III by pasteurization in AHC or F VIII and included in this study cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), poliovirus and vaccinia virus. Each of these viruses was efficiently inactivated by pasteurization although considerable differences were observed between the different viruses HTLV III was rapidly inactivated, becoming nondetectable within 30-60 min. Our findings indicate that pasteurized AHC or F VIII should have a high margin of safety regarding the transmission of AIDS or any other infectious disease caused by viruses such as those tested.

  12. Preparation and properties of BSA-loaded microspheres based on multi-(amino acid) copolymer for protein delivery.

    Science.gov (United States)

    Chen, Xingtao; Lv, Guoyu; Zhang, Jue; Tang, Songchao; Yan, Yonggang; Wu, Zhaoying; Su, Jiacan; Wei, Jie

    2014-01-01

    A multi-(amino acid) copolymer (MAC) based on ω-aminocaproic acid, γ-aminobutyric acid, L-alanine, L-lysine, L-glutamate, and hydroxyproline was synthetized, and MAC microspheres encapsulating bovine serum albumin (BSA) were prepared by a double-emulsion solvent extraction method. The experimental results show that various preparation parameters including surfactant ratio of Tween 80 to Span 80, surfactant concentration, benzyl alcohol in the external water phase, and polymer concentration had obvious effects on the particle size, morphology, and encapsulation efficiency of the BSA-loaded microspheres. The sizes of BSA-loaded microspheres ranged from 60.2 μm to 79.7 μm, showing different degrees of porous structure. The encapsulation efficiency of BSA-loaded microspheres also ranged from 38.8% to 50.8%. BSA release from microspheres showed the classic biphasic profile, which was governed by diffusion and polymer erosion. The initial burst release of BSA from microspheres at the first week followed by constant slow release for the next 7 weeks were observed. BSA-loaded microspheres could degrade gradually in phosphate buffered saline buffer with pH value maintained at around 7.1 during 8 weeks incubation, suggesting that microsphere degradation did not cause a dramatic pH drop in phosphate buffered saline buffer because no acidic degradation products were released from the microspheres. Therefore, the MAC microspheres might have great potential as carriers for protein delivery.

  13. Effects of Soy Protein Hydrolysates Prepared by Varying Subcritical Media on the Physicochemical Properties of Pork Patties.

    Science.gov (United States)

    Lee, Yun-Kyung; Ko, Bo-Bae; Davaatseren, Munkhtugs; Hong, Geun-Pyo

    2016-01-01

    This study investigated the effect of soy protein hydrolysates (SPH) prepared by varying subcritical media on the physicochemical properties of pork patties. For resource of SPH, two different soybean species (Glycine max Merr.) of Daewonkong (DWK) and Saedanbaek (SDB) were selected. SPH was prepared by subcritical processing at 190℃ and 25 MPa under three different of media (water, 20% ethanol and 50% ethanol). Solubility and free amino group content revealed that water was better to yield larger amount of SPH than ethanol/water mixtures, regardless of species. Molecular weight (Mw) distribution of SPH was also similar between two species, while slightly different Mw distribution was obtained by subcritical media. For pork patty application, 50% ethanol treatment showed clear red color comparing to control after 14 d of storage. In addition, ethanol treatment had better oxidative stability than control and water treatment based on thiobarbituric acid-reactive substances (TBARS) analysis. For eating quality, although 20% ethanol treatment in SDB showed slightly higher cooking loss than control, generally addition of SPH did not affect the water-binding properties and hardness of pork patties. Consequently, the present study indicated that 50% ethanol was the best subcritical media to produce SPH possessing antioxidant activity, and the SPH produced from DWK exhibited better antioxidant activity than that produced SDB. PMID:27499657

  14. Preparation and Primary Application of Monoclonal Antibodies against a Novel Ribosome-inactivating Protein Moschatin from Pumpkin Seeds

    Institute of Scientific and Technical Information of China (English)

    Heng-Chuan XIA; Wei-Guo HU; Xin-Xiu YANG; Feng LI; Zu-Chuan ZHANG

    2004-01-01

    Plant ribosome-inactivating proteins (RIPs) have multiple biological functions, and have beenwidely used in the studies on biomedical and agronomic applications. Moschatin is a novel single-chain RIPrecently purified from pumpkin seeds, and it has been successfully applied to construct the immunotoxin thatcan selectively kill the cultured human melanoma cells. Six stable strains of hybridomas (2H8, 4A8, 5B6,6F8, 4H 10 and 6C2) that can secrete high specific monoclonal antibodies against Moschatin have beensuccessfully prepared using hybridoma technique. The isotypes of these monoclonal antibodies areIgG1, IgG1, IgG1, IgG1, IgG2a and IgM. Their affinity constants were determined to be 1.42×108, 2.71×108,8.72×107, 2.06×108, 1.36×108 and 1.51×108M-1 in a sequent order, measured by non-competitive ELISA.The monoclonal antibody 4A8 has been used to detect Moschatin in Western blot. An immunoaffinity gel,which consisted ofa monoclonal antibody 4H10 and Sepharose 4B, was prepared and used to purify Moschatinfrom pumpkin seeds crude extract.

  15. Honeycomb-patterned films of polystyrene/poly(ethylene glycol):Preparation,surface aggregation and protein adsorption

    Institute of Scientific and Technical Information of China (English)

    WAN LingShu; KE BeiBei; LI XiaoKai; MENG XiangLin; ZHANG LuYao; XU ZhiKang

    2009-01-01

    Highly ordered honeycomb-patterned polystyrene (PS)/poly(ethylene glycol) (PEG) films were prepared by a water-assisted method using an improved setup,which facilitated the formation of films with higher regularity,better reproducibility,and larger area of honeycomb structures.Surface aggregation of hydrophilic PEG and adsorption of bovine serum albumin (BSA) on the honeycomb-patterned films were investigated.Field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) were used to observe the surface morphologies of the films before and after being rinsed with water.As confirmed by the FESEM images and the AFM phase images,PEG was enriched in the pores and could be gradually removed by water.The adsorption of fluorescence-labeled BSA on the films was studied in visual form using laser scanning confocal microscopy.Results clearly demonstrated that the protein-resistant PEG was selectively enriched in the pores.This water-assisted method may be a latent tool to prepare honeycomb-patterned biofunctional surfaces.

  16. Preparation and Primary Application of Monoclonal Antibodies against a Novel Ribosome-inactivating Protein Moschatin from Pumpkin Seeds

    Institute of Scientific and Technical Information of China (English)

    Heng-ChuanXIA; Wei-GuoHU; Xin-XiuYANG; FengLI; Zu-ChuanZHANG

    2004-01-01

    Plant ribosome-inactivating proteins (RIPs) have multiple biological functions, and have beenwidely used in the studies on biomedical and agronomic applications. Moschatin is a novel single-chain RIPrecently purified from pumpkin seeds, and it has been successfully applied to construct the immunotoxin thatcan selectively kill the cultured human melanoma cells. Six stable strains of hybridomas (2H8, 4A8, 5B6,6F8, 4H10 and 6C2) that can secrete high specific monoclonal antibodies against Moschatin have beensuccessfully prepared using hybridoma technique. The isotypes of these monoclonal antibodies areIgG1, IgG1, IgG1, IgG1, IgG2a and IgM. Their affinity constants were determined to be 1.42×108, 2.71×108,8.72×07, 2.06×l08, 1.36×108 and 1.51×108 M-1 in a sequent order, measured by non-competitive ELISA.The monoclonal antibody 4A8 has been used to detect Moschatin in Western blot. An immunoaffinity gel,which consisted ofa monoclonal antibody 4H 10 and Sepharose 4B, was prepared and used to purify Moschatinfrom pumpkin seeds crude extract.

  17. Cryogenic temperature effects and resolution upon slow cooling of protein preparations in solid state NMR

    Energy Technology Data Exchange (ETDEWEB)

    Linden, Arne H.; Franks, W. Trent; Akbey, Uemit; Lange, Sascha; Rossum, Barth-Jan van; Oschkinat, Hartmut, E-mail: oschkinat@fmp-berlin.de [Forschungsinstitut fuer Molekulare Pharmakologie (FMP) (Germany)

    2011-11-15

    X-ray crystallography using synchrotron radiation and the technique of dynamic nuclear polarization (DNP) in nuclear magnetic resonance (NMR) require samples to be kept at temperatures below 100 K. Protein dynamics are poorly understood below the freezing point of water and down to liquid nitrogen temperatures. Therefore, we investigate the {alpha}-spectrin SH3 domain by magic angle spinning (MAS) solid state NMR (ssNMR) at various temperatures while cooling slowly. Cooling down to 95 K, the NMR-signals of SH3 first broaden and at lower temperatures they separate into several peaks. The coalescence temperature differs depending on the individual residue. The broadening is shown to be inhomogeneous by hole-burning experiments. The coalescence behavior of 26 resolved signals (of 62) was compared to water proximity and crystal structure Debye-Waller factors (B-factors). Close proximity to the solvent and large B-factors (i.e. mobility) lead, generally, to a higher coalescence temperature. We interpret a high coalescence temperature as indicative of a large number of magnetically inequivalent populations at cryogenic temperature.

  18. CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations.

    Science.gov (United States)

    Liang, Huazheng; Akladios, Bassem; Canales, Cesar P; Francis, Richard; Hardeman, Edna H; Beverdam, Annemiek

    2016-01-01

    The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution. PMID:27584943

  19. Preparation and characterization of Protein A-immobilized PVDF and PES membranes

    Directory of Open Access Journals (Sweden)

    N. Akashi

    2015-01-01

    Full Text Available Polyvinylidene fluoride (PVDF and polyether sulfone (PES membranes were activated using low-temperature plasma at atmospheric pressure, and their surface characteristics were investigated. In the plasma-treated PVDF, the XPS data showed that defluorination and oxidation reactions proceeded to 18 and 31%, respectively, at ±4.0 kVp-p for 180 s. Hydroperoxide groups were detected on both the plasma-treated membranes. By decomposing the S2p spectrum, it was proven that the sulfide and sulfo groups were newly formed on the plasma-treated PES. Based on these findings, we proposed an activation mechanism. The SEM images showed that the macrovoid formations were maintained after the plasma treatment. Polyacrylic acid (PAA was grafted on both of the plasma-treated membranes by thermal treatments. Protein A, originating from Staphylococcus aureus, was immobilized on the membrane grafted with PAA using the EDC/Sulfo-NHS system. Adsorption isotherms with a human immunoglobulin G (IgG antibody were fitted with the monolayer Langmuir model, and the maximum binding capacity (qm and equilibrium association constant (Ka were obtained. The ligand densities of the PVDF (pore size 0.45 and 5.0 µm and PES (pore size 0.45 µm membranes were 0.98, 1.42 and 2.06 mg•mL–1, respectively.

  20. Characterization of stable, electroactive protein cage/synthetic polymer multilayer thin films prepared by layer-by-layer assembly

    Energy Technology Data Exchange (ETDEWEB)

    Uto, Koichiro [National Institute for Materials Science (NIMS), Biomaterials Unit, International Research Center for Materials Nanoarchitectonics (WPI-MANA) (Japan); Yamamoto, Kazuya [Graduate School of Science and Engineering, Kagoshima University, Department of Nanostructure and Advanced Materials (Japan); Kishimoto, Naoko [CREST, Japan Science and Technology Agency (Japan); Muraoka, Masahiro [Osaka Institute of Technology, Department of Applied Chemistry, Faculty of Engineering (Japan); Aoyagi, Takao, E-mail: aoyagi.takao@nims.go.jp [National Institute for Materials Science (NIMS), Biomaterials Unit, International Research Center for Materials Nanoarchitectonics (WPI-MANA) (Japan); Yamashita, Ichiro, E-mail: ichiro@ms.naist.jp [CREST, Japan Science and Technology Agency (Japan)

    2013-04-15

    We have fabricated electroactive multilayer thin films containing ferritin protein cages. The multilayer thin films were prepared on a solid substrate by the alternate electrostatic adsorption of (apo)ferritin and poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) (NIPAAm-co-CIPAAm) in pH 3.5 acetate buffer solution. The assembly process was monitored using a quartz crystal microbalance. The (apo)ferritin/poly(NIPAAm-co-CIPAAm) multilayer thin films were then cross-linked using a water-soluble carbodiimide, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. The cross-linked films were stable under a variety of conditions. The surface morphology and thickness of the multilayer thin films were characterized by atomic force microscopy, and the ferritin iron cores were observed by scanning electron microscopy to confirm the assembly mechanism. Cyclic voltammetry measurements showed different electrochemical properties for the cross-linked ferritin and apoferritin multilayer thin films, and the effect of stability of the multilayer film on its electrochemical properties was also examined. Our method for constructing multilayer films containing protein cages is expected to be useful in building more complex functional inorganic nanostructures.

  1. Preparation and rebinding properties of protein-imprinted polysiloxane using mesoporous calcium silicate grafted non-woven polypropylene as matrix.

    Science.gov (United States)

    Kan, Bohong; Feng, Lingzhi; Zhao, Kongyin; Wei, Junfu; Zhu, Dunwan; Zhang, Linhua; Ren, Qian

    2016-03-01

    Calcium silicate particle containing mesoporous SiO2 (CaSiO3@SiO2) was grafted on the surface of non-woven polypropylene. The PP non-woven grafted calcium silicate containing mesoporous SiO2 (PP-g-CaSiO3@SiO2) was used as the matrix to prepare bovine serum albumin (BSA) molecularly imprinted polysiloxane (MIP) by using silanes as the functional monomers and BSA as the template. PP non-woven grafted BSA-imprinted polysiloxane (PP-g-CaSiO3@SiO2 MIP) was characterized by scanning electron microscope (SEM), Fourier transform infrared spectometry (FTIR) and drilling string compensator (DSC). Influence factors on the rebinding capacity of the MIP were investigated, such as grafting degree, the pH in treating CaSiO3 and the type and proportion of silanes. The rebinding properties of BSA on PP-g-CaSiO3@SiO2 and MIP were investigated under different conditions. The results indicated that the rebinding capacity of MIP for BSA reached 56.32 mg/g, which was 2.65 times of NIP. The non-woven polypropylene grafted BSA-imprinted polysiloxane could recognize the template protein and the selectivity factor (β) was above 2.4 when using ovalbumin, hemoglobin and γ-globulin as control proteins. The PP-g-CaSiO3@SiO2 MIP has favorable reusability. PMID:25726930

  2. Characterization of stable, electroactive protein cage/synthetic polymer multilayer thin films prepared by layer-by-layer assembly

    International Nuclear Information System (INIS)

    We have fabricated electroactive multilayer thin films containing ferritin protein cages. The multilayer thin films were prepared on a solid substrate by the alternate electrostatic adsorption of (apo)ferritin and poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) (NIPAAm-co-CIPAAm) in pH 3.5 acetate buffer solution. The assembly process was monitored using a quartz crystal microbalance. The (apo)ferritin/poly(NIPAAm-co-CIPAAm) multilayer thin films were then cross-linked using a water-soluble carbodiimide, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. The cross-linked films were stable under a variety of conditions. The surface morphology and thickness of the multilayer thin films were characterized by atomic force microscopy, and the ferritin iron cores were observed by scanning electron microscopy to confirm the assembly mechanism. Cyclic voltammetry measurements showed different electrochemical properties for the cross-linked ferritin and apoferritin multilayer thin films, and the effect of stability of the multilayer film on its electrochemical properties was also examined. Our method for constructing multilayer films containing protein cages is expected to be useful in building more complex functional inorganic nanostructures.

  3. Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro

    Directory of Open Access Journals (Sweden)

    Lylo V. V.

    2014-11-01

    Full Text Available Aim. To study the effect of EMAP II, IFN-α2b and its medicinal preparations on the amount of O6-methylguanine-DNA methyltransferase (MGMT protein in human cells in vitro. Methods. The human cells of 4BL and Hep-2 lines were treated with the purified recombinant proteins EMAP II, IFN-α2b and its commercial me dicinal preparations. Changes in the MGMT gene expression were studied at a protein level by Western blot analysis. Results. Treatment of Hep-2 and 4BL cells with EMAP II at the concentrations of 0.02 mg/ml and 2 mg/ml respectively led to induction of the MGMT gene expression. EMAP II at the concentrations of 0.2–20 g/ml caused decrease of the MGMT protein amount in Hep-2 cells. The regulating activity of EMAP II was also observed for MARP (anti-Methyltransferase Antibody Recognizable Protein. IFN-α2b and Laferon-PharmBiotek with the activity of 200 and 2000 IU/ml were shown to cause an increase of the MGMT protein amount in Hep-2 cells. Conclusions. The purified recombinant proteins EMAP II and IFN-α2b which are substrates for the medicinal preparations influenced on the amount of MGMT protein in the human cell cultures in a concentration-dependent manner. At the same time the effect of medicinal preparations differs from that of the purified protein IFN-α2b. Possibly it depends on the presence of stabilizing components in their compositions.

  4. Food protein-stabilized nanoemulsions as potential delivery systems for poorly water-soluble drugs: preparation, in vitro characterization, and pharmacokinetics in rats

    Directory of Open Access Journals (Sweden)

    Zhiqiang Tian

    2011-03-01

    Full Text Available Wei He1, Yanan Tan1, Zhiqiang Tian1, Lingyun Chen2, Fuqiang Hu3, Wei Wu11Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, People's Republic of China; 2Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Alberta, Canada; 3Department of Pharmaceutics, School of Pharmacy, Zhejiang University, Hangzhou, Zhejiang, People's Republic of ChinaAbstract: Nanoemulsions stabilized by traditional emulsifiers raise toxicological concerns for long-term treatment. The present work investigates the potential of food proteins as safer stabilizers for nanoemulsions to deliver hydrophobic drugs. Nanoemulsions stabilized by food proteins (soybean protein isolate, whey protein isolate, ß-lactoglobulin were prepared by high-pressure homogenization. The toxicity of the nanoemulsions was tested in Caco-2 cells using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium-bromide viability assay. In vivo absorption in rats was also evaluated. Food protein-stabilized nanoemulsions, with small particle size and good size distribution, exhibited better stability and biocompatibility compared with nanoemulsions stabilized by traditional emulsifiers. Moreover, ß-lactoglobulin had a better emulsifying capacity and biocompatibility than the other two food proteins. The pancreatic degradation of the proteins accelerated drug release. It is concluded that an oil/water nanoemulsion system with good biocompatibility can be prepared by using food proteins as emulsifiers, allowing better and more rapid absorption of lipophilic drugs.Keywords: oil in water nanoemulsions, food proteins, poorly water-soluble drugs, biocompatibility, in vivo absorption

  5. Preparation and properties of BSA-loaded microspheres based on multi-(amino acid copolymer for protein delivery

    Directory of Open Access Journals (Sweden)

    Chen X

    2014-05-01

    Full Text Available Xingtao Chen,1 Guoyue Lv,1 Jue Zhang,2 Songchao Tang,2 Yonggang Yan,1 Zhaoying Wu,2 Jiacan Su,2 Jie Wei2 1College of Physical Science and Technology, Sichuan University, Chengdu, 2Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai, People’s Republic of China Abstract: A multi-(amino acid copolymer (MAC based on ω-aminocaproic acid, γ-aminobutyric acid, L-alanine, L-lysine, L-glutamate, and hydroxyproline was synthetized, and MAC microspheres encapsulating bovine serum albumin (BSA were prepared by a double-emulsion solvent extraction method. The experimental results show that various preparation parameters including surfactant ratio of Tween 80 to Span 80, surfactant concentration, benzyl alcohol in the external water phase, and polymer concentration had obvious effects on the particle size, morphology, and encapsulation efficiency of the BSA-loaded microspheres. The sizes of BSA-loaded microspheres ranged from 60.2 µm to 79.7 µm, showing different degrees of porous structure. The encapsulation efficiency of BSA-loaded microspheres also ranged from 38.8% to 50.8%. BSA release from microspheres showed the classic biphasic profile, which was governed by diffusion and polymer erosion. The initial burst release of BSA from microspheres at the first week followed by constant slow release for the next 7 weeks were observed. BSA-loaded microspheres could degrade gradually in phosphate buffered saline buffer with pH value maintained at around 7.1 during 8 weeks incubation, suggesting that microsphere degradation did not cause a dramatic pH drop in phosphate buffered saline buffer because no acidic degradation products were released from the microspheres. Therefore, the MAC microspheres might have great potential as carriers for protein delivery. Keywords: poly (amino acid copolymer, release, degradation

  6. Preparation of human tau exon-2- and -10-specific monoclonal antibodies for the recognition of brain tau proteins in various mammals.

    Science.gov (United States)

    Chen, Cao; Lv, Yan; Shi, Qi; Zhang, Bao-Yun; Chen, Li-Na; Xiao, Kang; Sun, Jing; Dong, Xiao-Ping

    2015-08-01

    The aggregations of tau protein in brain tissue have been described in a large number of neurodegenerative diseases; however, due to the lack of tau isoform- or exon-specific antibodies, the exact situations under which various brain tau isoforms can be found and their exact contributions during disease progression remain unknown. Therefore, in this study, we prepared tau exon-specific monoclonal antibodies (mAbs) that recognize different mammalian tau isoforms. Briefly, 3 Balb/c mice were separately immunized (3 mice per antigen) with the recombinant GST-fusion proteins, GST-tE2 and GST-tE10. Two hybridoma cell lines, 4A8 and 3E12, secreting antibodies against human tau exon-2 and -10 were established using the hybridoma technique. The sensitivity and specificity of the prepared mAbs were evaluated using indirect ELISA and western blot analysis. The ability of the prepared mAbs, 4A8 and 3E12, to recognize endogenous tau protein in the brain tissues of various mammals was estimated by immunoprecipitation. Based on the results of various verification methods, we found that the prepared mAbs, 4A8 and 3E12, not only specifically reacted with the individual recombinant GST tau exon fusion proteins, but also correctly recognized the recombinant human tau isoforms containing respective exon sequences, as shown by western blot analysis. Furthermore, western blot analysis and immunoprecipitation assays verified that the mAbs, 4A8 and 3E12, recognized endogenous tau proteins in human brain tissue, as well as tau proteins in a series of mammalian tissues, including goat, bovine, rabbit, hamster and mouse. Thus, in the present study, using the hybridoma technique, we successfully prepared the mAbs, 4A8 against tau exon-2 and 3E12 against tau exon-10, which provide useful tools for determining potential alternations of tau isoforms in neurodegenerative diseases. PMID:26046129

  7. Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Snakenborg, Detlef; Nielsen, Søren Skou;

    2011-01-01

    A new microfluidic sample-preparation system is presented for the structural investigation of proteins using small-angle X-ray scattering (SAXS) at synchrotrons. The system includes hardware and software features for precise fluidic control, sample mixing by diffusion, automated X-ray exposure co...

  8. A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

    Science.gov (United States)

    Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

    2016-08-01

    In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency. PMID:27321140

  9. The eff ect of addition of selected milk protein preparations on the growth of Lactobacillus acidophilus and physicochemical properties of fermented milk

    Directory of Open Access Journals (Sweden)

    Waldemar Gustaw

    2016-03-01

    Full Text Available Background. The intake of fermented milk products, especially yoghurts, has been systematically increasing for a few decades. The purpose of this work was to obtain milk products fermented with a mix of bacterial cultures (yoghurt bacteria and Lactobacillus acidophillus LA-5 and enriched with selected milk protein preparations. Secondly, the aim of the work was to determine physiochemical and rheological properties of the obtained products. Material and methods. The following additives were applied in the experiment: whey protein concen- trate (WPC 65, whey protein isolate (WPI, demineralised whey powder (SPD, caseinoglycomacropeptide (CGMP, α-lactalbumin (α-la, sodium caseinate (KNa and calcium caseinate (KCa. Milk was fermented using probiotic strain Lactobacillus acidophillus LA-5 and a typical yoghurt culture. The products were analysed in terms of the survivability of bacterial cells during refrigerated storage, rheological properties and syneresis. Fermented milk products were obtained using blends of bacterial strains: ST-B01:Lb-12 (1:1, ST-B01:Lb-12:LA-5 (1:1:2. Results. Milk beverages fermented with typical yoghurt bacteria and LA-5 strain showed intensive syner- esis. The addition of LA-5 strain caused formation of harder acid gels, comparing to typical yoghurts. Milk products which were prepared from skimmed milk possessed higher values of hardness and consistency coefficient. The increase of concentrations of milk preparations (except of WPI did not cause significant differences in the hardness of acidic gels obtained by fermentation of mixed culture with a probiotic strain. Conclusion. The applied preparations improved physiochemical properties of the milk beverages which were prepared with a probiotic strain. The increase of protein milk preparations concentration resulted in a gradual decrease of the secreted whey. Among the products that were made of full milk powder and were subjected to three weeks of refrigerated storage

  10. Comparison of heat and pressure treatments of skim milk, fortified with whey protein concentrate, for set yogurt preparation: effects on milk proteins and gel structure.

    Science.gov (United States)

    Needs, E C; Capellas, M; Bland, A P; Manoj, P; MacDougal, D; Paul, G

    2000-08-01

    Heat (85 degrees C for 20 min) and pressure (600 MPa for 15 min) treatments were applied to skim milk fortified by addition of whey protein concentrate. Both treatments caused > 90 % denaturation of beta-lactoglobulin. During heat treatment this denaturation took place in the presence of intact casein micelles; during pressure treatment it occurred while the micelles were in a highly dissociated state. As a result micelle structure and the distribution of beta-lactoglobulin were different in the two milks. Electron microscopy and immunolabelling techniques were used to examine the milks after processing and during their transition to yogurt gels. The disruption of micelles by high pressure caused a significant change in the appearance of the milk which was quantified by measurement of the colour values L*, a* and b*. Heat treatment also affected these characteristics. Casein micelles are dynamic structures, influenced by changes to their environment. This was clearly demonstrated by the transition from the clusters of small irregularly shaped micelle fragments present in cold pressure-treated milk to round, separate and compact micelles formed on warming the milk to 43 degrees C. The effect of this transition was observed as significant changes in the colour indicators. During yogurt gel formation, further changes in micelle structure, occurring in both pressure and heat-treated samples, resulted in a convergence of colour values. However, the microstructure of the gels and their rheological properties were very different. Pressure-treated milk yogurt had a much higher storage modulus but yielded more readily to large deformation than the heated milk yogurt. These changes in micelle structure during processing and yogurt preparation are discussed in terms of a recently published micelle model.

  11. Methoden zur Immobilisierung von Proteinen auf Polyurethan- und Goldoberflächen und ihr Einfluss auf Konformation und Aktivität der Proteine

    OpenAIRE

    Kreider, Alexej

    2014-01-01

    In recent years anti-freeze proteins became the focus of interest for materials science due to their ice-crystall-growth inhibiting properties, recrystallisation properties and ice-crystall structuring properties. The transfer of these properties to surfaces by means of a molecular biomimetic approach is the challenge as well as motivation of this work. Here, the molecular bionic approach is based on chemical immobilization methods of proteins to solid surfaces. Thus, the first question of th...

  12. Over-expression of 72 ku protein of wheat yellow mosaic virus in E.coli and preparation of its antiserum

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    By reverse transcription-polymerase chain reaction (RT-PCR),cDNA fragment of wheat yellow mosaic virus (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in prokaryotic cells.BL21(DE3) pLys S of E.coli transformed with the recombinant plasmid pETP72 containing the fragment has been induced to express the 72 ku protein on high level.The produced protein has been purified from sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for its antiserum preparation.In Western-blotting analysis,the antibodies reacted with the 72 ku protein expressed in E.coli.

  13. Simplified sample preparation method for protein identification by matrix-assisted laser desorption/ionization mass spectrometry: in-gel digestion on the probe surface

    DEFF Research Database (Denmark)

    Stensballe, A; Jensen, Ole Nørregaard

    2001-01-01

    Identification and detailed characterization of complex mixtures of proteins separated by polyacrylamide gel electrophoresis (PAGE) require optimized and robust methods for interfacing electrophoretic techniques to mass spectrometry. Peptide mapping by matrix-assisted laser desorption/ionization-......Identification and detailed characterization of complex mixtures of proteins separated by polyacrylamide gel electrophoresis (PAGE) require optimized and robust methods for interfacing electrophoretic techniques to mass spectrometry. Peptide mapping by matrix-assisted laser desorption...... for protein identification similar to that obtained by the traditional protocols for in-gel digestion and MALDI peptide mass mapping of human proteins, i.e. approximately 60%. The overall performance of the novel on-probe digestion method is comparable with that of the standard in-gel sample preparation...

  14. Antifreeze Proteins Enhance Survival of Cells in Cryopreservation - Substituting DMSO with RmAFP#1 in cryopreservation of cells

    OpenAIRE

    Henriksen, Beatriche L. E.; Kofod, Lotte; Gammeltoft, Karen A.; Christensen, Erik; Khan, Omar J.

    2015-01-01

    Cryopreservation is a useful method for preserving living cells and biological tissues. Dimethyl sulfoxide (DMSO) is considered the most effective cryoprotective agent (CPA) used in cryopreservation. DMSO helps to reduce ice crystallization within the cell and thus preventing cell death during the freezing and thawing process. However, DMSO has toxic effects on cells which are not only concentration dependent, but also temperature dependent. In this study, DMSO was substituted with an ins...

  15. Buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: from protein buffer capacity prediction to bioprocess applications.

    Science.gov (United States)

    Bahrenburg, Sven; Karow, Anne R; Garidel, Patrick

    2015-04-01

    Protein therapeutics, including monoclonal antibodies (mAbs), have significant buffering capacity, particularly at concentrations>50 mg/mL. This report addresses pH-related issues critical to adoption of self-buffered monoclonal antibody formulations. We evaluated solution conditions with protein concentrations ranging from 50 to 250 mg/mL. Samples were both buffer-free and conventionally buffered with citrate. Samples were non-isotonic or adjusted for isotonicity with NaCl or trehalose. Studies included accelerated temperature stability tests, shaking stability studies, and pH changes in infusion media as protein concentrate is added. We present averaged buffering slopes of capacity that can be applied to any mAb and present a general method for calculating buffering capacity of buffer-free, highly concentrated antibody liquid formulations. In temperature stability tests, neither buffer-free nor conventionally buffered solution conditions showed significant pH changes. Conventionally buffered solutions showed significantly higher opalescence than buffer-free ones. In general, buffer-free solution conditions showed less aggregation than conventionally buffered solutions. Shaking stability tests showed no differences between buffer-free and conventionally buffered solutions. "In-use" preparation experiments showed that pH in infusion bag medium can rapidly approximate that of self-buffered protein concentrate as concentrate is added. In summary, the buffer capacity of proteins can be predicted and buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions.

  16. Advanced fluidic handling and use of two-phase flow for high throughput structural investigation of proteins on a microfluidic sample preparation platform

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Snakenborg, Detlef; Møller, M.;

    2010-01-01

    Research on the structure of proteins can bring forth a wealth of information about biological function and can be used to better understand the processes in living cells. This paper reports a new microfluidic sample preparation system for the structural investigation of proteins by Small Angle X......-ray Scattering (SAXS). The system includes hardware and software features for precise fluidic control, synchrotron beamline control, UV absorbance measurements and automated data analysis. The precise fluidic handling capabilities are used to transport and precisely position samples as small as 500 nL into the...

  17. Effects of gamma irradiation on physicochemical properties of heat-induced gel prepared with chicken salt-soluble proteins

    International Nuclear Information System (INIS)

    The technological effects of gamma irradiation (0, 3, 7, and 10 kGy) on chicken salt-soluble meat proteins in a model system were investigated. There were no significant differences in protein, fat, and ash content, and sarcoplasmic protein solubility among all samples. The samples with increasing gamma irradiation levels had higher pH, lightness, yellowness, and apparent viscosity, whereas moisture content, water holding capacity, redness, myofibrillar protein solubility, total protein solubility, hardness, springiness, cohesiveness, gumminess, and chewiness were the highest in the unirradiated control. The result from meat products using gamma irradiation was intended to provide a basic resource processing technology. - Highlights: • The effect of gamma irradiation on salt-soluble meat proteins was investigated. • Gelling properties of salt-soluble protein affected by gamma irradiation. • Gamma irradiation of meat products provides a basic resource processing technology

  18. Research Progress of Preparation Technique of Peanut Protein%花生蛋白制备技术研究进展

    Institute of Scientific and Technical Information of China (English)

    林琳

    2015-01-01

    Peanut protein is rich in resources and nutritive value. Its prospect of development and utilization is extensive. In the article, it introduced the composition and nutritive value of peanut protein, discussed present preparation technique of peanut protein, including crushing(high temperature pressing and low temperature prepressing),organic solvent extraction,aqueous enzymatic method,alkali solu-ble acid sinking method,membrane separation technique,supercritical fluid extraction technique and reverse micelle extraction tech-nique. The aim is to offer a reference for peanut protein preparation industry.%花生蛋白资源丰富,营养价值高,拥有广泛的开发利用前景。介绍花生蛋白的构成及营养价值,探讨现有花生蛋白的制备技术,包括压榨法(高温压榨和低温预榨)、有机溶剂浸提法、水酶法、碱溶酸沉法、膜分离技术、超临界萃取技术及反胶束萃取技术,旨在为花生蛋白制备行业提供参考。

  19. When protein-based biomineralization meets hydrothermal synthesis: the nanostructures of the as-prepared materials are independent of the protein types.

    Science.gov (United States)

    Wang, Liqiang; Li, Xiangzhi; Jiang, Xingxing; Chen, Wansong; Hu, Lanshuang; Walle, Maru Dessie; Deng, Liu; Yang, Minghui; Liu, You-Nian; Kirin, Srećko I

    2015-12-14

    Proteins were proved to be type-independent templates for the biomineralization of iron ions into hematite mesocrystals with tunable structures and morphologies under hydrothemal conditions. Our finding could pave the way for the synthesis of mesocrystals with controlled stuctures and morphologies using templates of low-cost proteins.

  20. Preparation and properties of cottonseed protein/polyurethane composite%棉籽蛋白/聚氨酯复合材料的制备与性能

    Institute of Scientific and Technical Information of China (English)

    王长松; 陈磊

    2012-01-01

    为了开发棉籽蛋白在材料领域的应用,利用含肽键的棉籽蛋白与含酰胺基的聚氨酯预聚物共混改性反应制备复合材料,来改善棉籽蛋白的力学性能和耐水性,以保持其生物降解性.利用反应挤出技术,将棉籽分离蛋白与聚氨酯预聚物共混挤出,采用热压工艺,制备了聚氨酯预聚物交联的可降解棉籽蛋白复合材料.结果表明,该材料的加工性、力学性能和耐水性优良.随着聚氨酯组分的增加,材料的断裂伸长率增加,耐水性提高,其中,聚氨酯预聚物质量分数为50%的复合材料,其拉伸强度、断裂伸长率和耐水性分别达到7 MPa、150%和20%,是优良的可降解韧性复合材料.%In order to promote the application of cottonseed protein in material field,the blending modification reaction between cottonseed protein containing peptide bonds and polyurethane prepolymer containing amide groups was used for preparing the composites to improve the mechanical properties and water resistance of cottonseed protein and thus maintain the biodegradability of cottonseed protein.The reaction extrusion technology was applied to perform the blending extrusion of isolated cottonseed protein and polyurethane prepolymer.The degradable cottonseed protein composites with cross-linking of polyurethane prepolymer were prepared with hot extrusion technology.The results show that the workability,mechanical properties and water resistance of the prepared composites are excellent.With increasing the content of polyurethane prepolymer,the fracture elongation and water resistance of the composites get enhanced.The tensile strength,fracture elongation and water resistance of the composite with 50% of polyurethane prepolymer reach 7 MPa,150% and 20%,respectively.It is obvious that the prepared material is an excellent degradable ductile composite.

  1. Anti-freezing Piping Design and Analysis of Chemical Plants in North China%我国北方地区化工装置管道防冻设计分析

    Institute of Scientific and Technical Information of China (English)

    王轲

    2016-01-01

    对于建设在北方地区的化工装置来说,冬季管道防冻一直是各生产企业非常重视的问题,但由于在设计阶段重视不够,往往给各化工生产企业的生产安全埋下了不小的隐患。本文从防冻设计源头控制的理念出发,优化管道布置,增加管道伴热,并从多方面、全角度阐述化工装置管道防冻设计要点,力求做到防患于未然。%In construction of a chemical plant in North China, pipeline anti-freezing has been the issue that production enterprises pay attentions to.Be-cause of insufficient attention to pipe anti-freezing at design stage, hidden troubles are existing for chemical production enterprises.In this paper, the au-thor starts from source control of anti-freezing design, optimizes piping layout, adds pipe tracing, and elaborates the anti-freezing piping design highlights of chemical plants from diversified aspects and angles, and strives to accomplish a nip in the bud.

  2. 酶法水解制备植物蛋白肽粉的研究%Enzymatic preparation of peptides from vegetable protein

    Institute of Scientific and Technical Information of China (English)

    孔祥珍; 华欲飞; 张彩猛

    2013-01-01

    Soybean protein isolate and wheat gluten were hydrolyzed by Alcalase to prepare peptides. The enzymatic hydrolysis conditions (pH, temperature and time) were investigated. The results showed that the hydrolysis degree of wheat gluten was higher than that of soybean protein isolate under the same conditions, and the relative molecular mass distribution of wheat peptides was smaller than that of soybean peptides. Peptides prepared with the mixed proteins (soybean protein isolate and wheat gluten) had more balanced amino acid composition, which could meet the requirement recommended by FAO, and the content of free amino acids of the peptides was few. The surface hydrophobicity of the peptides was much lower than that of protein materials. The results provided the theoretical basis of preparing vegetable protein peptides by regulating proportion of protein materials.%以大豆分离蛋白和谷朊粉为原料,选择碱性蛋白酶(Alcalase)酶解制备植物蛋白肽粉.考察了pH、酶解温度、酶解时间对大豆分离蛋白、谷朊粉及二者混合物分别酶解的影响.结果表明:相同的条件下谷朊粉的酶解程度比大豆分离蛋白高,所得肽粉的相对分子质量整体分布更小一些;大豆分离蛋白和谷朊粉混合物酶解制备的肽粉,其氨基酸组成比例基本满足FAO推荐的必需氨基酸模式,且肽粉中游离氨基酸含量很少;肽粉的表面疏水性远小于大豆分离蛋白和谷朊粉.研究结果为通过调节蛋白原料比例(大豆分离蛋白、谷朊粉等)来制备具有合适氨基酸配比的植物蛋白肽粉提供了理论基础.

  3. Preparation of metallochelating microbubbles and study on their site-specific interaction with rGFP-HisTag as a model protein.

    Science.gov (United States)

    Lukáč, Róbert; Kauerová, Zuzana; Mašek, Josef; Bartheldyová, Eliška; Kulich, Pavel; Koudelka, Štěpán; Korvasová, Zina; Plocková, Jana; Papoušek, František; Kolář, František; Schmidt, Roland; Turánek, Jaroslav

    2011-04-19

    The histidine-metallochelating lipid complex is one of the smallest high affinity binding units used as tools for rapid noncovalent binding of histidine tagged molecules, especially recombinant proteins. The advantage of metallochelating complex over protein-ligand complexes (e.g., streptavidine-biotin, glutathiontransferase-glutathion) consists in its very low immunogenicity, if any. This concept for the construction of surface-modified metallochelating microbubbles was proved with recombinant green fluorescent protein (rGFP) containing 6His-tag. This protein is easy to be detected by various fluorescence techniques as flow cytometry and confocal microscopy. Microbubbles (MB) composed of DPPC with various contents of metallochelating lipid DOGS-NTA-Ni were prepared by intensive shaking of the liposome suspension under the atmosphere of sulfur hexafluoride. For this purpose, the instrument 3M ESPE CapMix was used. Various techniques (static light scattering, flow cytometry, and optical microscopy) were compared and used for the measurements of the size distribution of MB. All three methods demonstrated that the prepared MB were homogeneous in their size, and the mean diameter of the MB in various batches was within the range of 2.1-2.8 μm (the size range of 1-10 μm). The presence of large MB (8-10 μm) was marginal. Counting of MB revealed that the average amount of MB prepared of 10 mg of phospholipid equaled approximately 10(9) MB/mL. Lyophilized MB were prepared with saccharose as a cryoprotectant. These MB were shown to be stable both in vitro (the estimated half-live of the MB in bovine serum at 37 °C was 3-7 min) and in vivo (mouse). The stability of the MB was affected by molar content of DOGS-NTA-Ni. DPPC-based metallochelating MB provided a clear and very contrast image of the ventricular cavity soon after the injection. Site selective and stable binding of rGFP-HisTag (as a model of His-tagged protein) onto the surface of metallochelating MB was

  4. Vi capsular polysaccharide-protein conjugates for prevention of typhoid fever. Preparation, characterization, and immunogenicity in laboratory animals.

    Science.gov (United States)

    Szu, S C; Stone, A L; Robbins, J D; Schneerson, R; Robbins, J B

    1987-11-01

    The Vi has proven to be a protective antigen in two double masked, controlled clinical trials in areas with high rates of typhoid fever (approximately 1% per annum). In both studies the protective efficacy of the Vi was approximately 70%. Approximately 75% of subjects in these areas responded with a fourfold or greater rise of serum Vi antibodies. In contrast, the Vi elicited a fourfold or greater rise in 95-100% of young adults in France and the United States. Methods were devised, therefore, to synthesize Vi-protein conjugates in order to both enhance the antibody response and confer T-dependent properties to the Vi (and theoretically increase its protective action in populations at high risk for typhoid fever). We settled on a method that used the heterobifunctional crosslinking reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), to bind thiol derivatives of the Vi to proteins. This synthetic scheme was reproducible, provided high yields of Vi-protein conjugates, and was applicable to several medically relevant proteins such as diphtheria and tetanus toxoids. The resultant conjugates were more immunogenic in mice and juvenile Rhesus monkeys than the Vi alone. In contrast to the T-independent properties of the Vi, conjugates of this polysaccharide with several medically relevant proteins induced booster responses in mice and in juvenile Rhesus monkeys. Clinical studies with Vi-protein conjugates are planned. This scheme is also applicable to synthesize protein conjugates with other polysaccharides that have carboxyl functions.

  5. Preparation of magnetic chitosan and graphene oxide-functional guanidinium ionic liquid composite for the solid-phase extraction of protein

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Xueqin; Wang, Yuzhi, E-mail: wyzss@hnu.edu.cn; Wang, Ying; Pan, Qi; Chen, Jing; Huang, Yanhua; Xu, Kaijia

    2015-02-25

    Highlights: • A strategy for the solid-phase extraction of protein based on magnetic chitosan and graphene oxide-functional guanidinium ionic liquids. • Trypsin, lysozyme, ovalbumin and bovine serum albumin were used as the analyst. • The possibility of reusability and regeneration has been evaluated. - Abstract: A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been synthesized, and then magnetic chitosan graphene oxide (MCGO) composite has been prepared and coated with these functional guanidinium ionic liquids to extract protein by magnetic solid-phase extraction. MCGO-functional guanidinium ionic liquid has been characterized by vibrating sample magnetometer, field emission scanning electron microscopy, X-ray diffraction spectrometer and Fourier transform infrared spectrometer. After extraction, the concentrations of protein were determined by measuring the absorbance at 278 nm using an ultra violet visible spectrophotometer. The advantages of MCGO-functional guanidinium ionic liquid in protein extraction were compared with magnetic chitosan, graphene oxide, MCGO and MCGO-ordinary imidazolium ionic liquid. The proposed method has been applied to extract trypsin, lysozyme, ovalbumin and bovine serum albumin. A comprehensive study of the adsorption conditions such as the concentration of protein, the amount of MCGO-functional guanidinium ionic liquid, the pH, the temperature and the extraction time were also presented. Moreover, the MCGO-functional guanidinium ionic liquid can be easily regenerated, and the extraction capacity was about 94% of the initial one after being used three times.

  6. Preparation, characterization and protein sorption of photo-crosslinked cell membrane-mimicking chitosan-based hydrogels.

    Science.gov (United States)

    Zhao, Yunfei; Ma, Liubo; Zeng, Rong; Tu, Mei; Zhao, Jianhao

    2016-10-20

    Photocrosslinkable biomimetic chitosan derivative, glycidyl methacrylate-phosphorylcholine-chitosan (PCCs-GMA) was synthesized through the combination of Atherton-Todd reaction for coupling phosphorylcholine and ring opening reaction of epoxides for attaching GMA, and confirmed by (1)H and (31)P NMR and Fourier transform infrared (FTIR) spectroscopy. The photo-crosslinking reaction of PCCs-GMA with different degree of substitution (DS) of GMA allowed the formation of biomimetic hydrogels with tunable mechanical and swelling properties. Cold crystallization behaviors ascribed to their restrained freezing bound water were investigated using differential scanning calorimetry (DSC). The rheological and swelling behaviors, hemolysis as well as protein sorption of PCCs-GMA hydrogels were investigated in terms of the DS of GMA, using fibrinogen, bovine serum albumin and lysozyme as model proteins. Low irreversible protein sorption and non hemolytic results indicated that photo-crosslinked PCCs-GMA hydrogels may offer a promising candidate material with resistance to protein fouling in biomedical applications. PMID:27474563

  7. EFFECTS OF CORDYCEPS SINENSIS PREPARATION ON BODY PROTEIN AND AMINO ACID METABOLISM IN PATIENTS AND RATS WITH CHRONIC RENAL FAILURE

    Institute of Scientific and Technical Information of China (English)

    朱淳; 刘强; 左静南; 朱汉威; 马济民

    2002-01-01

    Objective To study the effects of Cordyceps sinensis (CS) on the metabolism of body protein and intra-extracellular amino acids in patients with chronic renal failure( CRF) , and on the rates of protein synthesis in rats with CRF. Methods In patients with CRF, free amino acid concentrations in plasma and skeletal muscle before and after CS treatment were measured by the LKB-4400 amino acid automatic analytical instrument and the changes of body protein metabolism were observed by the method of 15 N-labeled glycine.Meanwhile, the rates of protein synthesis in liver ( SL % /d ) and muscle (SM%/d) of rats with CRF were determinedd by 3f-phenylalanine radioactive tracer. Results After patients with CRF were treated by CS, the Leu, lie, Thr , Lys, Cys, Tyr concentrations in plasma approached the normal levels. In one sample of skeletal muscle the Thr and Lys concentrations approached the normal, whereas both the intracellular and extracellular Val concentrations were still remarkably decreased as compared with the normal controls. Moreover, the nitrogen flow rate (Q) , rates of protein synthesis (S) and catabolism ( C) , and amino nitrogen utilization ratio (S/Q) in patients with CRF and the SL % /d and SM%/d in rats with CRF were significantly increased as compared with those before CS treatment. Conclusion CS can notably improve the amino acid metabolism, promote the body protein synthesis in patients with CRF , and increase the rates of SL % /d and SM%/d in rats with CRF.

  8. Ethylene glycol assisted preparation of Ti(4+)-modified polydopamine coated magnetic particles with rough surface for capture of phosphorylated proteins.

    Science.gov (United States)

    Ma, Xiangdong; Ding, Chun; Yao, Xin; Jia, Li

    2016-07-27

    The reversible protein phosphorylation is very important in regulating almost all aspects of cell life, while the enrichment of phosphorylated proteins still remains a technical challenge. In this work, polydopamine (PDA) modified magnetic particles with rough surface (rPDA@Fe3O4) were synthesized by introduction of ethylene glycol in aqueous solution. The PDA coating possessing a wealth of catechol hydroxyl groups could serve as an active medium to immobilize titanium ions through the metal-catechol chelation, which makes the fabrication of titanium ions modified rPDA@Fe3O4 particles (Ti(4+)-rPDA@Fe3O4) simple and very convenient. The spherical Ti(4+)-rPDA@Fe3O4 particles have a surface area of 37.7 m(2) g(-1) and superparamagnetism with a saturation magnetization value of 38.4 emu g(-1). The amount of Ti element in the particle was measured to be 3.93%. And the particles demonstrated good water dispersibility. The particles were used as adsorbents for capture of phosphorylated proteins and they demonstrated affinity and specificity for phosphorylated proteins due to the specific binding sites (Ti(4+)). Factors affecting the adsorption of phosphorylated proteins on Ti(4+)-rPDA@Fe3O4 particles were investigated. The adsorption capacity of Ti(4+)-rPDA@Fe3O4 particles for κ-casein was 1105.6 mg g(-1). Furthermore, the particles were successfully applied to isolate phosphorylated proteins in milk samples, which demonstrated that Ti(4+)-rPDA@Fe3O4 particles had potential application in selective separation of phosphorylated proteins.

  9. Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein.

    Science.gov (United States)

    Zhang, Yongning; Wu, Shaoqiang; Song, Shanshan; Lv, Jizhou; Feng, Chunyan; Lin, Xiangmei

    2015-08-01

    Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that predominantly infects livestock such as cattle, sheep, and goats. Its nucleocapsid (N) protein is an ideal target antigen for SBV diagnosis. In this study, a stable BHK-21 cell line, BHK-21-EGFP-SBV-N, constitutively expressing the SBV N protein was obtained using a lentivector-mediated gene transfer system combined with puromycin selection. To facilitate the purification of recombinant SBV N protein, the coding sequence for a hexa-histidine tag was introduced into the C-terminus of the SBV N gene during construction of the recombinant lentivirus vector pLV-EGFP-SBV-N. The BHK-21-EGFP-SBV-N cell line was demonstrated to spontaneously emit strong enhanced green fluorescent protein (EGFP) signals that exhibited a discrete punctate distribution throughout the cytoplasm. SBV N mRNA and protein expression in this cell line were detected by real-time RT-PCR and western blot, respectively. The expressed recombinant SBV N protein carried an N-terminal EGFP tag, and was successfully purified using Ni-NTA agarose by means of its C-terminal His tag. The purified SBV N protein could be recognized by SBV antisera and an anti-SBV monoclonal antibody (mAb) 2C8 in an indirect enzyme-linked immunosorbent assay and western blot analyses. Indirect immunofluorescence assays further demonstrated that the stable cell line reacts with SBV antisera and mAb 2C8. These results suggest that the generated cell line has the potential to be used in the serological diagnosis of SBV. PMID:26013296

  10. Ethylene glycol assisted preparation of Ti(4+)-modified polydopamine coated magnetic particles with rough surface for capture of phosphorylated proteins.

    Science.gov (United States)

    Ma, Xiangdong; Ding, Chun; Yao, Xin; Jia, Li

    2016-07-27

    The reversible protein phosphorylation is very important in regulating almost all aspects of cell life, while the enrichment of phosphorylated proteins still remains a technical challenge. In this work, polydopamine (PDA) modified magnetic particles with rough surface (rPDA@Fe3O4) were synthesized by introduction of ethylene glycol in aqueous solution. The PDA coating possessing a wealth of catechol hydroxyl groups could serve as an active medium to immobilize titanium ions through the metal-catechol chelation, which makes the fabrication of titanium ions modified rPDA@Fe3O4 particles (Ti(4+)-rPDA@Fe3O4) simple and very convenient. The spherical Ti(4+)-rPDA@Fe3O4 particles have a surface area of 37.7 m(2) g(-1) and superparamagnetism with a saturation magnetization value of 38.4 emu g(-1). The amount of Ti element in the particle was measured to be 3.93%. And the particles demonstrated good water dispersibility. The particles were used as adsorbents for capture of phosphorylated proteins and they demonstrated affinity and specificity for phosphorylated proteins due to the specific binding sites (Ti(4+)). Factors affecting the adsorption of phosphorylated proteins on Ti(4+)-rPDA@Fe3O4 particles were investigated. The adsorption capacity of Ti(4+)-rPDA@Fe3O4 particles for κ-casein was 1105.6 mg g(-1). Furthermore, the particles were successfully applied to isolate phosphorylated proteins in milk samples, which demonstrated that Ti(4+)-rPDA@Fe3O4 particles had potential application in selective separation of phosphorylated proteins. PMID:27251945

  11. Preparation and characterization of folate-poly(ethylene glycol)-grafted-trimethylchitosan for intracellular transport of protein through folate receptor-mediated endocytosis.

    Science.gov (United States)

    Zheng, Yu; Song, Xiangrong; Darby, Michael; Liang, Yufeng; He, Ling; Cai, Zheng; Chen, Qiuhong; Bi, Yueqi; Yang, Xiaojuan; Xu, Jiapeng; Li, Yuanbo; Sun, Yiyi; Lee, Robert J; Hou, Shixiang

    2010-01-01

    To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins to specific tumor cells, folate-poly(ethylene glycol)-grafted-trimethylchitosan (folate-PEG-g-TMC) was synthesized. Nano-scaled spherical polyelectrolyte complexes between the folate-PEG-g-TMC and fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) were prepared under suitable weight ratio of copolymer to FITC-BSA by ionic interaction between the positively charged copolymers and the negatively charged FITC-BSA. Intracellular uptake of FITC-BSA was specifically enhanced in SKOV3 cells (folate receptor over-expressing cell line) through folate receptor-mediated endocytosis compared with A549 cells (folate receptor deficient cell line). Folate-PEG-g-TMC shows promise for intracellular transport of negatively charged therapeutic proteins into folate receptor over-expressing tumor cells.

  12. Preparation of Monoclonal Antibodies Against SpiC Protein Secreted by T3SS-2 of Salmonella spp.

    Science.gov (United States)

    Geng, Shizhong; Qian, Shanshan; Pan, Zhiming; Sun, Lin; Chen, Xiang; Jiao, Xinan

    2015-12-01

    SpiC protein, a member of Salmonella spp. type III secretion system (T3SS)-2, is necessary for the survival of Salmonella within macrophages, and it plays a vital role in Salmonella pathogenesis. To develop and test monoclonal antibodies (MAbs) against SpiC protein, two recombinant proteins, rHis-SpiC and rGST-SpiC, were expressed in vitro in the prokaryotic expression vectors pET-30(a) and pGEX-6p-1, respectively, and rHis-SpiC protein used to immunize mice. Hybridomas were generated from the splenocytes of these mice and the monoclonal antibodies produced by these cells were assessed using indirect enzyme-linked immunosorbent assay (ELISA) with rGST-SpiC as the coating antigen. An immunoblotting analysis indicated that all seven of the MAbs developed in this study could specifically recognize the SpiC protein. These MAbs will be very useful in the study of SpiC function and for use in the immunodiagnosis of Salmonella infection.

  13. Preparation of Monoclonal Antibodies Against SpiC Protein Secreted by T3SS-2 of Salmonella spp.

    Science.gov (United States)

    Geng, Shizhong; Qian, Shanshan; Pan, Zhiming; Sun, Lin; Chen, Xiang; Jiao, Xinan

    2015-12-01

    SpiC protein, a member of Salmonella spp. type III secretion system (T3SS)-2, is necessary for the survival of Salmonella within macrophages, and it plays a vital role in Salmonella pathogenesis. To develop and test monoclonal antibodies (MAbs) against SpiC protein, two recombinant proteins, rHis-SpiC and rGST-SpiC, were expressed in vitro in the prokaryotic expression vectors pET-30(a) and pGEX-6p-1, respectively, and rHis-SpiC protein used to immunize mice. Hybridomas were generated from the splenocytes of these mice and the monoclonal antibodies produced by these cells were assessed using indirect enzyme-linked immunosorbent assay (ELISA) with rGST-SpiC as the coating antigen. An immunoblotting analysis indicated that all seven of the MAbs developed in this study could specifically recognize the SpiC protein. These MAbs will be very useful in the study of SpiC function and for use in the immunodiagnosis of Salmonella infection. PMID:26683183

  14. Preparation of cottonseed protein concentrate by enzymolysis and alcohol extraction%酶解醇洗法制备棉籽浓缩蛋白

    Institute of Scientific and Technical Information of China (English)

    潘晶; 陈思思; 张晖; 郭晓娜

    2011-01-01

    The pre-pressed cottonseed meal as material was firstly hydrolyzed by pentosan compound enzyme and glucoamylase, and then washed by ethanol to preparate protein concentrate. The result illustrated as that:Under the condition of adding glucoamylase 1 600 U/g, pentosan enzyme 0. 60 fbg/g, solvent/solid ratio of 1:10, temperature 50℃, the cottonseed meal was hydrolyzed for 140 min, and then washed by 70% (v/v) ethanol at 60℃ dipped for 2 times(once for 45 min), with solvent/solid ratio of 1 :7. The protein content of cottonseed protein concentrate prepared was 64 %, and the yield of protein was 78 %.%以预榨浸提棉籽粕为原料,先选用戊聚糖复合酶、糖化酶等植物水解酶对棉籽粕进行作用,再进一步用乙醇溶液浸洗制备棉籽浓缩蛋白.结果表明:在添加糖化酶1600 U/g、戊聚糖复合酶0.60 fbg/g、料液比1 ∶ 10和温度50℃条件下酶作用140 min后,再以料液比1∶7、体积分数70%乙醇溶液和温度60℃条件下醇洗2次,每次45 min,得到产品浓缩蛋白的蛋白质质量分数为64%,蛋白质收率为78%.

  15. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Wei, Shi-Cheng, E-mail: kqsc-wei@bjmu.edu.cn [Peking University School of Stomatology, Beijing 100081 (China); Liang, Yu-He, E-mail: kqsc-wei@bjmu.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China)

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  16. Preparation and In Vitro Release of Drug-Loaded Microparticles for Oral Delivery Using Wholegrain Sorghum Kafirin Protein

    Directory of Open Access Journals (Sweden)

    Esther T. L. Lau

    2015-01-01

    Full Text Available Kafirin microparticles have been proposed as an oral nutraceutical and drug delivery system. This study investigates microparticles formed with kafirin extracted from white and raw versus cooked red sorghum grains as an oral delivery system. Targeted delivery to the colon would be beneficial for medication such as prednisolone, which is used in the management of inflammatory bowel disease. Therefore, prednisolone was loaded into microparticles of kafirin from the different sources using phase separation. Differences were observed in the protein content, in vitro protein digestibility, and protein electrophoretic profile of the various sources of sorghum grains, kafirin extracts, and kafirin microparticles. For all of the formulations, the majority of the loaded prednisolone was not released in in vitro conditions simulating the upper gastrointestinal tract, indicating that most of the encapsulated drug could reach the target area of the lower gastrointestinal tract. This suggests that these kafirin microparticles may have potential as a colon-targeted nutraceutical and drug delivery system.

  17. Quantitative analysis of plasma proteins in whole blood-derived fresh frozen plasma prepared with three pathogen reduction technologies.

    Science.gov (United States)

    Larrea, Luis; Ortiz-de-Salazar, María-Isabel; Martínez, Patricia; Roig, Roberto

    2015-06-01

    Several plasma pathogen reduction technologies (PRT) are currently available. We evaluated three plasma PRT processes: Cerus Amotosalen (AM), Terumo BCT riboflavin (RB) and Macopharma methylene blue (MB). RB treatment resulted in the shortest overall processing time and in the smallest volume loss (1%) and MB treatment in the largest volume loss (8%). MB treatment retained the highest concentrations of factors II, VII, X, IX, Protein C, and Antithrombin and the AM products of factor V and XI. Each PRT process evaluated offered distinct advantages such as procedural simplicity and volume retention (RB) and overall plasma protein retention (MB).

  18. Antifreeze gene and its application in gene engineering%抗冻基因及其在基因工程中的应用研究进展

    Institute of Scientific and Technical Information of China (English)

    郝凤; 刘晓静; 周爱琴; 于铁峰

    2009-01-01

    低温是限制植物分布与生长的重要因素,低温伤害是一种严重的自然灾害,全球每年因此造成农作物的损失高达数千亿美元.本文综述了抗寒基因研究中一些已分离和鉴定出的低温诱导表达基因,对抗冻基因的功能特性和作用机制进行了全面的回顾,总结了抗冻基因工程的研究方向,对典型抗冻基因的表达效果进行了比较分析,并提出此领域尚存在的一些问题及发展前景.%Hypothermia is to limit the distribution and growth of plants important factor,Low-temperature injury is a serious natural disasters,The world′s crops each year resulting in the loss of up to hundreds of billions of dollars. Antifreeze genetic engineering is the field of biotechnology has been one of the hot research. Cold resistance gene has attracted wide attention from many countries scholars since it was been discovered. Many crops,especially fruits and vegetables,not only during cultivation in the fields,but also in post-harvest cold storage,frozen storage and frozen will be encountered during the processing of frozen injury. However, using conventional breeding methods to improve the cold tolerance of crops will encounter many difficulties. With the development of molecular biology,gene cloning technology involved in the progressive study of plant cold-hardiness,In this paper,resistance gene has been isolated and identified some of the low-temperature-induced gene expression,function and antifreeze mechanisms of cold resistance gene are reviewed,frost summed up the direction of genetic engineering research,on the typical effects of antifreeze genes in a comparative analysis of expression,and put forward in this field are still some problems and prospects.

  19. Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins.

    NARCIS (Netherlands)

    Lu, J.; Boeren, J.A.; Vries, de S.C.; Valenberg, van H.J.F.; Vervoort, J.J.M.; Hettinga, K.A.

    2011-01-01

    Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as

  20. Developing immunological methods for detecting Macrobrachium rosenbergii nodavirus and extra small virus using a recombinant protein preparation.

    Science.gov (United States)

    Wang, C-S; Chang, C-Y; Wen, C-M

    2016-06-01

    Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) have been identified as the causative agents for white tail disease (WTD) of M. rosenbergii. In this study, the gene sequences encoding MrNV and XSV capsid proteins were separately ligated into the pGEX-4T-3 expression vector and transformed into Escherichia coli. After induction, glutathione-S-transferase (GST)-tagged MrNV and XSV fusion proteins were obtained with molecular masses of 68 and 43 kDa, respectively. Specific polyclonal antibodies for MrNV and XSV against viral recombinant proteins and infected prawn tissues were verified using Western blotting. According to immunodot blot assay results, the detection sensitivities of antibodies were approximately 5 ng μL(-1) for both recombinant proteins GST-MrNV and GST-XSV. In additional, MrNV and XSV were detected at dilution levels of 1:2560 and 1:640 in the infected prawn tissues, respectively. No cross-reactions with white spot syndrome virus or grouper nervous necrosis virus were observed using immunodot blot assays. MrNV and XSV in infected muscle tissues were detected using immunohistochemistry. Although the detection limit of the immunodot blot assay was lower than that of nested reverse transcription polymerase chain reaction, these polyclonal antibodies can be useful for confirming MrNV and XSV infections in field tests.

  1. Lack of immunogenicity of ice structuring protein type III HPLC12 preparation administered by the oral route to human volunteers

    DEFF Research Database (Denmark)

    Crevel, R W R; Cooper, K J; Poulsen, Lars K.;

    2007-01-01

    background against which to interpret the results. Nevertheless, the absence of an immune response using a protocol which could have been expected to result in a response with a strongly immunogenic protein, confirms the conclusions of earlier published work, and attests to the lack of allergenicity of ISP...

  2. Preparation and characterization of protein loaded microspheres based on a hydroxylated aliphatic polyester, poly (lactic-co-hydroxymethyl glycolic acid)

    NARCIS (Netherlands)

    Ghassemi, A.H.; van Steenbergen, M.J.; Talsma, H.; van Nostrum, C.F.; Jiskoot, W.; Crommelin, D.J.A.; Hennink, W.E.

    2009-01-01

    The purpose of this study was to investigate the suitability of a novel hydroxylated aliphatic polyester, poly (lactic-co-hydroxymethyl glycolic acid) (PLHMGA), as controlled release system for pharmaceutical proteins. Dextran Blue (as a macromolecular model compound) and lysozyme-loaded PLHMGA and

  3. Induction of heat shock protein (hsp)60 in Isochrysis galbana exposed to sublethal preparations of dispersant and Prudhoe Bay crude oil

    Energy Technology Data Exchange (ETDEWEB)

    Wolfe, M.F.; Olsen, H.E.; Gasuad, K.A.; Tjeerdema, R.S. [University of California, Santa Cruz (United States). Dept. of Chemistry and Biochemistry; Sowby, M.L. [California Dept. of Fish and Game, Sacramento (United States). Office of Spill Prevention and Response

    1999-11-01

    Adaptation to sublethal exposure to crude oil by phytoplankton is poorly understood. Use of chemical dispersants for oil spill remediation increases petroleum hydrocarbon concentrations in water, while exposing marine organisms to potentially toxic concentrations of dispersant. Heat shock proteins (hsps) have been found to serve as an adaptive and protective mechanism against environmental stresses. The objective of this project was to examine the induction of hsps in Isochrysis galbana, a golden-brown algae, following exposure to the water-accommodated fraction (WAF) of Prudhoe Bay crude oil (PBCO) and PBCO chemically dispersed with Corexit 9527 (dispersed oil: DO). Initial experiments using {sup 35}S-labelled amino acids and 2-dimensional electrophoresis with subsequent western blotting identified and confirmed hsp60, a member of the chaperonin family of stress proteins, as being efficiently induced by heat shock in this species. One-dimensional SDS PAGE and western blotting, with hsp60 antibodies and chemiluminescence detection, were used to quantitate hsp60 following exposure to a range of environmental temperatures and concentrations of WAF and DO preparations. Results of this study are consistent with previous studies in other species documenting increases in hsp60 levels with exposure to xenobiotics. Further studies are investigating the protective function of hsp60 against the toxic effects of exposure to WAF and DO preparations. (author)

  4. Trehalose limits BSA aggregation in spray-dried formulations at high temperatures: implications in preparing polymer implants for long-term protein delivery.

    Science.gov (United States)

    Rajagopal, Karthikan; Wood, Joseph; Tran, Benjamin; Patapoff, Thomas W; Nivaggioli, Thierry

    2013-08-01

    Polymer implants are promising systems for sustained release applications but their utility for protein delivery has been hindered because of concerns over drug stability at elevated temperatures required for processing. Using bovine serum albumin (BSA) as a model, we have assessed whether proteins can be formulated for processing at elevated temperatures. Specifically, the effect of trehalose and histidine-HCl buffer on BSA stability in a spray-dried formulation has been investigated at temperatures ranging from 80°C to 110°C. When both the sugar and buffer are present, aggregation is suppressed even when exposed to 100°C, the extrusion temperature of poly(lactide-co-glycolide) (PLGA), a bioresorbable polymer. Estimation of aggregation rate constants (k) indicate that though both trehalose and histidine-HCl buffer contribute to BSA stability, the effect because of trehalose alone is more pronounced. BSA-loaded PLGA implants were prepared using hot-melt extrusion process and in vitro release was conducted in phosphate buffered saline at 37°C. Comparison of drug released from implants prepared using four different formulations confirmed that maximal release was achieved from the formulation in which BSA was least aggregated. These studies demonstrate that when trehalose and histidine-HCl buffer are included in spray-dried formulations, BSA stability is maintained both during processing at 100°C and long-term residence within implants.

  5. Effect of compound preparation Tongqiao Jiannao capsules on neural cell apoptosis and Bcl-2 and Bax protein levels in a rat model of brain ischemia/reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Rui Wang; Guanglai Li; Wei Wang; Huanying Li

    2008-01-01

    BACKGROUND: Pharmacological studies have demonstrated that compound preparation Tongqiao Jiannao capsules composed of Zexie, Baizhu, Honghua, Danshen, and Shexiang can supplement qi,activate blood circulation, relieve blood stasis, induce resuscitation for alleviating pain, relieve pain, anddilate blood vessels.OBJECTIVE: To observe the effects of Tongqiao Jiannao capsules on the levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax, and verify the mechanism of action.DESIGN, TIME AND SETTING: Randomized, controlled animal experiment, performed in the Laboratory of Biochemistry and Molecular Biology, Shanxi Medical University between June 2001 and December 2002.MATERIALS: The right middle cerebral arteries of 24 healthy adult Sprague Dawley rats were occluded by the suture method. The primary Chinese herbal medicinal ingredients of Tongqiao Jiannao capsules are Zexie. Baizhu, Honghua, Danshen, and Shexiang, which were purchased from Shanxi Provincial Medicinal Material Company, China, and prepared into condensed granules in the Room for Chinese Herbal Medicine Preparation, Second Hospital, Shanxi Medical University. Bcl-2 and Bax immunohistochemical staining kits, a 3,3-diaminobenzidine(DAB) kit, and an in situ apoptosis detection kit were purchased from Wuhan Boster Bioengineering Co., Ltd., China.METHODS: Twenty-four rats were randomly and evenly divided into three groups: (1) sham-operated rats in which sutures were inserted and immediately pulled out; (2) Tongqiao Jiannao capsule-treated rats that were intragastrically administered 6.5 g/kg/d Tongqiao Jiannao capsule preparation for seven successive days prior to middle cerebral artery occlusion (MCAO); and (3) MCAO rats without any other treatments.MAIN OUTCOME MEASURES: The levels of neural cell apoptosis and Bcl-2 and Bax proteins at 24 hours post-surgery.RESULTS: In the MCAO group, the numbers of apoptotic cells and Bax-positive cells were significantly increased, while the numbers of

  6. 活性染料蛋白类防沾色剂的制备%The Preparation of Protein Anti-staining Agent for Reactive Dyes

    Institute of Scientific and Technical Information of China (English)

    徐华凤; 王雪燕

    2015-01-01

    以明胶和自制的阳离子交联改性剂WLS为原料,制备了一种阳离子明胶蛋白助剂,并与聚乙烯基吡咯烷酮( PVP)复配,制成一种新型防沾色助剂,应用于活性染料染色棉织物皂洗后处理。以白布沾色K/S值和色布耐皂洗色牢度为评价指标,运用正交试验确定了有利于改善活性染料染色棉织物皂洗防沾色的阳离子明胶蛋白助剂的合成条件,其为:m(明胶蛋白)∶m (WLS)=1∶10,NaOH用量为WLS用量的1.8%,反应温度70℃,反应时间3h;PVP与阳离子明胶蛋白复配质量比为1:50。%Taking gelatin and self-made cationic crosslinking modifier WLS as raw materials, a kind of cat-ionic gelatin protein additive was prepared and was compounded with PVP to prepare a new anti-staining agent for after-soaping treatment of dyed cotton fabric with reactive dyes. Taking K/S value of stained cloth and color fast-ness to soaping of dyed cloth as evaluation indicators, the preparation conditions of cationic gelatin protein addi-tives was determined by using orthogonal test, which could improve soaping anti-staining property of cotton fabric dyed with reactive dyes. The preparation conditions were: m ( gelatin): m ( WLS) was 1∶14, the dosage of NaOH was1. 8% of that of WLS, the reaction temperature was 70℃ and time was 3h, the quality ratio of PVP to cationic gelatin protein was 1∶50 .

  7. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of Smu.1475c from caries pathogen Streptococcus mutans.

    Science.gov (United States)

    Zhou, Yan-Feng; Mi, Wei; Li, Lanfen; Zhang, Xiaoyan; Liang, Yu-He; Su, Xiao-Dong; Wei, Shicheng

    2006-02-01

    The gene smu.1475c encodes a putative protein of 211 residues in Streptococcus mutans, a primary pathogen for human dental caries. In this work, smu.1475c was cloned into pET28a and expressed in good amount from the E. coli strain BL21 (DE3). Smu.1475c protein was purified to homogeneity in a two-step procedure of Ni2+ chelating and size exclusion chromatography. Crystals were obtained by hanging-drop vapor-diffusion method and diffracted to 2.7 angstroms resolution. The crystal belongs to orthorhombic space group P2(1)2(1)2(1) with cell dimension of a = 68.3 angstroms, b = 105.9 angstroms, c = 136.2 angstroms. The asymmetric unit is expected to contain four molecules with solvent content of 49.4%.

  8. Identification of a Protein Subset of the Anthrax Spore Immunome in Humans Immunized with the Anthrax Vaccine Adsorbed Preparation

    OpenAIRE

    Kudva, Indira T.; Griffin, Robert W.; Garren, Jeonifer M.; Calderwood, Stephen B.; John, Manohar

    2005-01-01

    We identified spore targets of Anthrax Vaccine Adsorbed (AVA)-induced immunity in humans by screening recombinant clones of a previously generated, limited genomic Bacillus anthracis Sterne (pXO1+, pXO2−) expression library of putative spore surface (spore-associated [SA]) proteins with pooled sera from human adults immunized with AVA (immune sera), the anthrax vaccine currently approved for use by humans in the United States. We identified 69 clones that reacted specifically with pooled immu...

  9. Preparation of Sulfobetaine-Grafted PVDF Hollow Fiber Membranes with a Stably Anti-Protein-Fouling Performance

    Directory of Open Access Journals (Sweden)

    Qian Li

    2014-04-01

    Full Text Available Based on a two-step polymerization method, two sulfobetaine-based zwitterionic monomers, including 3-(methacryloylamino propyl-dimethyl-(3-sulfopropyl ammonium hydroxide (MPDSAH and 2-(methacryloyloxyethyl ethyl-dimethyl-(3-sulfopropyl ammonium (MEDSA, were successfully grafted from poly(vinylidene fluoride (PVDF hollow fiber membrane surfaces in the presence of N,N′-methylene bisacrylamide (MBAA as a cross-linking agent. The mechanical properties of the PVDF membrane were improved by the zwitterionic surface layers. The surface hydrophilicity of PVDF membranes was significantly enhanced and the polyMPDSAH-g-PVDF membrane showed a higher hydrophilicity due to the higher grafting amount. Compared to the polyMEDSA-g-PVDF membrane, the polyMPDSAH-g-PVDF membrane showed excellent significantly better anti-protein-fouling performance with a flux recovery ratio (RFR higher than 90% during the cyclic filtration of a bovine serum albumin (BSA solution. The polyMPDSAH-g-PVDF membrane showed an obvious electrolyte-responsive behavior and its protein-fouling-resistance performance was improved further during the filtration of the protein solution with 100 mmol/L of NaCl. After cleaned with a membrane cleaning solution for 16 days, the grafted MPDSAH layer on the PVDF membrane could be maintain without any chang; however, the polyMEDSA-g-PVDF membrane lost the grafted MEDSA layer after this treatment. Therefore, the amide group of sulfobetaine, which contributed significantly to the higher hydrophilicity and stability, was shown to be imperative in modifying the PVDF membrane for a stable anti-protein-fouling performance via the two-step polymerization method.

  10. Sample preparation of membrane proteins suitable for solid-state MAS NMR and development of assignment strategies

    OpenAIRE

    Hiller, Matthias

    2009-01-01

    Although the basic structure of biological membranes is provided by the lipid bilayer, most of the specific functions are carried out by membrane proteins (MPs) such as channels, ion-pumps and receptors. Additionally, it is known, that mutations in MPs are directly or indirectly involved in many diseases. Thus, structure determination of MPs is of major interest not only in structural biology but also in pharmacology, especially for drug development. Advances in structural biology of membrane...

  11. The effect of the application of protein and cellulose preparations as iodine carriers on stability of thiamine in processed meats

    OpenAIRE

    Krystyna Szymandera-Buszka; Katarzyna Waszkowiak; Marzanna Hęś; Anna Jędrusek-Golińska

    2011-01-01

      Fortification of processed meat with iodised table salt was shown to increase thiamine losses, both during thermal processing and storage. Taking into consideration the fact, as well as the recommendation for reduction of consumption of table salt, alternative iodine carriers need to be searched for. Thus the aim of the study was to determine the effect of soy protein isolate (SPI) and wheat fibre (WF) as iodine salts’ (potassium iodide and iodate) carriers on thiamine stabil...

  12. 防冻剂对自燃煤矸石混凝土力学性能影响研究%Study of Effects of Antifreezing Agents on Mechanical Properties of Spontaneous Combustion Coal Gangue Concrete

    Institute of Scientific and Technical Information of China (English)

    贾宝新; 李庆文; 梁鹏飞; 李桂秀

    2016-01-01

    为探究防冻剂对煤矸石混凝土力学性能的影响,以防冻剂种类、不同掺量和养护温度等为因素进行对比试验,分析其对煤矸石混凝土强度影响水平和作用机制。结果表明:通过单掺不同防冻剂标养煤矸石混凝土强度分析,早强效果显著性为氯化钙>硝酸钙>乙二醇;养护环境由负温度到正温度,基准煤矸石混凝土的抗压强度损失率与养护温度呈正相关;在-5℃和-10℃养护环境中,不同掺量3种防冻剂对负温煤矸石混凝土均起一定防冻效果,其显著性体现为氯化钙>硝酸钙>乙二醇。%In order to investigate thoroughly the effects of antifreezing agents on mechanical properties of coal gangue concrete, in consideration of various factors such as the kind of antifreezing agents, dosages of antifreezing agents and curing temperature, contrast test is conducted to analyze the inlfuence of these factors on coal gangue concrete strength and mechanism of action. The results show that the signiifcance of early strength, through mixing just one type of antifreezing agent each time, by analyzing compressive strength of coal gangue concrete under standard curing condition, is as follows: calcium chloride > calcium nitrate > ethylene glycol. The compressive strength loss rate of ifducial coal gangue concrete has positive correlation with curing temperature as curing environment varies from negative temperature to positive temperature. In addition, under different curing environment of -5℃ and -10℃, three types of antifreezing agents with different dosages exert antifreezing effect on negative temperature coal gangue concrete to some extent. The signiifcance of early strength is as follows: calcium chloride > calcium nitrate > ethylene glycol.

  13. The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

    Directory of Open Access Journals (Sweden)

    Cheng Xu

    2014-08-01

    Full Text Available Follicle-stimulating hormone receptor (FSHR, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein (FSHR-57aa as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a(+-FSHR-57aa plasmid was constructed and expressed in Escherichia coli strain BL21 Star TM (DE3 and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 (16 weeks after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

  14. Preparation of denatured protein bone sterilized with gamma radiation; Preparacion de hueso desproteinizado esterilizado con radiacion gamma

    Energy Technology Data Exchange (ETDEWEB)

    Luna Z, D. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)]. e-mail: dlz@nuclear.inin.mx

    2005-07-01

    The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

  15. Preparation and testing of a Vi conjugate vaccine using pneumococcal surface protein A (PspA) from Streptococcus pneumoniae as the carrier protein.

    Science.gov (United States)

    Kothari, Neha; Genschmer, Kristopher R; Kothari, Sudeep; Kim, Jeong Ah; Briles, David E; Rhee, Dong Kwon; Carbis, Rodney

    2014-09-29

    In the current study pneumococcal surface protein A (PspA) was conjugated to Vi capsular polysaccharide from Salmonella Typhi to make available a vaccine against typhoid fever that has the potential to also provide broad protection from Streptococcus pneumoniae. High yielding production processes were developed for the purification of PspAs from families 1 and 2. The purified PspAs were conjugated to Vi with high recovery of both Vi and PspA. The processes developed especially for PspA family 2 could readily be adapted for large scale production under cGMP conditions. Previously we have shown that conjugation of diphtheria toxoid (DT) to Vi polysaccharide improves the immune response to Vi but can also enhance the response to DT. In this study it was shown that conjugation of PspA to Vi enhanced the anti-PspA response and that PspA was a suitable carrier protein as demonstrated by the characteristics of a T-cell dependent response to the Vi. We propose that a bivalent vaccine consisting of PspA from families 1 and 2 bound to Vi polysaccharide would protect against typhoid fever and has the potential to also protect against pneumococcal disease and should be considered for use in developing countries.

  16. 实验室去污评价用蛋白污布的制备%Preparation of protein-based soiled cloth for laboratorial evaluation of detergency

    Institute of Scientific and Technical Information of China (English)

    乔建芬; 严方; 高越胜; 郭朝华; 夏建明

    2012-01-01

    从减少指示剂用量、稀释污垢原液、减少蛋白加入量、调整轧车压力和污布老化处理等方面人手,对实验室洗涤剂去污性能评价中采用的蛋白污布进行改进,试验采用标准洗衣粉与不同加酶浓度的洗涤剂对污布进行去污效果测试,发现减少蛋白加入量、调整轧车压力和污布老化处理均为有效的改进措施.%Preparation of protein-based soiled cloth used in laboratorial detergency evaluation is improved, such as reducing the dosages of indicator and protein, diluting dirt liquor, adjusting padder pressure and ageing treatment. The detergency of standard powder and detergents with different enzyme concentrations are tested. The results show that reducing protein dosage, adjusting padder pressure and ageing treatment are effective ways.

  17. Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    CHEN Yang; YANG Su-juan; FU Yao; WANG Jia-peng; ZHAO Zhi-zhuang; FU Xue-qi

    2008-01-01

    A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPa.

  18. Preparation of protein-like silver-cysteine hybrid nanowires and application in ultrasensitive immunoassay of cancer biomarker.

    Science.gov (United States)

    Chen, Wenjuan; Zheng, Liyan; Wang, Meilan; Chi, Yuwu; Chen, Guonan

    2013-10-15

    Novel protein-like silver-cysteine hybrid nanowires (p-SCNWs) have been synthesized by a green, simple, nontemplate, seedless, and one-step aqueous-phase approach. AgNO3 and l-cysteine were dissolved in distilled water, forming Ag-cysteine precipitates and HNO3. Under vigorous stirring, the pH of the solution was rapidly adjusted to 9.0 by addition of concentrated sodium hydroxide solution, leading to quick dissolution of the Ag-cysteine precipitates and sudden appearance of white precipitates of p-SCNWs. The p-SCNWs are monodispersed nanowires with diameter of 100 nm and length of tens of micrometers, and have abundant carboxyl (-COOH) and amine (-NH2) groups at their surfaces, large amounts of peptide-linkages and S-bonding silver ions (Ag(+)) inside, making them look and act like Ag-hybrid protein nanostructures. The abundant -COOH and -NH2 groups at the surfaces of p-SCNWs have been found to facilitate the reactions between the p-SCNWs and proteins including antibodies. Furthermore, the fact that the p-SCNWs contain large amounts of silver ions enables biofunctionalized p-SCNWs to be excellent signal amplifying chemiluminescence labels for ultrasensitive and highly selective detection of important antigens, such as cancer biomarkers. In this work, the immunoassay of carcinoembryonic antigen (CEA) in human serum was taken as an example to demonstrate the immunoassay applications of antibody-functionalized p-SCNWs. By the novel p-SCNW labels, CEA can be detected in the linear range from 5 to 400 fg/mL with a limit of detection (LOD) of 2.2 fg/mL (at signal-to-noise ratio of 3), which is much lower than that obtained by commercially available enzyme-linked immunosorbent assay (ELISA). Therefore, the synthesized p-SCNWs are envisioned to be an excellent carrier for proteins and related immunoassay strategy would have promising applications in ultrasensitive clinical screening of cancer biomarkers for early diagnostics of cancers.

  19. Preparation of Polyclonal Antibody against Crystalline Inclusion Protein of Entomopathogenic Bacterium%昆虫病原细菌Cip蛋白多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    游娟; 黄建林; 曹莉; 韩日畴

    2013-01-01

    Entomopathogenic Photorhabdus bacteria produce two types of intracellular crystalline inclusion proteins,CipA and CipB,as the nutrient resource for the symbiotic nematode.Based on constructed high-level prokaryotic expression system,recombinant Cip proteins were purified with a warm extract method and used as the antigens to immune New Zealand rabbits,respectively.The polyclonal antibodies with the titers of 1:6400 were raised and monitored by indirect enzyme-linked immunosorbent assay (ELISA).Western blot assay confirmed that the antibodies could specifically recognize the inclusion proteins in Photorhabdus bacteria.Preparation of Cip polyclonal antibodies will be helpful to develop the immunological assay method of these proteins.%昆虫病原发光杆菌属(Photorhabdus)细菌产生2种胞内晶体蛋白CipA和CipB,为其共生线虫的生长发育提供营养.基于Cip蛋白已在大肠杆茵原核表达系统中得到稳定且高水平表达,以纯化的重组Cip蛋白为抗原,免疫新西兰大耳兔制备多克隆抗体,ELISA测定2种蛋白抗血清效价,均可达到1∶6400.Western blot分析显示,制备的抗体均可特异识别发光杆菌的胞内晶体蛋白.Cip蛋白多克隆抗体的制备为建立该类蛋白的免疫学检测方法奠定了基础.

  20. Evaluation of protein structural changes and water mobility in chicken liver paste batters prepared with plant oil substituting pork back-fat combined with pre-emulsification.

    Science.gov (United States)

    Xiong, Guoyuan; Han, Minyi; Kang, Zhuangli; Zhao, Yingying; Xu, Xinglian; Zhu, Yingying

    2016-04-01

    Protein structural changes and water mobility properties in chicken liver paste batters prepared with plant oil (sunflower and canola oil combinations) substituting 0-40% pork back-fat combined with pre-emulsification were studied by Raman spectroscopy and low-field nuclear magnetic resonance (NMR). Results showed that pre-emulsifying back-fat and plant oil, including substituting higher than 20% back-fat with plant oil increased the water- and fat-binding (pproperties, formed more even and fine microstructures, and gradually decreased the NMR relaxation times (T21a, T21b and T22), which was related to the lower fluid losses in chicken liver paste batters. Raman spectroscopy revealed that compared with a control, there was a decrease (poil combined with pre-emulsification. Pre-emulsification and plant oil substitution changed tryptophan and tyrosine doublet hydrophobic residues in chicken liver paste batters.

  1. Studies on the Renaturation with Simultaneous Purification of Recombinant Human Proinsulin with Unit of Simultaneous Renaturation and Purification of Protein in Semi-preparative Scale

    Institute of Scientific and Technical Information of China (English)

    Quan BAI; Yu KONG; Xin Du GENG

    2003-01-01

    The renaturation and purification of recombinant human proinsulin (rh-proinsulin) expressed in E. Coli with the unit of simultaneous renaturation and purification of protein (USRPP) in semi-preparative scale was studied. The result shows that rh-proinsulin extracted with 8.0 mol/L urea can be renatured and purified simultaneously in 45 minutes with the USRPP (10×50 mm ID). The purity of rh-proinsulin was found to be more than 90% and the mass recovery to be more than 80%. The renaturation effect of rh-proinsulin with the USRPP was tested by enzyme cleavage for obtaining insulin. In addition, the result was further confirmed with RPLC, SDS-PAGE electrophoresis, and MALDI-TOF, respectively.

  2. Booster immunization with a partially purified citrus tristeza virus (CTV) preparation after priming with recombinant CTV coat protein enhances the binding capacity of capture antibodies by ELISA.

    Science.gov (United States)

    Bar-Joseph, M; Filatov, V; Gofman, R; Guang, Y; Hadjinicolis, A; Mawassi, M; Gootwine, E; Weisman, Y; Malkinson, M

    1997-08-01

    Groups of rabbits and young lambs were immunized subcutaneously and intramuscularly with a recombinant citrus tristeza virus (CTV) coat protein (rCTV-CP) antigen. Three weeks after primary immunization the animals were divided into two groups that were boosted either with rCTV-CP or with a partially purified preparation of CTV particles (ppCTV). Twelve and 15 days after the last injection, the animals were bled and the binding capacity of the antisera for CTV detection was examined for capture antibodies by the indirect ELISA. Considerably higher ELISA titers were obtained from animals that were boosted with ppCTV than with rCP. Boosting with partially purified native antigens after priming with recombinant antigens is expected to extend the applicability of the antisera for detecting other structural and non-structural viral antigens by trapping ELISA. PMID:9274814

  3. Evaluation of protein structural changes and water mobility in chicken liver paste batters prepared with plant oil substituting pork back-fat combined with pre-emulsification.

    Science.gov (United States)

    Xiong, Guoyuan; Han, Minyi; Kang, Zhuangli; Zhao, Yingying; Xu, Xinglian; Zhu, Yingying

    2016-04-01

    Protein structural changes and water mobility properties in chicken liver paste batters prepared with plant oil (sunflower and canola oil combinations) substituting 0-40% pork back-fat combined with pre-emulsification were studied by Raman spectroscopy and low-field nuclear magnetic resonance (NMR). Results showed that pre-emulsifying back-fat and plant oil, including substituting higher than 20% back-fat with plant oil increased the water- and fat-binding (pfluid losses in chicken liver paste batters. Raman spectroscopy revealed that compared with a control, there was a decrease (poil combined with pre-emulsification. Pre-emulsification and plant oil substitution changed tryptophan and tyrosine doublet hydrophobic residues in chicken liver paste batters. PMID:26593506

  4. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

    Science.gov (United States)

    Drori, Ran; Celik, Yeliz; Davies, Peter L; Braslavsky, Ido

    2014-09-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

  5. Preparation and functional properties of two kinds of oilseed protein%两种油料蛋白制备及其功能性研究

    Institute of Scientific and Technical Information of China (English)

    王楠; 冯志彪

    2012-01-01

    以脱脂紫苏籽粕和南瓜籽粕为原料,采用碱溶酸沉法分别制备紫苏籽分离蛋白和南瓜籽分离蛋白.结果表明,制备的紫苏籽分离蛋白和南瓜籽分离蛋白中蛋白质含量分别为89.64%和90.39%,氨基酸组成平衡,必需氨基酸组成模式符合FAO/WHO标准.SDS- PAGE显示,紫苏籽分离蛋白和南瓜籽分离蛋白分别由4条、8条带组成.两种分离蛋白与大豆分离蛋白功能性质比较表明,紫苏籽分离蛋白和南瓜籽分离蛋白的溶解性、乳化性和乳化稳定性、起泡性和泡沫稳定性均较好,3种分离蛋白的持水性和持油性相差不大.%The perilla seed protein isolates ( PEPI) and pumpkin seed protein isolates ( PUPI) were prepared from defatted perilla seed meal and defatted pumpkin seed meal by alkali extraction - acid precipitation, and their nutritional value and functional properties were investigated. The protein contents of PEPI and PUPI were 89. 64% and 90. 39% , respectively; their amino acids compositions were balancea-ble and their composition pattern of essential amino acids met FAO/WHO standards. Moreover, compared with soy protein isolates, the solubility, emulsification capacity, emulsion stability, foaming ability and foam stability of the both protein isolates were better, but the water resistance and oil resistance of the three kinds of protein were almost the same.

  6. Preparation of a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine) by surface-initiated atom transfer radical polymerization and analysis of protein adsorption resistance.

    Science.gov (United States)

    Inoue, Yuuki; Onodera, Yuya; Ishihara, Kazuhiko

    2016-05-01

    The purpose of this study was to prepare a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) and assess its resistance to protein adsorption from the dissolved state of poly(MPC) chains in an aqueous condition. The thick poly(MPC) brush layer was prepared through the surface-initiated atom transfer radical polymerization (SI-ATRP) of MPC with a free initiator from an initiator-immobilized substrate at given [Monomer]/[Free initiator] ratios. The ellipsometric thickness of the poly(MPC) brush layers could be controlled by the polymerization degree of the poly(MPC) chains. The thickness of the poly(MPC) brush layer in an aqueous medium was larger than that in air, and this tendency became clearer when the polymerization degree of the poly(MPC) increased. The maximum thickness of the poly(MPC) brush layer in an aqueous medium was around 110 nm. The static air contact angle of the poly(MPC) brush layer in water indicated a reasonably hydrophilic nature, which was independent of the thickness of the poly(MPC) brush layer at the surface. This result occurred because the hydrated state of the poly(MPC) chains is not influenced by the environment surrounding them. Finally, as measured with a quartz crystal microbalance, the amount of protein adsorbed from a fetal bovine serum solution (10% in phosphate-buffered saline) on the original substrate was 420 ng/cm(2). However, the poly(MPC) brush layer reduced this value dramatically to less than 50 ng/cm(2). This effect was independent of the thickness of the poly(MPC) brush layer for thicknesses between 20 nm and about 110 nm. These results indicated that the surface covered with a poly(MPC) brush layer is a promising platform to avoid biofouling and could also be applied to analyze the reactions of biological molecules with a high signal/noise ratio.

  7. The preparation and culture of washed human sperm:A comparison of a suite of protein-free media with media containing human serum albumin

    Institute of Scientific and Technical Information of China (English)

    Kelli L Peirce; Peter Roberts; Jaffar Ali; Phillip Matson

    2015-01-01

    Objective:To compare two suites of culture media (one with HSA and one protein-free (PF) supplemented with methylcellulose) for washing human sperm in IVF.Methods:Semen samples (n=41) underwent parallel density gradient preparation using PF or HSA-supplemented culture medium and subsequent yield, survival, morphology and motility were compared.Results:The PF medium resulted in a significantly higher sperm yield (P<0.0001), but similar sperm morphology (P=0.822) and 24-hour survival (P=0.11). There was, however, a lower percentage of progressively motile sperm (P<0.0001) and a higher proportion of sperm demonstrating non-progressive motility (P<0.0001) in the PF medium when observed on a Makler Chamber, apparently an artefact as a similar sperm motility index was measured using a Sperm Quality Analyser (P=0.83). Attachment of sperm in PF medium to the glass chamber reduced with time and any differences had disappeared after 6 minutes on the counting chamber.Conclusion:These results support the use of PF media supplemented with methylcellulose as an alternative to HSA, although a modification to the manufacturer’s protocol of 6-minutes pre-incubation before assessing sperm motility must be used. Further studies should investigate the function of such sperm prepared in PF medium.

  8. Preparation and characterization of soy protein films with a durable water resistance-adjustable and antimicrobial surface.

    Science.gov (United States)

    Li, Shuzhao; Donner, Elizabeth; Xiao, Huining; Thompson, Michael; Zhang, Yachuan; Rempel, Curtis; Liu, Qiang

    2016-12-01

    A water resistant surface was first obtained by immobilizing hydrophobic copolymers, poly (styrene-co-glycidyl methacrylate) (PSG), with functional groups on soy protein isolate (SPI) films. XPS and AFM results showed that PSG copolymers were immobilized on the film by chemical bonding, and formed a rough surface with some bumps because of the segregation of two different phases on PSG copolymers. Water resistance of the modified films could be adjusted dramatically by further immobilizing different amounts of guanidine-based antimicrobial polymers, poly (hexamethylene guanidine hydrochloride) (PHMG) on the resulting hydrophobic surface. The introduction of hydrophilic PHMG on the resulting surface generated many micropores, which potentially increased the water uptake of the modified films. Furthermore, the modified SPI films showed higher thermostability compared to native SPI film and broad-spectrum antimicrobial activity by contact killing, attributed to the presence of PHMG on the surface. The modified SPI film with a multi-functional surface showed potential for applications in the packaging and medical fields. PMID:27612790

  9. 月见草籽粕分离蛋白的制备%Study on preparation of protein isolation from primrose meal

    Institute of Scientific and Technical Information of China (English)

    马涛; 吕品

    2009-01-01

    Based on primrose meal, the process conditions and functional properties of protein isolated was prepared on alkali-extraction and acid-isolation.Through orthogonal tests,the best conditions of extraction were: the pH 12,the temperature 45℃, the times of extraction 3, the proportion of the material to liquid 1:12, 1:10, 1:8 respectively and protein deposited at pH6.0, pH3.8.In the product gained through spray drying process the content were 75.5%.%以月见草冷榨浸出粕为材料,初步探索碱提酸沉法制备月见草籽粕分离蛋白的工艺条件及其功能特性通过正交实验得到提取的最佳工艺条件为:pH12,温度45℃,提取3次,料液比分别1:12、1:10、1:8;调pH6.0、3.8二次沉淀,喷雾干燥后产品的粗蛋白含量达75.5%.

  10. Pressure-assisted dissociation and degradation of "proteinase K-resistant" fibrils prepared by seeding with scrapie-infected hamster prion protein.

    Science.gov (United States)

    Akasaka, Kazuyuki; Maeno, Akihiro; Murayama, Taichi; Tachibana, Hideki; Fujita, Yuzo; Yamanaka, Hitoki; Nishida, Noriyuki; Atarashi, Ryuichiro

    2014-01-01

    The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP(C), into a fibrous pathogenic form, PrP(Sc), which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP(Sc) into PrP(C) is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP(Sc) from PrP(C) eventually in vivo. Here we show that, by applying pressures at kbar range, the "proteinase K-resistant" fibrils (rHaPrP(res)) prepared from hamster prion protein (rHaPrP [23-231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP(res) fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP(Sc) from PrP(C) is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP(Sc) from various systems of pathogenic concern.

  11. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes.

    Science.gov (United States)

    Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L; Toyoda, Hiroo

    2011-12-01

    Arsenic trioxide (arsenite, As(III)) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As(III) on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As(III) on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As(III)-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As(III) were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As(III) than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As(III) in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As(III)-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As(III) cytotoxicity between these cells.

  12. Preparation of silver carp protein powder by enzymatic hydrolysis and spray drying%酶法制备白鲢鱼蛋白粉的研究

    Institute of Scientific and Technical Information of China (English)

    王丽; 姜启兴; 许艳顺; 夏文水

    2013-01-01

    Silver carp protein powder was prepared by enzymatic hydrolysis and spray drying.The effect on different temperature,papain dosage,hydrolysis time,substrate concentration and pH on the degree of hydrolysis of silver carp protein were determined.And the effects of the conditions of spray drying on the physicochemical properties and water-soluble properties of the protein powder were studied.The results showed that the optimum conditions of hydrolysis by papain were temperature 50℃,enzyme dosages 4000U/g protein,hydrolysis pH6.5,hydrolysis time 4h.The degree of hydrolysis was as high as 29.82%.The hydrolysate was concentrated to 25%~30% and it was dried to protein powder by spray drying which operating conditions were inlet air temperature t70~180℃ and outlet air temperature 80~90℃.The content of which the relative molecular weight were below 1000 accounted for 90.90%.The solubility of the fish protein powder was 98.52%.The product with high nutritional value and watersoluble properties were thus potential protein functional food ingredients.%对白鲢鱼肉进行酶水解经喷雾干燥以制备水溶性高的鱼蛋白粉.研究了木瓜蛋白酶的添加量、温度、时间、底物浓度和pH水解条件对鱼蛋白水解度的影响以及喷雾干燥条件对蛋白粉物性和水溶性的影响.结果表明:木瓜蛋白酶对鲢鱼蛋白的最适水解条件是温度50℃、加酶量4000U/g蛋白质、底物浓度为5.5%、pH6.5、时间4h,水解度达到29.82%;该水解液经浓缩至浓度25% ~30%时,通过控制进风温度170~180℃、出风温度80~90℃用喷雾干燥得到鱼蛋白水解产物,相对分子量在1000以下组分占90.90%,溶解度达98.52%,是一种营养价值高和溶解好的蛋白质功能性食品配料.

  13. Ice-active proteins from New Zealand snow tussocks, Chionochloa macra AND C. rigida.

    Science.gov (United States)

    Wharton, D A; Selvanesan, L; Marshall, C J

    2010-01-01

    The ice active protein profile of New Zealand snow tussocks Chionochloa macra and C. rigida consisted of ice nucleation activity but no antifreeze or recrystallization inhibition activity. The ice nucleation activity was similar in the two species, despite them being collected at different altitudes and at different times. The activity is intrinsic to the plant and is associated with the surface of the leaves. Snow tussocks collect water from fog. Nucleation sites on the surface of their leaves may aid the efficiency of this process. PMID:20919453

  14. Investigation of in-situ poly(lactic acid)/soy protein concentrate composites: Composite preparation, properties and foam application development

    Science.gov (United States)

    Liu, Bo

    2011-12-01

    In this study, soy protein (SP), the residue of oil crushing, was used for preparation of value-added thermoplastics. Novel poly(lactic acid) (PLA)/soy protein concentrate (SPC) blends were investigated and foaming of the resulting blends was developed. PLA/SPC blends were prepared by twin-screw extrusion and test specimens by injection molding. Unlike the practice elsewhere SP was used as a filler in mixing with other polymers, SPC was processed as a plastic component in blending process in this work. Processing SPC as plastic component, water played an important role in terms of the deformability and the morphology of SP thus the properties of the blends. Plasticization of SP, compatibilization of the blends and structure-property relationship of the PLA/SPC blends were studied. In the literature water and glycerol were often used together in preparing SP plastics or plastic blends, but this study found that this traditional combination did not provide the best results in terms of morphology and mechanical properties. Water is only recommended in plasticizing SP in the blends. This study showed water as a plasticizer was a domain factor on control of morphology and properties of PLA/SPC blends. The due to the evaporation of water after extrusion, SP domain lost its deformability thus resulted in in-situ composites. Interconnected SPC phase structure was achieved by control water content in the pre-formulated SPC and SPC content in the blends. A novel dual compatibilization method was developed to improve the properties of PLA/SPC blends. Poly(2-ethyl-2-oxazoline) was used to improve the dispersion of SPC in the blending stage, and polymeric methylene diphenyl diisocyanate was used to improve the interfacial adhesion between SPC and PLA in the subsequent processing. The result showed excellent mechanical properties and improved thermal properties of PLA/SPC blends. Using processing aids is an effective way to decrease processing temperature and thermal degradation

  15. 改性豆基蛋白胶粘剂的制备及应用%Preparation and application of modified soy-based protein adhesive

    Institute of Scientific and Technical Information of China (English)

    邱盈盈; 张越; 李城; 张世锋; 李建章

    2012-01-01

    以改性异氰酸酯作为交联剂,制备改性豆基蛋白胶粘剂.探讨了交联剂、乳化剂和热压工艺条件等因素对该胶粘剂耐水胶接强度的影响.结果表明:当w(交联剂)=6%、w(乳化剂)=1.5%、热压时间为60s/mm、热压压力为1.0Mpa和热压温度为120℃时,胶合板的耐水胶接强度为1.21Mpa,完全满足GB/T9846.3-2004标准中Ⅱ类胶合板的使用要求,并且改性豆基蛋白胶粘剂的适用期超过60h.%With modified isocyanate as cross linker, a modified soy-based protein adhesive was prepared. The influences of cross linker, emulsifier, hot-pressing process conditions and other factors were discussed on the water—resistance bonding strength of the adhesive. The results showed that the water—resistance bonding strength of plywood(1.21 Mpa) could fully meet application requirements of II-type plywood in GB/T 9846.3—2004 standard, and the working life of the modified soy-based protein adhesive was more than 60 h when mass fractions of cross linker and emulsifier were 6% and 1.5% respectively, hot-pressing time, hot-pressing pressure and hot-pressing temperature were 60 s/mm,1.0 Mpa and 120 ℃ respectively.

  16. Preparation of Protein Hydrolysates with Waste Brewer's Yeast%啤酒废酵母蛋白水解物的制取工艺

    Institute of Scientific and Technical Information of China (English)

    张晓鸣; 袁信华; 徐柔; 章克昌

    2001-01-01

    Protein hydrolysates were prepared by enzymatic hydrolysis ofpretreated waste brewer's yeast. The hydrolysis parameters were specified by response surface analysis. The Parameters are as follows: initial yeast concentration 15%, temperature 55~60 ℃, the β-glucanase-substrate ratio 15~20 U/g, and pH 6.6~6.8. When hydrolysed for 7 hour, more than 82% of the Kjeldahl protein in yeast was extracted. Measured by high-performance liquid chromatography(HPLC), 65.26% of the hydrolysates had molecular weights of 210~635 Dalton, which corresponded to dipeptides and tripeptides.%利用啤酒废酵母资源,通过破壁预处理与复合酶降解相结合的工艺,制取特定相对分子质量范围的蛋白水解物.响应面分析结果表明,在温度55~60℃,pH6.6~6.8,β-葡聚糖酶用量15~20U/g(以酵母计)的条件下,有最高的肽提取率.酶解7h,蛋白质提取率大于82%,经HPLC测定,水解产物中65%组分的相对分子质量在210~635之间,处于二肽和三肽相对分子质量范围.

  17. Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

    Science.gov (United States)

    Zhang, Zhenbin; Sun, Liangliang; Zhu, Guijie; Cox, Olivia F; Huber, Paul W; Dovichi, Norman J

    2016-01-01

    A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method. PMID:26670623

  18. Photo-crosslinked networks prepared from fumaric acid monoethyl ester-functionalized poly(D,L-lactic acid) oligomers and N-vinyl-2-pyrrolidone for the controlled and sustained release of proteins

    NARCIS (Netherlands)

    Jansen, Janine; Tibbe, Martijn P.; Mihov, George; Feijen, Jan; Grijpma, Dirk W.

    2012-01-01

    Photo-crosslinked networks were prepared from fumaric acid monoethyl ester-functionalized poly (D,L-lactic acid) oligomers and N-vinyl-2-pyrrolidone. Two model proteins, lysozyme and albumin, were incorporated into the network films as solid particles and their release behavior was studied. By varyi

  19. Preparation and scaling up of a low phenylalanine enzymatic hydrolysate of bovine whey proteins Preparação e escalonamento de um hidrolisado enzimático de proteínas do soro de leite bovino

    OpenAIRE

    Marilisa Guimarães Lara; Clarice Izumi; Lewis Joel Greene; Luciano Vilela; Osvaldo Freitas

    2005-01-01

    We describe the preparation of pancreatic enzymes hydrolysate of milk whey proteins containing low levels of aromatic amino acids. Pancreatin and trypsin/chymotrypsin (6.3% w/w protein) when used to hydrolyze whey proteins for 27 h at 37±2 ºC, released 74% of the Phe, 100% of the Tyr and 100% of the Trp as free amino acids. Most of the free aromatic amino acids present in 2 kg hydrolysate were separated from the remaining peptides and other amino acids by gel filtration on a 15 liter Sephadex...

  20. Preparation of an aptamer based organic-inorganic hybrid monolithic column with gold nanoparticles as an intermediary for the enrichment of proteins.

    Science.gov (United States)

    Zhao, Jin-Cheng; Zhu, Qing-Yun; Zhao, Ling-Yu; Lian, Hong-Zhen; Chen, Hong-Yuan

    2016-08-01

    A novel strategy for the preparation of an aptamer based organic-inorganic hybrid affinity monolithic column was developed successfully using gold nanoparticles (GNPs) as an intermediary for a sandwich structure to realize the functional modification of the surface of the monolithic matrix. This monolithic matrix was facilely pre-synthesized via one-step co-condensation. Due to the high surface-to-volume ratio of GNPs and the large specific surface area of the hybrid matrix, the average coverage density of aptamers on the hybrid monolith reached 342 pmol μL(-1). With the combination of an aptamer based hybrid affinity monolithic column and enzymatic chromogenic assay, the quantitation and detection limits of thrombin were as low as 5 nM and 2 nM, respectively. These results indicated that the GNPs attached monolith provided a novel technique to immobilize aptamers on an organic-inorganic hybrid monolith and it could be used to achieve highly selective recognition and determination of trace proteins. PMID:27307035

  1. Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-△SHP-1 Antibodies

    Institute of Scientific and Technical Information of China (English)

    LI Wan-nan; ZHUANG Yan; LI He; SUN Ying; FU Yao; WU Xiao-xia; ZHAO Zhi-zhuang; FU Xue-qi

    2008-01-01

    This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated △SHP-1) and the preparation of its polyelonal antibodies.A cDNA fragment encoding △SHP-1 was amplified by PCR and then cloned into the pT7 expression vector.The recombinant pT7-△SHP-1 plasmid was used to transform Rosetta(DE3) E.coll cells.△SHP-1 was distributed in the exclusion body of E.coll cell extracts and was purified through a two-column chromatographic procedure.The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns.It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases.To generate polyclonal anti-△SHP-1 antibodies,purified recombinant △SHP-1 was used to immunize a rabbit.The resultant anti-serum was subjected to purification on △SHP-1 antigen affinity chromatography.The purified polyclonal antibody displayed a high sensitivity and specificity toward △SHP-1.This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.

  2. Effects of L- and iso-ascorbic acid on meat protein hydrolyzing activity of four commercial plant and three microbial protease preparations.

    Science.gov (United States)

    Ha, Minh; Bekhit, Alaa El-Din; Carne, Alan

    2014-04-15

    The present study investigated the effects of both l- and iso-ascorbic acid (AA) on the activity of four plant proteases (papain, bromelain, actinidin and zingibain) and three microbial proteases (Bacterial Protease G, Fungal 31,000 and Fungal 60,000) preparations using fluorescent-labelled casein, meat myofibrillar and connective tissue extracts to explore their effects on meat structure components upon treatment with individual proteases. While l-AA in the range 0.8-3.2mM inhibited the activity of papain, bromelain and zingibain, iso-AA acted as an inhibitor of papain but as an activator of zingibain and had no significant effect on bromelain. Both AA isoforms acted as an activator of the actinidin protease and the concentration of AA isoforms appeared to affect the level of activation of the protease. The effect of the two AA isoforms on collagen and myofibrillar protein hydrolyzing activity varied depending on the concentration of the two AA isoforms. The results indicate the ability to up and down regulate the activity of the investigated proteases by using an appropriate concentration of the AA isoform. PMID:24295669

  3. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshino, Yuta [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Yuan, Bo, E-mail: yuanbo@toyaku.ac.jp [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Kaise, Toshikazu [Laboratory of Environmental Chemodynamics, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Takeichi, Makoto [Yoneyama Maternity Hospital, 2-12 Shin-machi, Hachioji, Tokyo 192-0065 (Japan); Tanaka, Sachiko; Hirano, Toshihiko [Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Kroetz, Deanna L. [Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Toyoda, Hiroo [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)

    2011-12-15

    Arsenic trioxide (arsenite, As{sup III}) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As{sup III} on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As{sup III} on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As{sup III}-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As{sup III} were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As{sup III} than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As{sup III} in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As{sup III}-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As{sup III} cytotoxicity between these cells. -- Highlights: Black-Right-Pointing-Pointer Examination of effect of As{sup III} on primary cultured chorion (C) and amnion

  4. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides.

    Science.gov (United States)

    Mitchell, Daniel E; Gibson, Matthew I

    2015-10-12

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs.

  5. Ultrastructural and Extracellular Protein Changes in Cell Suspension Cultures of Populus euphratica Associated with Low Temperature-induced Cold Acclimation

    Institute of Scientific and Technical Information of China (English)

    Dai Huanqin; Lu Cunfu; Zhang Hui; Zhang Xujia

    2003-01-01

    Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of-17.5 ℃) in cell suspension at 4-5 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of-12.5 ℃ in nonacclimated cells to LT50 of-17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.

  6. Antifreeze effect and mechanism of sodium poly-γ-glutamate on backers' yeast cells%γ-聚谷氨酸钠对面包酵母的抗冻作用及其机理

    Institute of Scientific and Technical Information of China (English)

    时晓剑; 缪冶炼; 卫昊; 徐虹; 王冀宁

    2012-01-01

    γ-聚谷氨酸钠(γ—PGA钠)是由Y-聚谷氨酸与钠离子结合而成的水溶性物质,可以食用,无色无味。γ—PGA钠作为面包酵母抗冻剂,具有较强的实用性。在0.03~0.40℃/min的冷冻速率、-7~-60℃的冻藏温度条件下,探讨了γ—PGA钠对面包酵母的抗冻作用及其机理。实验结果表明,酵母细胞存活率在冻藏初期逐渐下降,6d后趋于稳定。γ—PGA钠对酵母的抗冻作用明显高于葡萄糖和谷氨酸。γ—PGA钠抑制了冷冻和冷藏过程中大冰晶的产生、以及冰晶的重结晶。在冷冻速率大于0.27℃/min、冻藏温度低于-30℃、冻藏时间6f1的条件下,添加1%的γ—PGA钠使酵母细胞存活率从无γ—PGA钠时的36.5%上升到67.5%。γ—PGA钠、葡萄糖和谷氨酸的抗冻活性分别为2.48、2.03和1.42。细胞存活率(η,%)随抗冻活性(Aa,-)的增加呈线性上升。γ—PGA钠具有较大抗冻活性的机理是,γ—PGA钠的解离度较大,并且Na+能够固定较多的水分子。%Sodium poly-γ-glutamate (Na-γ-PGA), a water-soluble polyamide composed of poly-γ- glutamic acid and sodium ion, is edible, tasteless, colorless and odorless. It is considered as a potential cryoprotectant for backers' yeast. In the present study, the antifreeze effect of Na-γ-PGA on bakers' yeast cells was investigated in the freezing rate range of 0.03-0.40 ℃/min and the storage temperature range of -7--60 ℃. The mechanism of the antifreeze effect was also discussed. The experimental results showed that the survival ratio of yeast cells decreased at the beginning, and was constant after 6 dduring frozen storage. Na-γ-PGA had greater antifreeze effect on the yeast cells than glutamic acid and glucose. It inhibited both the generation of big-size ice crystals during freezing and the recrystallization of ice crystals during freezing storage. Under the condition of a freezing rate at 0

  7. Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata.

    Science.gov (United States)

    Wharton, D A; Pow, B; Kristensen, M; Ramløv, H; Marshall, C J

    2009-01-01

    Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (approximately 2 degrees C) and the gut contents somewhat lower levels (approximately 0.6 degrees C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization. PMID:18955061

  8. Improvement and application of sodium sulfite type concrete antifreeze%亚钠型混凝土防冻剂的改进与应用

    Institute of Scientific and Technical Information of China (English)

    付饶

    2015-01-01

    In order to solve the cold freezing problems of early age concrete at the cold season in the northeast , northwest an north of Chi-na, a small amount of vitamin C,vitamin D,fluorocarbon surfactantFN -3 were introduced in the sodium type concrete antifreeze and ap-plied to the winter construction of concrete .The fluidity loss is less than 30% within 2 hours.The capillary channel and the pervious of concrete significantly reduced .While the anti permeability and anti freeze properties improved obviously .This research reached the goal of improving the strength of concrete ,promoting and optimizing the formation of early age concrete slurry structure and reinforcing later strength of the concrete .%为解决在东北、华北、西北地区寒冷季节早期混凝土的冻害问题,在通用的亚钠型混凝土防冻剂中引入少量的维生素C、维生素D、氟碳表面活性剂FN-3,加入到冬季施工的混凝土中,2h的流动度损失小于30%,混凝土的毛细通道显著减少,降低了混凝土的透水性,其抗渗和防冻性能明显提高。达到提高混凝土早期防冻能力,促进、优化早期混凝土浆体结构的形成,增大混凝土后期强度的目的。

  9. 晚期氧化蛋白产物的制备及纯化%Preparation and purification of advanced oxidation protein products

    Institute of Scientific and Technical Information of China (English)

    田梅; 易维京; 余荣杰; 吴雄飞

    2009-01-01

    BACKGROUND: Advanced oxidation protein products (AOPPs) are a crucial pathogenic link to such long-term uremic complications in hemodialysis patients as immune system dysregulation, accelerated atherosclerosis, dialysis-related amyloldosis and so on. However, basic studies on AOPPs are relatively few, and one of the main reasons is the fact that it is difficult to obtain AOPPs with high pudty and biological activity.OBJECTIVE: To prepare, pudfy and indentify AOPPs, with the hope of searching for a method of preparing AOPPs with high purity and biological activity.DESIGN, TIME AND SETTING: A single sample observation was completed in the Clinical Biochemistry Section of Ecsomatics Department, Third Military Medical University of Chinese PLA from September to November in 2008. MATERIALS: Human serum albumin (HSA) was provided by Chengdu Rongsheng Company Ltd. Hitrap 26/60 sephacryl S-300 was purchased from GE Healthcare.METHODS: Hypochloric acid was used in the oxidation of purified HSA to prepare in vitro the AOPPs-modified HSA (AOPPs-HSA), which was then isolated by Hitrap 26/60 sephacryl S-300. Relative molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE), native polyacrylamide gel electrophoresis (PAGE) and molecular weight standards. Structural features and biological activities were identified in the experiment of tumor necrosis factor α (TNF-α) release from monocytes.MAIN OUTCOME MEASURES: ①The purification and gel electrophoresis results of HSA. ②The purification and gel electrophoresis results of AOPPs. ③The dose-effect relationship between AOPPs-HSA and TNF-α release from monocytes. RESULTS: The relative molecular mass of AOPPs-HSA was 670 000 according to SDS-PAGE, native PAGE and molecular weight standards. Moreover, AOPPs-HSA could encourage the release of TNF-α from monocytes. The time effects showed that TNF-α release volume significantly increased after 6 hours of stimulation by AOPPs-HSA (1

  10. Preparation and application of sericin protein modifier on polyester fabrics%丝胶改性剂的制备及对涤纶的改性研究

    Institute of Scientific and Technical Information of China (English)

    戴杰; 郭晓玲; 周青青; 本德萍; 崔贞; 申国栋

    2014-01-01

    以环氧氯丙烷、丝胶蛋白为原料,在碱性条件下制备新型丝胶改性剂.丝胶改性剂与涤纶织物上的羧基发生酯化反应并固着于涤纶织物上,改善涤纶织物的抗静电性、吸湿性等性能.对整理后涤纶织物的抗静电性、透气性、毛细效应、接触角、强力等进行测试.结果表明:改性涤纶织物的抗静电性得到改善,表面电荷由原来的0.036μC变为0.001μC;多次水洗后,涤纶织物表面电荷基本稳定,说明丝胶改性剂与涤纶织物上的羧基发生酯化反应,并固着在涤纶织物上;透气性由原来的19.43 mm/s变为46.29 mm/s.%A novel sericin modifier was prepared using epichlorohydrin and sericin protein as raw materi-als under alkaline condition. The sericin modifier reacted with the carboxyl groups on polyester fabric through esterification reaction and fixed onto the fabrics, improving the antistatic and hygroscopic properties of polyes-ter fabrics. The antistatic, gas permeability, capil ary effect, contact angle and strength of finished polyester fab-ric were tested. The results showed that the antistatic property of modified polyester fabric was improved and the surface charge changed from 0.036 μC to 0.001 μC. After repeated launderings, the surface charge on the finished polyester remained steady showing the esterification reaction between the sericin modifier with the carboxyl groups on polyester fabrics. The gas permeability also increased from 19.43 mm/s to 46.29 mm/s.

  11. 米糠蛋白抗氧化活性肽的制备%Preparation of Anti-oxidation Bioactive Peptide of Rice Bran Protein

    Institute of Scientific and Technical Information of China (English)

    周梅; 张敏

    2012-01-01

    This study screened the most suitable prolease oi preparation of bioactive peptide with the indexes of Hydrolysis (DH% ) and the eliminating rate of DPPH free radical. In order to find the most suitable zymohydrolysis condition of prolease,effects of concentration of substrate,pH, addition of protease and dissociation-time on the hydrolysis (DH% ) and the elirninating rate of DPPH freeradical in hydrolyzing were investigated. Based on the single-factor experiment,the optimum conditions of extraction of bioactive peptide from rice bran by the Box-Behnken response surface methodology design of Design-Expert 7.0 were determined as follows; addition amount of enzyme 13970.82 U/g, dissociation-time 3 h and concentration 4.97%. The results showed that degree of hydrolysis of rice bran protein was 23.67% and the eliminating rate of DPPH free radical could reach 64.26% under the optimum conditions.%以水解度(DH%)和对DPPH自由基清除率为指标,筛选出制备米糠蛋白抗氧化活性肽的最适蛋白酶.研究最适蛋白酶的酶解条件,探讨底物浓度、蛋白酶的加入量、pH值、酶解时间等因素对水解度(DH%)和DPPH自由基清除率的影响;在单因素基础上采用Box-Behnken响应曲面中心组合设计法,对酶解米糠蛋白的工艺进行优化.试验结果表明,在加酶量13970.82 U/g,时间3.05h,底物浓度4.97%的水解条件下,米糠蛋白的水解度能够达到23.67%,活性肽对DPPH自由基清除率达到64.26%.

  12. Preparation Technology of Hydrolysis Protein of Tilapia Meat by Alcalase%罗非鱼肉水解蛋白Alcalase酶解工艺优化

    Institute of Scientific and Technical Information of China (English)

    方细娟; 曾庆祝; 许庆陵; 战宇; 刘鹏; 林金鹰; 顾采琴

    2013-01-01

      Preparation technology of tilapia protein hydrolysates by built single factor test and response surface methodology(RSM)were defined with the index degree of dydrolysis(DH), TCA-soluble nitrogen index (TCA-NSI) and yield of acid-solubility peptide (YASP). The effect of hydrolysis temperature, pH, the quantity of Alcalase, the ratio of Liquid-to-solid on DH, TCA-NSI, YASP were studied by RSM, and Regression model was obtained by response surface analysis. Regression model indicated that the optimal condition hydrolyzed Animal Protein of Tilapia Meat by Proteinase was described in the following:hydrolysis temperature 54.30℃, pH 8.77, the quantity of Alcalase 2.4 L was 3 702 U/g, the ratio of Liquid-to-solid was 3.06∶1, hydrolysis time 1hour. under such condition, the DH (%), TCA-NSI (%) and YASP (%) reached 33.63%, 22.10%, 64.55%respectively. Experiment result showed that the DH(%), TCA-NSI(%) and YASP(%) were 33.57%, 22.09%, 64.52%, the predictive value and experimental value have small deviation , indicated that Regression model was reasonable and reliable.%  以水解度(DH)、三氯乙酸蛋白质氮溶解指数(TCA-NSI)、酸溶肽得率(YASP)为指标,通过单因素试验及响应面优化法确定碱性蛋白酶水解罗非鱼肉蛋白的最佳工艺条件。结果表明,温度、pH、加酶量、液固比等因素对DH、TCA-NSI、YASP均有影响,获得了罗非鱼肉水解蛋白制备工艺的回归模型,并通过回归模型确定的最佳工艺条件为:温度54.30℃、pH为8.77、加酶量为3702 U/g(以罗非鱼计)、液固比3.06∶1、水解时间1 h。在此条件下,DH、TCA-NSI、YASP的模型预测值为33.63%、22.10%、64.55%, DH、TCA-NSI、YASP的模型实测值为33.57%、22.09%、64.52%。预测值和实测值偏差较小,回归模型合理可靠。

  13. 双亲灭活原生质体融合技术在草菇耐低温菌株选育上的应用%Application of Inactivated Parental Strain Protoplasts Fusion Technology in Selection of Volvariella volvacea with Higher Antifreeze Capacity

    Institute of Scientific and Technical Information of China (English)

    陈建中; 祝子坪; 吴潇; 何建华; 蒋玮; 王金斌; 唐雪明

    2013-01-01

    为了选育耐低温草菇菌株,以草菇V23、V3552为亲本,采用酶解法制备原生质体,用紫外诱变、化学诱变两种方法对草菇菌株进行诱变,筛选到耐低温的突变株;然后利用紫外灭活(20 W,30 cm,110 s)和热灭活(50℃3 min)的双亲灭活标记法对突变株进行化学融合,结果表明在400 g/L的PEG6000、pH 8.0、融合时间30 min和融合温度32℃的条件下融合率最高,达到0.517%,共获得200个融合子。经过0℃低温筛选,最终获得15株草菇耐低温菌株,菌丝在0℃的耐冻能力提高了4.5倍。经出菇实验证明其子实体与出发菌株相比具有明显的耐低温性,液化现象明显推迟,说明该方法筛选出的菌株具有进一步应用开发的价值。%The protoplasts of Volvariella volvacea V23 and V3552 were prepared by enzymatic method, and then were taken mutagenesis by UV and chemical 2 methods. A fusant displaying higher antifreeze capacity, as compared to that of its parents, was obtained by PEG-induced fusion between UV-inactivated protoplasts of V3552 EMS and heat-inactivated protoplasts of V23 UV through low temperature ( 0℃) selection. The best fusion condition is gained at 400g/LPEG6000, pH 8.0, for 30min fusion time, at 32℃ fusion temperature. The fusion rate could be as high as 0�517%. And 200 fusants were obtained. Through screening with 0℃ low temperature, 15 Volvariella volvacea cold tolerant bacterial strains were obtained. The cold tolerant ability of hypha at 0℃ was improved by 4. 5 times. The results proved that the fruit displayed higher antifreeze capacity, as compared with bacterial strains; and the liquefaction phenomenon was obviously postponed. This indicated that strains screened by this method were of further development value.

  14. Anti-freezing System's Status and Improvement of Sluice Gate in Huangbizhuang Reservoir%黄壁庄水库闸门防冻系统的状况及改进

    Institute of Scientific and Technical Information of China (English)

    郭文宇

    2013-01-01

    Itcombinedwiththeoperationpracticecomparedthreekindsofanti-freezingmeasures(diggingiceditch,anti-freezingsystemofsub-mersible pump, and anti-freezing system of air compressor) of the sluice gate in winter. It puts forward the improvement measures and practica-bility of anti-freezing system of submersible pump in Huangbizhuang reservoir, and puts forward some ideas for the further improvement.%结合黄壁庄水库冬季闸门运行实践,对闸门的三种防冰冻措施(开凿冰沟、潜水泵防冰冻系统、压缩空气泡防冰冻系统)进行了比较。结合潜水泵式防冰冻系统在黄壁庄水库的应用实践,提出了此系统对于北方水库的实用性改进措施,并根据实际应用中积累的经验提出了进一步的改进设想。

  15. Research on high temperature stability of including salt anti-freezing asphalt mixture%蓄盐类抑制冻结沥青混合料高温性能研究

    Institute of Scientific and Technical Information of China (English)

    钟科

    2014-01-01

    In order to evaluate the high temperature performance of including salt anti-freezing asphalt mixture,ordinary asphalt mixture,anti-freezing asphalt mixture and anti-freezing asphalt mixture with rubber particles were chosen to carry out high temperature rutting test and immer-sion rutting test. The results show that whether there is the role of water or not,anti-freezing asphalt mixture with rubber particles shows more ex-cellent high temperature performance,compared with other two asphalt mixture.%为了研究蓄盐类抑制冻结沥青混合料的高温抗车辙性能,选取普通沥青混合料、抑制冻结沥青混合料及掺橡胶颗粒的抑制冻结沥青混合料,分别进行干法车辙试验和浸水车辙试验,结果表明:无论干法车辙或是浸水车辙,掺橡胶颗粒的抑制冻结沥青混合料都拥有良好的高温性能,优于其他两种沥青混合料。

  16. 干混抗冻混凝土配合比设计及抗冻性能研究%Dry-mixed antifreeze concretes design of mixing proportion and performance of winter

    Institute of Scientific and Technical Information of China (English)

    罗憨; 李延和; 夏永清; 范贤玉

    2014-01-01

    随着建筑市场的飞速发展,混凝土冬季施工在所难免。且我国北方地区气候寒冷,普通商品混凝土不能满足建筑工程要求。介绍一种特殊的混凝土---干混抗冻混凝土,通过模拟冬季施工环境,调整混凝土配合比,在满足混凝土强度要求前提下,找出最适宜的干混抗冻混凝土配合比。%With the rapid development of the construction market,concrete construction in winter is inevitable.Since the weather is cold,ordinary concrete construction can not meet the requirements.This paper describes a special concrete-dry-mixed concrete antifreeze. By simulating winter construction environment,adjust the concrete mix,concrete strength to meet the requirements of the premise,we want to identify the most suitable dry-mixed antifreeze concrete mix.

  17. Influence of frozen dough processing conditions on the activity of antifreeze yeast in fermented dough%冷冻工艺条件对发酵面团中抗冻酵母活性的影响

    Institute of Scientific and Technical Information of China (English)

    周璐; 孙冰玉; 石彦国; 梁金钟

    2011-01-01

    研究了冷冻面团的冷冻终结温度、冻藏温度、冻藏时间对抗冻酵母活性的影响,得出最有利于抗冻酵母活性保持的冷冻工艺条件:冷冻终结温度-18℃,冻藏温度应与冷冻终结温度保持一致,贮存时间不超过30d为宜。%The effects of the end of freezing temperature,freeze preservation temperature and freeze preservation time of frozen dough on the activity of antifreeze yeast were researched.The most beneficial freezing process conditions to keep the activity of antifreeze yeast were found.The end of freezing temperature was-18℃,freeze preservation temperature should be consistent with the end of the freezing temperature.And it's better to keep the freeze preservation time less than 30 days.

  18. An immunocytochemical study of pulpal responses to cavity preparation by laser ablation in rat molars by using antibodies to heat shock protein (Hsp) 25 and class II MHC antigen.

    Science.gov (United States)

    Suzuki, Takeshi; Nomura, Shuichi; Maeda, Takeyasu; Ohshima, Hayato

    2004-03-01

    Initial responses of odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars. In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused the destruction of the odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site. Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation. By 3-5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp. The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity. These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.

  19. Testing antifreeze protein from the longhorn beetle Rhagium mordax as a kinetic gas hydrate inhibitor using a high-pressure micro differential scanning calorimeter

    DEFF Research Database (Denmark)

    Daraboina, Nagu; Perfeldt, Christine Malmos; von Solms, Nicolas

    2015-01-01

    Low dosage kinetic hydrate inhibitors are employed as alternatives to expensive thermodynamic inhibitors to manage the risk of hydrate formation inside oil and gas pipelines. These chemicals need to be tested at appropriate conditions in the laboratory before deployment in the field. A high...

  20. 防冻剂对混凝土引气剂气泡稳定性能影响研究∗%Impact study of anti-freezing agent on bubble stability performance of concrete air-entraining agent

    Institute of Scientific and Technical Information of China (English)

    张向东; 李庆文; 李广华; 李桂秀

    2015-01-01

    为探究防冻剂对混凝土引气剂气泡稳定性能的影响,考虑4种防冻剂及不同掺量等敏感性因素进行水泥稀浆摇泡实验,分析其对引气剂起泡与稳泡能力影响及作用机理。结果表明,建立摇泡实验的气泡体积衰减指数模型,其参数能表征不同引气剂起泡与稳泡能力;硝酸钙对皂苷类引气剂稳泡能力有正作用,防冻剂对其起泡能力均有负作用;乙二醇对苯磺酸盐类引气剂起泡能力有正作用,对其稳泡能力有正作用,表现为亚硝酸钙>硝酸钙>乙二醇;4种防冻剂对掺皂苷类引气剂的新拌混凝土含气量均有负影响,其显著性为乙二醇最强,亚硝酸钙最弱,钙盐类防冻剂对掺苯磺酸盐类引气剂均有负作用,乙二醇对其有正作用。%In order to explore the influence of anti-freezing agent on bubble stability performance of concrete air-entraining agent,the cement slurry bubble test was carried out considering sensitivity factors which was four kinds of anti-freezing agents and different dosages,and analyzed its effects on foaming ability,stabilizing foam ability and mechanism.The results revealed that bubble volume attenuation index model of shake bubble test was set up,which parameters characterized the foaming ability and stabilizing foam ability of different air-en-training agents.Calcium nitrate had a positive effect on stabilizing foam ability of the saponins air-entraining agent,and anti-freezing agents had a negative effect on it.Ethylene glycol had a positive effect on foaming abili-ty of the benzene sulfonate air-entraining agent,the significance of improving the stabilizing foam ability was calcium nitrite>calcium nitrate>ethylene glycol.Four kinds of anti-freezing agents all had a negative impact on the air content of fresh concrete mixed with the saponins air-entraining agent,the most significant of it was ethylene glycol,and the least significant was calcium nitrite

  1. Tumor-directed lymphocyte-activating cytokines: refolding-based preparation of recombinant human interleukin-12 and an antibody variable domain-fused protein by additive-introduced stepwise dialysis

    International Nuclear Information System (INIS)

    Integration of lymphocyte-activating cytokines (e.g., interleukin-12: IL-12) to tumor cells offers promise for cancer immunotherapy, but the preparation of such heterodimeric proteins by refolding is difficult because of subunit instability. We achieved the refolding of Escherichia coli-expressed human IL-12 by a stepwise dialysis method, preventing the formation of insoluble aggregates by adding a redox reagent and an aggregation suppressor. We also constructed a tumor-specific IL-12 protein, each subunit of which was fused with one chain of variable domain fragment (Fv) of anticarcinoembryonic antigen (CEA) antibody T84.66 (aCEA-IL12). Fusion of IL-12 with Fv greatly increased the yield of functional heterodimer. Several assays have indicated that the Fv domain and IL-12 domain of the fused protein had cognate biological activities, and it enhanced the cytotoxicity of T-LAK cells for the cancer cell line

  2. Study on preparation technology of rice bran protein hydrolysate chelated with zinc%米糠蛋白酶解物锌螯合物的制备工艺研究

    Institute of Scientific and Technical Information of China (English)

    张强; 王松华; 孙玉军; 马玉涵; 史芳芹

    2011-01-01

    Chelation process of rice bran protein hydrolyses with the zinc was studied.Three main factors including preparation methods of the rice bran protein hydrolysate, mass ratio of rice bran protein hydrolysate to zinc and pH were investigated by using single factor analysis method. On this basis, the optimal chelation technology was obtained by orthogonal experiment. The experimental results showed that the best technical conditions for preparation of chelate were as follows: pepsin combined with neutral protease was used for preparation of rice bran hydrolysate,mass ratio of rice bran protein hydrolysate to zinc was 2 to 1, pH 8.0, under this conditions the yield rates and chelate ratio was 94.93% and 94.85% respectively.%研究了米糠蛋白酶解物锌螯合物的制备工艺.通过单因素实验分析了米糠蛋白酶解物的制备方法、米糠蛋白酶解物与锌质量比和pH对螯合反应的影响,在此基础上利用正交实验优化螯合物的制备工艺.结果表明,米糠蛋白酶解物锌螯合物的最佳制备工艺为:米糠蛋白酶解物的制备方法为胃蛋白酶结合中性蛋白酶法,米糠蛋白酶解物与锌质量比2:1,pH 8.0,此条件下螯合物得率和螯合率分别为94.93%和94.85%.

  3. 登革病毒4型E蛋白的表达与多克隆抗体的制备%Expression of the envelope protein of Dengue virus type 4 and preparation of the polyclonal antibody

    Institute of Scientific and Technical Information of China (English)

    李竹石; 俞永新; 李玉华; 林华; 杨健; 杨会强; 曾献武; 赵宇; 刘俐; 刘莉娜; 康庄

    2012-01-01

    目的 在原核细胞中表达登革病毒4型E蛋白及E蛋白Ⅲ区,分别以两种蛋白免疫家兔,获得可检测登革病毒的多克隆抗体.方法 将登革病毒4型E蛋白与E蛋白Ⅲ区编码序列克隆到pET-32a(+)质粒中,分别构建两种蛋白的表达载体,以IPTG诱导其在Rosetta细胞中的大量表达,并进行SDS-PAGE检测.分别以两种蛋白免疫家兔,制备出针对E蛋白与E蛋白Ⅲ区的多克隆抗体,并对其进行Western blot鉴定.结果 登革病毒4型E蛋白与E蛋白Ⅲ区在Rosetta细胞中以包涵体的形式大量表达;利用所制备的多克隆抗体对登革病毒进行检测,出现了预期的条带.结论 本研究制备获得的多克隆抗体可用于登革病毒的检测,为登革病毒相关研究奠定了基础.%Objective To express full length (DENV4E) and domain III (DENV4EIII) of the envelope protein of Dengue virus type 4 in procaryotic cells respectively, and to prepare the polyclonal antibody against dengue virus by immunizing rabbits with the expressed proteins. Methods The sequences encoding DENV4E and DENV4EIII proteins were cloned into pET-32a ( + ) plasmids respectively to construct expression vectors. Then DENV4E or DENV4EIII proteins were expressed in Rosetta cells by IPTG induction and detected by SDS-PAGE. Polyclonal antibody against DENV4E or DENV4EIII proteins was obtained by immunizing rabbits with the expressed proteins and was identified by Western blot. Results DENV4E and DENV4EIII proteins were both successfully expressed in Rosetta cells mainly in the form of inclusion body. The prepared polyclonal antibody formed bands as expected when reacting with the dengue virus. Conclusions The polyclonal antibody is prepared and may be used in the detection of dengue virus.

  4. Study on the Anti-freezing Technology to Mountain Tea Plantations%山地茶园防冻技术研究

    Institute of Scientific and Technical Information of China (English)

    王烨军; 黄建琴; 罗仲兴; 张必桦; 徐奕鼎

    2009-01-01

    Six kinds of technical measures (pruning, covering couch grass, mulching plastic film, sprinkling irri-gation antifreeze etc.) were used to mountain tea plantations to improve the capacity of frost resistance and study the relationship between the enzyme activity of SOD, POD and freezing rates. The results showed that: (1) with freezing rates increase, the enzyme activity of SOD also increases accordingly, in a certain range to various technical measures. This is because tea plants have the self-repairing ability; (2) to mountain tea plantations; the technical measures used directly to crown of tea plant have better effect than used to environment and soil because of wind-chill effect. The result of six kinds of the technical measures is: mulching plastic film > cover-ing couch grass > CK> no pruning>irrigating roots> spraying crown; (3) It's beneficial to tea plant to reduce freezing injury with suitable pruning in autumn.%研究了修剪、铺草、覆膜、喷灌防冻剂等6项技术措施对山地茶园茶树的抗寒性影响及SOD、POD酶活性与受冻率之间的关系,结果表明:①各处理随着受冻率的增加,在一定范围内SOD酶活性也相应增加,这与茶树自身的修复功能有关;②对于山地茶园,受风冻影响,直接应用于茶树树冠的技术措施要比应用于环境及土壤的技术措施效果好.6种处理效果为:覆膜>铺草>对照>未修剪>灌根>喷冠;③秋天进行适当的修剪,对降低茶树冻害有利.

  5. Preparation and characterization of antisera against recombinant E2 protein of bovine viral diarrhea virus%牛病毒性腹泻病毒E2蛋白的多克隆抗体制备及鉴定

    Institute of Scientific and Technical Information of China (English)

    高欲燃; 朱远茂; 康健; 史鸿飞; 李娇; 任宪刚; 冯军科; 于作; 薛飞

    2011-01-01

    To prepare the polyclonal antibody against recombinant E2 protein of bovine viral diarrhea virus (BVDV) in rabbits, E. coli BL21 (DE3) was transformed with the recombinant plasmid pET30a-E2. The recombinant E2 protein was expressed in E. coli after cultivation and induction. The purified recombinant E2 protein could be recognized by specific BVDV antisera in western blot. Then the purified recombinant E2 protein was used as antigen for immunizing rabbit to prepare polyclonal antibody against the recombinant E2 protein. The result of virus nentralization test showed that the titer of the polyclonal antibody to neutralize BVDV was 1:2,048. The polyclonal antibody against the recombinant E2 protein of BVDV also had highly reactivity and specialty in immunofluorescence analysis and western blot. The polyclonal antibody against recombinant E2 protein of BVDV developed in rabbits could be used in detection of BVDV in China and provided a good basis for establishing an ELISA for detecting of E2 protein of BVDV.%为制备牛病毒性腹泻病毒(BVDV)重组E2蛋白的兔源多克隆抗体,本研究利用表达BVDV E2蛋白的重组质粒pET30a-E2转化E.coli BL21(DE3),经诱导表达获得重组E2蛋白.Western blot检测显示纯化蛋白能够与BVDV参考阳性血清反应.以纯化的重组E2蛋白免疫新西兰白兔制备多克隆抗体,病毒中和试验测定其中和效价为1:2 048,间接免疫荧光和western blot试验表明其具有良好的反应性和特异性.本研究制备的BVDV重组E2蛋白兔源多克隆抗体可应用于BVDV的检测,同时为进一步建立检测BVDV E2蛋白的ELISA方法奠定基础.

  6. Preparation and functional characterization of human vascular endothelial growth factor-melittin fusion protein with analysis of the antitumor activity in vitro and in vivo.

    Science.gov (United States)

    Wang, Dingding; Hu, Lili; Su, Manman; Wang, Ju; Xu, Tianmin

    2015-09-01

    Vascular endothelial growth factor and its tyrosine kinase receptors have been identified as key mediators of the regulation of pathologic blood vessel growth and maintenance in the promotion of angiogenesis and tumor growth. Therefore, an alternative approach to destroying tumor endothelium would be to make this tissue particularly sensitive to VEGF-mediated drug delivery. To verify this hypothesis, we generated a protein containing VEGF165 fused to melittin. Melittin is a small linear peptide composed of 26 amino acid residues that can exert toxic or inhibitory effects on many types of tumor cells. This protein is a cytolytic peptide that attacks lipid membranes, leading to significant toxicity. In the present study, the Pichia pastoris expression system was used to express the fusion protein. Under optimal conditions, stable VEGF165-melittin production was achieved using a series of purification steps. The activity of VEGF165-melittin fusion protein was compared with melittin for its ability to suppress the growth of tumor cell line in vitro. The fusion toxin selectively inhibited growth of human hepatocellular carcinoma HepG-2 cell line with high expression of VEGFR-2. We found that sensitivity of VEGFR-2 transfected 293 cells to VEGF165-melittin enhanced as the cellular VEGFR-2 density increased. In an in vivo initial experiment, the fusion protein inhibited tumor growth in xenografts assays. Furthermore, successful expression and characterization of the fusion protein demonstrated its efficacy for use as a novel treatment strategy for cancer. PMID:26166416

  7. 素混凝土抗冻性能试验研究%Experimental investigation on anti-freeze behavior of plain concrete

    Institute of Scientific and Technical Information of China (English)

    方从启; 张俊萌; 段桂珍

    2014-01-01

    In order to further study the antifreeze performance difference of confined concrete and plain concrete,in confined concrete model for reference,making the same ratio of plain concrete cubic block and under the same conditions were also tested under freeze-thaw cycle of different numbers as 0,20,40,60,100 and 120 times,As the second part,mainly analyzed the compressive strength of plain concrete under freeze-thaw test,mass loss of samples and water absorption performance changing with the number of freezing and thawing,and establish the freeze-thaw damage prediction model of concrete compressive strength and mass loss ratio.Experiment showed that with the increase of freeze-thaw cycles,the compressive strength of plain concrete decreased parabola,drying quality and full water mass linearly decreased,while water absorption decreased by double line.From the aspects of material level,fly ash had a greater influence on the loss of concrete compressive strength,mass loss rate and water absorption,while water cement ratio had little influence on the above indicators.The loss of compressive strength of mixing 15%fly ash concrete was about 20%to 30%higher than mixing no fly ash concrete;Mass loss rate was about 5%to 6%larger than not mixing fly ash ,fly ash is not conducive to the antifreeze performance of concrete.As the same fly ash,the loss of compressive strength,mass and bibulous rate in low water-cement ratio concrete changed lager than high water cement ratio.When using fly ash concrete,it is easy to reduce the water cement ratio.%为了进一步研究约束混凝土和无约束混凝土的抗冻性能差异,以约束混凝土模型为参照,制作相同配合比的素混凝土立方体块并在相同条件下进行0、20、40、60、100、120次的冻融腐蚀试验。主要分析冻融腐蚀下素混凝土试块抗压强度,质量损失以及吸水率等性能随冻融次数的变化规律,并建立素混凝土抗压强度和质量损失冻融损伤预测

  8. Fast preparation of antioxidant proteins from ginkgo and soybean by using affinity chromatography%亲和层析快速提取白果和大豆的抗氧化蛋白

    Institute of Scientific and Technical Information of China (English)

    王桂珍; 凌辉生; 谢丽思; 李任强

    2012-01-01

    After Sephadex G-25 polymerbeads were activated by using epichlorohydrin,hemin was bound with them to prepare an immobilized hemin affinity chromatography column,which was used to prepare antioxidant proteins from ginkgo and soybean.Equilibrated with deionized water and eluted with pH3.6,0.2mol/L NaAc-HAc buffer,those proteins obtained from this column were demonstrated to possess high antioxidative activity(AA) after the measurement with linoleic acid-potassium thiocyanate.AA of crude proteins of ginkgo was 7.92% and two polypeptides of them were obtained with AA 17.45% after purification.In soybean,several polypeptides with AA 30.72% were obtained from the crude proteins with AA 10.52%.This method was a novel,rapid and effective method for preparation of antioxidant proteins from ginkgo and soybean.%用环氧氯丙烷活化法把血红素结合于惰性分子筛填料SephadexG-25上作为配基做成亲和层析柱,用于从白果或大豆中提取具有抗氧化活性的蛋白。此层析柱以去离子水为平衡液、0.2mol/L的NaAc-HAc缓冲液(pH3.6)为洗脱液,层析得到的蛋白经亚油酸-硫氰酸钾抗氧化活性测定证明具有较高的抗氧化活性。白果全蛋白的抗氧化活性为7.92%,经此方法提纯后得到2种抗氧化多肽,活性达到17.45%;而大豆中的抗氧化蛋白经纯化则得到多种,抗氧化活性由全蛋白时的10.52%提高至30.72%。这是一个新的、只需一个步骤即可从白果或大豆蛋白提取液中提取到具有高抗氧化活性蛋白的方法,具有成本低,快速高效的特点。

  9. Preparation and Physicochemical Properties of Whey Protein Isolate-soluble Starch Conjugate%乳清分离蛋白-可溶性淀粉接枝物的制备及其理化性质

    Institute of Scientific and Technical Information of China (English)

    罗志刚; 卢静静; 孙炜炜

    2011-01-01

    To provide a reference on the study of protein-starch conjugate, the formation and secondary structure of whey protein isolate-soluble starch(SS) conjugate prepared by dry-heating method were investigated. The indexes of significant changes in A294 , browning intensity, free amino groups content and SDS-PAGE showed that WPI-SS conjugate was successfully prepared based on Maillard reaction under dry-heating. The results also showed that more incubation time significantly promoted glycosylation in the WPI-SS mixture. Meanwhile, the secondary structure of whey protein isolate had a considerable loss due to the covalent attachment of high molecular weight starch ; the surface hydrophobicity of whey protein was reduced.%研究了干热法处理条件下乳清分离蛋白-可溶性淀粉接枝物的制备及其二级结构分析,为蛋白质-淀粉接枝物的研究提供参考。A294、褐变、游离氨基含量变化、电泳图谱等证实乳清分离蛋白与可溶性淀粉在干热处理下确实发生了以美拉德反应为基础的接枝反应,且反应天数的延长能够显著促进乳清分离蛋白-可溶性淀粉接枝物的生成。由于大分子淀粉的共价接入,乳清分离蛋白的二级结构遭到破坏;蛋白质表面疏水性指数降低。

  10. In vivo protein biotinylation and sample preparation for the proteomic identification of organ- and disease-specific antigens accessible from the vasculature.

    Science.gov (United States)

    Roesli, Christoph; Neri, Dario; Rybak, Jascha-N

    2006-01-01

    Targeted delivery of bioactive molecules to diseased organs or tissues by means of binding molecules specific to markers of diseases represents a promising area of pharmaceutical intervention. The availability of markers of pathology, ideally accessible from the vasculature, is crucial for such strategies. To this aim, here we present a protocol based on terminal perfusion of mice with a reactive ester derivate of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ or tissue extracts are (i) purified on streptavidin resin in the presence of strong detergents, (ii) digested on the resin and (iii) subjected to proteomic analysis. This technology is applicable to comparative proteomic investigations of differentially expressed, accessible proteins in numerous animal models having different physiological and pathological processes. PMID:17406232

  11. Preparation, crystallization and preliminary X-ray diffraction analysis of two intestinal fatty-acid binding proteins in the presence of 11-(dansylamino)undecanoic acid

    International Nuclear Information System (INIS)

    Intestinal fatty-acid binding proteins from human and rat have been crystallized in complex with the fluorescent probe 11-(dansylamino)undecanoic acid. Diffraction data for the crystals were collected to 1.8 Å resolution (human) and 1.6 Å resolution (rat). Fatty-acid binding proteins (FABPs) are abundantly expressed proteins that bind a range of lipophilic molecules. They have been implicated in the import and intracellular distribution of their ligands and have been linked with metabolic and inflammatory responses in the cells in which they are expressed. Despite their high sequence identity, human intestinal FABP (hIFABP) and rat intestinal FABP (rIFABP) bind some ligands with different affinities. In order to address the structural basis of this differential binding, diffraction-quality crystals have been obtained of hIFABP and rIFABP in complex with the fluorescent fatty-acid analogue 11-(dansylamino)undecanoic acid

  12. Silica as a Matrix for Encapsulating Proteins: Surface Effects on Protein Structure Assessed by Circular Dichroism Spectroscopy

    Directory of Open Access Journals (Sweden)

    Genet H. Zemede

    2012-08-01

    Full Text Available The encapsulation of biomolecules in solid materials that retain the native properties of the molecule is a desired feature for the development of biosensors and biocatalysts. In the current study, protein entrapment in silica-based materials is explored using the sol-gel technique. This work surveys the effects of silica confinement on the structure of several model polypeptides, including apomyoglobin, copper-zinc superoxide dismutase, polyglutamine, polylysine, and type I antifreeze protein. Changes in the secondary structure of each protein following encapsulation are monitored by circular dichroism spectroscopy. In many cases, silica confinement reduces the fraction of properly-folded protein relative to solution, but addition of a secondary solute or modification of the silica surface leads to an increase in structure. Refinement of the glass surface by addition of a monosubstituted alkoxysilane during sol-gel processing is shown to be a valuable tool for testing the effects of surface chemistry on protein structure. Because silica entrapment prevents protein aggregation by isolating individual protein molecules in the pores of the glass material, one may monitor aggregation-prone polypeptides under solvent conditions that are prohibited in solution, as demonstrated with polyglutamine and a disease-related variant of superoxide dismutase.

  13. 高纯菜籽蛋白的制备及其相关性质研究%Study on preparation of high-purity rapeseed protein and its properties

    Institute of Scientific and Technical Information of China (English)

    周俊; 潘丽军; 韩智宏

    2012-01-01

    Protein was extracted from coldpressed rapeseed meal which was detoxicated firstly,then the extract was ultrafiltered to obtain high-purity rapeseed protein,chemical components,amino acid and function of proteins were also studied.Extraction rate of protein from rapeseed meal after detoxication was 67.97%,rapeseed protein of different purity from 70% to 90% could be prepared by ultrafiltration.Phytic acid,glucosinolate were not detected in proteins,and content of polyphenol was decreased obviously with rise of protein purity.Sulphur-containing amino acid(Methionine+Cysteine) was the first limiting amino acid.Chemical scores of the other essential amino acids were higher than FAO/WHO standard,but most were inferior to standard of whole egg protein.Capacities of water-holding,fat-absorbing,emulsion,emulsion stability,foaming and foam stability correlated positively with purity of rapeseed protein.%对脱毒后的冷榨菜籽粕进行蛋白提取,超滤纯化提取液获得高纯度的蛋白成品,并研究分析了蛋白的成分、氨基酸及其功能特性。脱毒后菜籽粕中蛋白提取率为67.97%,超滤后蛋白纯度可达70%~90%。成品蛋白中植酸与硫苷未检出,多酚含量随蛋白纯度增加而显著下降,甲硫氨酸+半胱氨酸是第一限制性氨基酸,其余必需氨基酸化学评分均高于FAO/WHO标准,却大多低于全蛋蛋白标准。菜籽蛋白的持水性、吸油性、乳化性及乳化稳定性、起泡性及起泡稳定性均与蛋白纯度呈正相关。

  14. Research on the structure of antifreeze asphalt pavement made from rubber particles%橡胶颗粒防冻性沥青路面结构组合研究

    Institute of Scientific and Technical Information of China (English)

    郭平; 吴德军; 田伟平; 刘雨迪; 张岁应

    2011-01-01

    为研究橡胶颗粒沥青混合料作为沥青路面不同层位与ATB、AC结构组合下沥青路面的防冻性能的变化规律,通过ANSYS有限元软件模拟冰层的受力过程,分析不同路面结构组合下的荷载响应效果.结果表明:橡胶颗粒沥青混合料作为上面层时具有较好的防冻性能;作为中面层时可明显提高路面抗车辙性能,但破冰性能降低.最后,建议采用ATB作下面层与橡胶颗粒沥青混合料作中、上面层搭配的组合结构可有效改善沥青面层的永久变形及破冰性能.%In order to study the variation of anti-freezing capability of the asphalt pavement which is made from the mixture of asphalt and rubber particles and composed of different layers and ATB/AC structure, responses of the pavement to loads were analyzed by simulating the stressed process of the ice layer with ANSYS. The results indicate that when the asphalt mixed with rubber particles was used as the surface layer, the anti-freezing capability of the pavement was enhenced. When the mixture was used as the middle layer, the anti-rutting performance could be improved significantly but the ice-breaking performance was reduced. Finally, the combination of ATB as the bottom layer and the asphalt mixture of rubber particles as the middle and surface layer is advised to be adopted,which can improve effectively the permanent deformation and ice-breaking performance.

  15. A comparative in vitro study of the digestibility of heat- and high pressure-induced gels prepared from industrial milk whey proteins

    Science.gov (United States)

    He, Jin-Song; Mu, Tai-Hua; Wang, Juan

    2013-06-01

    We undertook this study to compare the digestibility of heat- and high pressure-induced gels produced from whey protein isolate (WPI). To simulate in vivo gastrointestinal digestion of WPI gels, a pepsin-trypsin digestion system was used. The in vitro protein digestibility of WPI gels induced by high pressure (400 MPa and 30 min; P-gel) and those induced by heat (80°C and 30 min; H-gel) was compared using a protein concentration of 0.14 g mL-1. The in vitro protein digestibility of P-gels was significantly greater than that of H-gels (p<0.05). The size-exclusion chromatography profiles of the hydrolysates showed that the P-gel generated more and smaller peptides than natural WPI and H-gels. Furthermore, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed some soluble disulfide-mediated aggregation in the P-gel, while there was more insoluble aggregation in the H-gel than the P-gel. The P-gel was more sensitive to proteinase than the H-gel, which was related to the content of S-S bonds, and this in turn could be attributed to the differences in the gelation mechanism between the H-gel and P-gel.

  16. Assessment of a radioisotopic assay for vitamin B12 using an intrinsic factor preparation with R proteins blocked by vitamin B12 analogues

    OpenAIRE

    Bain, Barbara; Broom, GN; Woodside, Jackie; Litwinczuk, RA; Wickramasinghe, SN

    1982-01-01

    A competitive protein binding radioassay kit for serum vitamin B12 has been assessed. Precision, linearity, sensitivity, and specificity have been found to be satisfactory. Falsely-normal assay results in patients with vitamin B12 deficiency have not been observed.

  17. Process of peanut protein milk powder by dry preparation%花生蛋白奶粉的干法生产工艺研究

    Institute of Scientific and Technical Information of China (English)

    章宝; 单杨; 李高阳

    2011-01-01

    以去壳花生仁为原料,经低温烘烤、脱红衣、冷榨脱脂、超微粉碎等工艺生产出花生蛋白粉,将其与全脂奶粉混合生产花生蛋白奶粉,所得产品溶解度高、冲调性好、口感细腻、营养均衡,同时具有奶味和花生特有的香味。%Peanut protein milk powder was made by mixing whole milk powder with peanut protein powder,which was made by the technologies of low-temperature baking,taking the peanut red skin off,cold-pressed degreasing and ultra-fine pulverizatio by using shelled pea

  18. Study on the preparation and application of hydrolyzed eel head protein by enzymatic methods%酶法制备水解鳗鱼头蛋白及其应用的研究

    Institute of Scientific and Technical Information of China (English)

    余杰; 陈美珍

    2001-01-01

    The composition of waste eel head from rosast eel was determined.The hydrolysis of eel head by enzymes to prepare fish protein hydrolyzate and its application in seafood condiment were studied. And the best hydrolytic condition was: temper ature 50℃,pH7.0, enzyme amount 60u/g∶40u/g (proteinase:papain), hydrolysis ti me 4.5h and solid/liquid ratio 1∶1.1~1.5. The content of crude protein in f ish protein hydrolysate amounted to 87.63% and the recovery of protein was 37. 1%. The optimum recipe of condiment made on the basis of hydrolyzed fish protein was determined, and the product had characters of high nutrition and intense de licious seafood flavor.%测定了烤鳗下脚料鳗鱼头的成分,研究了酶解鳗鱼头提取水解鱼蛋白及其在海鲜调味料中的应用。双酶水解的最适条件为:温度50℃,pH7.0,加酶量为中性蛋白酶:木瓜蛋白酶60u/g∶40u/g水解时间4.5h,料液比1∶1.1~1.5,水解鱼蛋白中粗蛋白含量达87.63%,蛋白质收率37.1%。确定了以水解鱼蛋白为基料制备风味调料的最适配方,该产品具有营养价值高,海鲜风味浓郁的特点。

  19. Evaluation of a Native Preparation of HCV Core Protein (2-122 For Potential Applications in Immunization, Diagnosis and Mab Production

    Directory of Open Access Journals (Sweden)

    MR Aghasadeghi

    2006-05-01

    Full Text Available Infection with hepatitis C virus (HCV is a worldwide problem. Among HCV proteins, core antigen (Ag, besides its importance for diagnostic application is a prime candidate for component of a vaccine. Herein, we report results of studies on production of the hydrophilic domain of core Ag (2-122 in native conformation by an arabinose induction system in E.coli and the primary characterization of this recombinant protein for applications in diagnosis, immunization and mAb production. Recombinant core (r-Core was able to detect anti-core antibodies in HCV positive serum samples in a dilution rate of 1/3200. It was also capable to elicit a potent anti-HCV humoral immune response in BALB/c mice. Finally, we established two stable clones of hybridoma which shown to produce specific and sensitive mAbs against the core protein. HCV core was able to elicit a broad range of antibody specificities depending on the immunogen conformation. Therefore, it may be possible to get new mAbs with higher affinities towards native conformation of core Ag.

  20. Comparison of two chemical cleavage methods for preparation of a truncated form of recombinant human insulin-like growth factor I from a secreted fusion protein.

    Science.gov (United States)

    Forsberg, G; Baastrup, B; Brobjer, M; Lake, M; Jörnvall, H; Hartmanis, M

    1989-12-01

    We have produced a naturally occurring variant of human insulin-like growth factor I, truncated by three amino acids at the amino terminus. The polypeptide is obtained as a fusion protein in Escherichia coli. The fusion partner is a synthetic IgG-binding peptide. During fermentation the fusion protein is secreted into the medium, and is purified on IgG--Sepharose prior to cleavage. Two different genes for the fusion protein were used, allowing chemical cleavage at either a tryptophan linker or a methionine linker between the fusion partner and the growth factor, using N-chlorosuccinimide (NCS) or cyanogen bromide (CNBr) respectively. A partial CNBr cleavage yielded the native peptide, whereas the NCS cleavage yielded a product in which the single methionine had been oxidized to the sulfoxide. The forms from both cleavage methods exhibited biological activity and were characterized after purification to homogeneity. Both cleavage methods gave products having correct N- and C-terminal ends. The purified product had a biological activity equal to that of corresponding material from natural sources, 15 000 U/mg. Modified forms of truncated IGF-I were also identified, purified and characterized. Modifications such as proteolysis and misincorporation of norleucine for methionine occurred during biosynthesis, while oxidation of methionine took place during both fermentation and chemical cleavage. PMID:2696476

  1. Extraction of phytic acid and preparation of protein isolates from rapeseed meal%菜籽粕植酸提取和分离蛋白的制备

    Institute of Scientific and Technical Information of China (English)

    潘丽军; 周俊; 姜绍通; 孙汉巨; 罗水忠; 韩智宏

    2011-01-01

    植酸和蛋白是菜籽粕中2种极具经济价值的成份.为提高菜籽粕的综合利用效果,该文以双低冷榨菜籽粕为原料,采用醋酸溶液提取植酸,在膜分离精制植酸粗提液过程中同时回收蛋白;再对植酸提取后的残余物进行蛋白分离,超滤纯化后获得高纯度的蛋白成品.响应面优化的植酸最适提取条件为:醋酸质量分数0.7%,提取温度48℃,液料比10 mL/g,提取时间1.6 h,该条件下植酸得率为1.865%.植酸粗提液中回收出的蛋白和损失植酸分别占菜籽粕的3.63%和0.395%.超滤精制的分离蛋白可达到70%~90%不同纯度的要求,蛋白中多酚含量显著减少,且植酸与硫苷未检出.%Phytic acid and protein are two kinds of valuable components in rapeseed meal. To improve comprehensive utilization in this research, phytic acid was extracted with acetum from double-low coldpressed rapeseed meal, and protein was recovered from the crude extract of phytic acid by membrane separating technology. The meal residue was dried to extract protein, which was then purified by ultrafiltration to obtain high purity product. Extraction conditions of phytic acid were optimized by response surface methodology (RSM) as follows: mass fraction of acetic acid 0.7%,extraction temperature 48℃, liquid-to-solid ratio 10 mL/g, extraction time 1.6 h. Under this condition, extraction yield was 1.865%. Protein recovered and phytic acid loss accounting for rapeseed meal were 3.63% and 0.395% respectively in crude extract of phytic acid. Purity of the protein refined by ultrafiltration was between 70% and 90%, in which the content ofpolyphenols was significantly reduced and no phytic acid and glucosinolates could be detected.

  2. Solution preparation

    International Nuclear Information System (INIS)

    Reviewed in this statement are methods of preparing solutions to be used in laboratory experiments to examine technical issues related to the safe disposal of nuclear waste from power generation. Each approach currently used to prepare solutions has advantages and any one approach may be preferred over the others in particular situations, depending upon the goals of the experimental program. These advantages are highlighted herein for three approaches to solution preparation that are currently used most in studies of nuclear waste disposal. Discussion of the disadvantages of each approach is presented to help a user select a preparation method for his particular studies. Also presented in this statement are general observations regarding solution preparation. These observations are used as examples of the types of concerns that need to be addressed regarding solution preparation. As shown by these examples, prior to experimentation or chemical analyses, laboratory techniques based on scientific knowledge of solutions can be applied to solutions, often resulting in great improvement in the usefulness of results

  3. 菠萝蛋白酶水解泥鳅蛋白制备ACE抑制肽的研究%Preparation of ACE Inhibitory Peptides by Bromelain Hydrolysis of Loach(Misgurnus anguillicaudatus) Protein

    Institute of Scientific and Technical Information of China (English)

    姚东瑞; 盘赛昆; 周鸣谦; 王淑军; 胡金玲

    2012-01-01

    为了探讨利用泥鳅蛋白制备功能性肽的可能性,采用高效液相色谱法测定泥鳅肉水解物对血管紧张素转换酶(ACE)的抑制作用,从胰蛋白酶、胃蛋白酶、菠萝蛋白酶、复合风味蛋白酶、复合蛋白酶5种酶中筛选出菠萝蛋白酶作为酶解泥鳅肉制备具有降血压活性水解物的适宜水解酶。在单因素试验的基础上,采用L9(34)正交试验设计对该酶的酶解条件进行优化。结果表明最佳水解条件为:温度55℃,固液比1:3,pH6.5,加酶量1000U/g pro,水解时间90min。在该条件下,水解物的ACE抑制率IC50值为0.0184mg/mL,ACE抑制肽的相对分子质量主要集中在924左右。%The present deals with the enzymatic preparation of angiotensin-converting enzyme(ACE) inhibitory active peptides from loach(Misgurnus anguillicaudatus) protein.An L9(34) orthogonal array design methods based on single factor experiments was used to optimize the hydrolysis conditions for achieving maximum ACE inhibitory activity of loach protein hydrolysates.HPLC was used to determine ACE inhibitory activity of loach protein hydrolysates.Bromelain was found to be more suitable to prepare ACE inhibitory active peptides than trypsin,pepsin,flavourzyme and protamex.The optimal hydrolysis conditions were temperature of 55 ℃,solid-to-liquid ratio of 1:3,pH of 6.5,bromelain dose of 1000 U/g protein and hydrolysis duration of 90 min.The IC50 of the loach protein hydrolysate obtained under these conditions was 0.0184 mg/mL and the relative molecular weight distribution of ACE inhibitory peptides in it was mainly concentrated around 924.

  4. 人博卡病毒VP2蛋白单克隆抗体的制备%Preparation of the monoclonal antibody against human Bocavirus VP2 protein

    Institute of Scientific and Technical Information of China (English)

    赵智慧; 薛鹏浩; 魏建民; 张骞; 郑文芝; 麻粉莲; 袁武梅; 郑丽舒

    2012-01-01

    Objective To express and purify HBoV VP2 protein,and the monoclonal antibody against HBoV VP2 protein was prepared with hybridoma technique.Methods The HBoV VP2 cloned into vector pET-30a was expressed in E.coil.After purified by immobilized metal affinity chromatography,the BALB/c mouse was immunized with purified protein as antigen.The positive hybridoma cells were screened with hybridoma technique and ELISA assay. Isotype and titer of the monoclonal antibody were detected.Results The recombinant HBoV VP2 protein was expressed and purified,and then the monoclonal antibody was obtained with hybridoma technique.The titer of the IgG monoclonal antibody was up to 1 ∶ 4 × 105.Conclusion Monoclonal antibody against recombinant HBoV VP2 protein was prepared and the antibody titer was high.This work may provide a new method in rapid diagnosis and study of HBoV.%目的 表达纯化人博卡病毒( human Bocavirus,HBoV) VP2蛋白,采用杂交瘤方法制备抗HBoV VP2蛋白的单克隆抗体.方法 应用原核表达载体pET-30a和大肠埃希菌表达VP2蛋白,并用固定化金属亲和层析,纯化后的蛋白作为抗原免疫BALB/c小鼠,利用杂交瘤技术和间接ELASA方法筛选阳性的杂交瘤细胞,并对单克隆抗体进行分型检测和滴度测定.结果 表达并纯化获得了重组HBoV VP2蛋白,利用杂交瘤技术得到单克隆IgG抗体,抗体效价达到1∶4×105.结论 利用HBoV VP2蛋白免疫制备了单克隆抗体,并具有较高的效价.本研究为快速诊断和研究HBoV打下基础.

  5. 响应面法超声波辅助提取核桃蛋白工艺优化%Optimization of the preparation of walnut protein with ultrasonic-assisted extraction method

    Institute of Scientific and Technical Information of China (English)

    敬思群

    2012-01-01

    Walnut protein was obstained by ultrasonic-assisted extraction method with the defatted walnut meal as raw material. The preparation process of walnut protein was optimized according to Box- Behnken central composition design principle on foundation of single factor experiment, the method of response surface analysis with 3 factors and 3 levels combined with Minitab15.0-statistical data analysis software was adopted with the leaching rate of walnut protein as the response value. The effect of extraction time, temperature, liquid to solid ratio and pH on the leaching rate of walnut protein were studied in this paper. According to the above results, the optimum preparation conditions were obtained as follows: extraction time 19 min, reaction time 90 min, temperature 46 %, ratio of solvent: meal at 20:1 and pHS.6. Under these conditions, the leaching rate of walnut protein reached 18.66%.%以脱脂核桃粕为原料,利用超声波辅助提取制备核桃蛋白。在单因素实验的基础上,根据Box-Behnken的中心组合实验设计原理,运用Minitab15.0数据统计分析软件,采用3因素3水平的响应面分析法,以核桃蛋白浸出率为响应值,研究了超声时间、超声温度、液料比和pH对核桃蛋白浸出率的影响,并优化了提取工艺。确定了超声辅助提取核桃蛋白的最佳工艺条件为:超声时间为19 min,超声温度为46℃,液料比为20:1,pH为8.6,在此条件下核桃蛋白的浸出率达到68.98%。

  6. Expression of mouse calreticuin protein and preparation of its polyclonal antibodies%小鼠钙网蛋白的原核表达和多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    汪龚泽; 刘朝奇; 杨建林; 聂纪芹

    2011-01-01

    In order to investigate the biological properties of calreticulin(CRT), a protein present in the mammalian cells with highly conservative and multiple biological properties, a short fragment of mouse CRT was amplified through RT-PCR method and then was cloned into prokaryotic expression vector pDET28a( + ). The recombinant protein CRT was induced by IPTG in E.coli BL2KDE3) and the expressed protein was detected by Western blotting. The purified protein was used to immune mouse to prepare polyclonal antibody and the specificity and titer of the antibody were detected by ELISA, Western blotting and FCM. It was demonstrated that the recombinant prokaryotic expression plasmid pET28a( + ) /CRT was successfully con structed and the CRT protein was expressed in E. Coli BL2KDE3) with high efficiency. Western blot assay showed that this re combinant protein was characterized with its antibody. Mouse immunized with the purified protein produced high titer of anti body. Western blot assay displayed that the CRT protein was highly expressed in some of eukaryotic cells and could specifically combine with the antibody. FCM assay displayed that the antibody could also specifically combine with the membrane extracel lular region of CRT. It is evident that the preparation of recombinant CRT and its polyclonal antibodies have a strong specificity to match with the CRT protein from mouse and human.%钙网蛋白(calreticulin,CRT)是存在于哺乳动物细胞具有高度保守性和多种生物学活性的蛋白,为了更好的研究其生物学活性,本课题组通过PCR方法扩增CRT截短基因,将其克隆到原核表达载体PET-28a(+)中,经大肠杆菌表达并纯化CRT蛋白.以纯化的CRT蛋白抗原免疫BALB/c小鼠,制备多克隆的CRT血清.进一步采用Western blot、ELISA、流式细胞术等技术对制备的抗体进行初步鉴定.结果显示:原核表达重组质粒在大肠杆菌中能高效表达CRT蛋白;获得多抗血清应用Western blot鉴定几种

  7. Preparation of Anti-malaria Antibodies with a Way of Peptide-protein Conjugation%用多肽-蛋白偶联方法制备抗恶性疟原虫抗体

    Institute of Scientific and Technical Information of China (English)

    钱锋

    2012-01-01

    目的 介绍一种多肽-蛋白偶联的流程,并用多肽-蛋白偶联产物制备抗疟原虫抗体. 方法 用化学连接剂Sulfo-EMCS在作为载体蛋白的铜绿假单胞菌重组去毒外毒素(rEPA)上加马来酰亚胺基团,用间接Ellman反应测定载体蛋白上所加的马来酰亚胺基团数量.用马来酰亚胺修饰的载体蛋白滴定Pfs48/45-158多肽[含恶性疟原虫表面蛋白48/45(Pfs48/45)第158~~173氨基酸序列,其N末端带有一个半胱氨酸残基],绘制滴定曲线并用线性回归进行曲线拟合,根据滴定曲线确定理论滴定终点,计算多肽与载体蛋白的偶联比(每摩尔载体蛋白所能结合的多肽的摩尔数).用过量的Pfs48/45-158多肽与马来酰亚胺修饰的rEPA进行反应,规模制备Pfs48/45-158-rEPA多肽-蛋白偶联物,偶联物用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定.用所制备的Pfs48/45-158-rEPA偶联物免疫BALA/c小鼠,制备免疫血清.用ELISA测定免疫小鼠血清抗Pfs48/45-158多肽的抗体效价,用免疫荧光试验(IFA)测定免疫血清识别疟原虫的能力. 结果 通过化学连接剂在每摩尔rEPA上添加约6.94摩尔的马来酰亚胺基团.制备了偶联比约为7.33的Pfs48/45-158-rEPA多肽-蛋白偶联物.偶联物免疫小鼠激发出抗Pfs48/45-158多肽的高抗体应答,免疫血清抗Pfs48/45-158多肽的效价为12 500 ELISA单位(即吸光度A405值为1时的血清稀释度倒数),同时免疫血清可识别疟原虫. 结论 多肽-蛋白偶联是一种可用于制备抗疟原虫抗体的便捷方法,间接Ellman检测、滴定反应和SDS-PAGE分析构成了多肽-蛋白偶联物制备的质控方法,可更好地保证多肽-蛋白偶联物的质量和稳定性.%Objective To introduce a procedure of peptide-protein conjugation and prepare anti-malaria antibodies using a peptide-protein conjugate. Methods The recombinant atoxic form of Pseudomonas aeruginosa exotoxin A (rEPA) was used as carrier

  8. 活性蛋白肉在速冻调理食品中的应用技术研究%Study on application of active protein meat alternatives in quick-frozen prepared food

    Institute of Scientific and Technical Information of China (English)

    尚丹; 史九根

    2015-01-01

    将大豆分离蛋白制备成蛋白肉并在各类含馅类速冻调理食品中使用。首先是利用大豆分离蛋白和胶凝剂制备活性蛋白肉,以蛋白肉为原料添加在各种调理食品中。以速冻饺子、鸡肉丸为例,代替10%、15%、20%、30%猪肉的蛋白肉,结果显示,在猪肉饺子中添加10%的活性蛋白肉产品口感更好,咀嚼感更强;另活性蛋白肉的添加有效吸收了饺子馅料在包制、成型和储存过程中析出的水分等液体汤料,从而降低了饺子皮的破皮率,提高饺子出品率;在鸡肉丸中加入5%、10%、15%、20%的蛋白肉,添加的鸡胸肉比例相应减少,结果显示,鸡肉丸中加入5%的活性蛋白肉和不添加蛋白肉的产品口感和质构接近,但在煮制过程中,添加活性蛋白肉的鸡肉丸出油少,保油效果更好。%Soy protein isolate was made into protein meat alternatives. And they were used in vari⁃ous quick-frozen food containing stuffing. Firstly soy protein isolate and gelling agent were used to pre⁃pare active protein meat alternatives. Protein meat alternatives were taken as material to add in all kinds of prepared food. Quick -frozen dumplings and chicken meatballs were taken as examples, replacing 10%, 15%, 20% and 30% pork with protein meat alternatives. The results showed that when the pork dumplings added with 10% active protein meat alternatives, the taste and crunch would be better. Be⁃sides the addition of active protein meat alternatives could efficiently absorb the water and soup separating from stuffing during making, shaping and storage process. Therefore the broken rate of dumpling wrapper decreased, and the output rate increased. The chicken meatballs were added with 5%, 10%, 15% and 20% protein meat alternatives, respectively. The addition proportion of chicken breast reduced corre⁃spondingly. The results showed that the chicken meatballs added with 5% protein meat

  9. High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization

    Directory of Open Access Journals (Sweden)

    Tanaka Shigeyasu

    2009-06-01

    Full Text Available Abstract Background Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR that displayed human (prorenin receptor (hPRR connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC. Results A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR. A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i., but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i.. Conclusion The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.

  10. Preparation and Preliminary Separation of Antioxidant Peptide of Rice Bran Protein%米糠蛋白抗氧化肽的制备及初步分离

    Institute of Scientific and Technical Information of China (English)

    李喆; 翟爱华

    2012-01-01

    试验以米糠蛋白为原料,制备米糠蛋白抗氧化肽。以DPPH自由基清除率为指标,采用正交试验,优化碱性蛋白酶酶解米糠蛋白制备抗氧化肽的工艺条件;采用超滤、SephadesG-25柱层析分离纯化米糠蛋白抗氧化肽。试验结果表明,制备米糠蛋白抗氧化肽的最优工艺条件为:酶解温度45℃,pH9.0,时间60rain,米糠蛋白底物浓度3%,碱性蛋白酶加酶量3000U·g-1。在米糠蛋白酶解产物中,Mrs〈5KDa组分的抗氧化活性最强,其对DPPH自由基清除率为68.7%。通过SephadexG-25凝胶层析柱进一步分离得到4个混合组分,混合组分Ⅱ活性最强,其DPPH自由基清除率为75.83%。%Antioxidative peptides was produced using rice bran protein as raw material. Clearance rate of 1,1-diphenyl-2- picrylhydrazyl (DPPH) radical was taken as index,using orthogonal test to optimize technological conditions of alkaline protease technology for production of antioxidant peptides from rice bran protein which were separated and purificated by ultrafiltration and Sephades G-25column chromatography.The results showed that the optimal technological conditions were as following: enzymolysis temperature 45 ~C,pH 9.0,reaction time 60 rain ,protein substrate concentration 3%,addition level of alkaline protease 3 000 U'g-l. The fraction of relative molecular weight ~〈5KDa,which was one of hydrolysis product of the rice bran protein,had the highest antioxidant activity and its clearance rate of DPPH reached to 68.7%. Four fix fractions were obtained from Sephadex G-25 column chromatography and the second fix fraction had the highest antioxidant activity,whose clearance rate of DPPH was 75.83%.

  11. Preparation and characterization of rosemary incorporated fish protein edible films%迷迭香添加鱼肉蛋白可食膜的制备与特性

    Institute of Scientific and Technical Information of China (English)

    翁武银; 陈亨莉; 刘光明; 苏文金; 曹敏杰

    2011-01-01

    Edible fish protein films based on silver carp meat were successfully prepared.The effect of rosemary antioxidant incorporation of film-forming solutions on the properties of edible fish protein films was investigated.As a result,white and transparent protein films could be produced using silver carp meat.The mechanical properties and water vapor permeability(WVP)of fish protein films were dependent mainly on the content of fish myofibrillar proteins while no obvious variation was observed between different fish species.The effect of incorporation of rosemary extract on the mechanical properties and WVP of films were not significant.However,the addition of rosemary extract led to the formation of yellow protein films with increased UV obstructing ability as well as antioxidant capacity.Eel meats packed with protein films were stored for 12h at 37℃ in the dark.Compared with the samples without packaging,the increase of eel meat POV and TBARS was markedly inhibited during the storage at 37℃.The inhibitory effect was more significant by adding rosemary extract into films.%以鲢鱼肉为主要原料制备蛋白可食膜,测定了迷迭香抗氧化剂添加对膜理化性质和抗氧化性能的影响。结果表明,利用鲢鱼肉可以制备成无色透明的蛋白可食膜,鱼肉蛋白膜的机械性质和水蒸气透过性(WVP)主要由肌原纤维蛋白含量决定,在鱼种间的差异并不显著。添加迷迭香抗氧化剂对鱼肉蛋白膜的机械性质和WVP影响不显著,但对膜的阻隔紫外线能力和抗氧化性能有提高作用。将蛋白膜包装的鳗鱼肉在37℃下放置12h,与未包装样品相比,鳗鱼肉的POV和TBARS的增加明显得到抑制,而添加迷迭香会进一步提高其抑制效果。

  12. TT病毒重组蛋白单克隆抗体的制备%Preparation and identification of monoclonal antibodies against recombinant protein of TT virus

    Institute of Scientific and Technical Information of China (English)

    刘树玲; 周育森; 陈万荣; 王海涛

    2001-01-01

    采用杂交瘤技术,获得了4株稳定分泌抗TTV重组蛋白的 单克隆抗体杂交瘤细胞株,1株属IgG2b λ链、1株属IgG1 κ链、2株属IgG2a κ链。4株杂 交瘤细胞培养上清液效价为1∶80~1∶1 280,腹水效价为1∶32万~1∶160万。%Monoclonal antibodies (McAb) against recombinant pr otein of TTV were produced by fusing SP2/0 myeloma cells with spleen cells from Bal b/c mice immunized with TTV recombinant proteins. Four hybridoma cell lines secreting specific antibodies against TTV recombinant proteins were obtained by screening hybridoma culture supernatants using ELISA with TTV recombinant protei ns on solid phase. One of McAbs was IgG2bλ, One of McAbs was IgG1κ, Others wer e IgG2aκ, The titers of hybridoma culture supernatants ranged from 1∶80~1∶1 280 and from 1∶320 000~1∶1 600 000 in ascites.

  13. Water-soluble gold nanoclusters prepared by protein-ligand interaction as fluorescent probe for real-time assay of pyrophosphatase activity.

    Science.gov (United States)

    Deng, Hao-Hua; Wang, Fei-Fei; Shi, Xiao-Qiong; Peng, Hua-Ping; Liu, Ai-Lin; Xia, Xing-Hua; Chen, Wei

    2016-09-15

    This paper reports a new and facile method for the synthesis of water-soluble thiolate-protected AuNCs via protein-ligand interaction. Using 3-mercaptopropionic acid (MPA) as a model ligand and bovine serum albumin (BSA) as a model protein, water-soluble AuNCs (BSA/MPA-AuNCs) with intense orange-yellow fluorescent emission (quantum yield=16%) are obtained. Results show that AuNCs produced with this method have hydrophobic interactions with BSA. The synthetic strategy is then successfully extended to produce water-soluble AuNCs protected by other thiolates. Moreover, a sensitive and eco-friendly sensing system is established for detection of the activity of inorganic pyrophosphatase (PPase), which relies on the selective coordination of Fe(3+)with BSA/MPA-AuNCs, the higher affinity between pyrophosphate (PPi) and Fe(3+), and the hydrolysis of PPi by PPase. A good linearity between the fluorescence intensity and PPase activity within the range from 0.1 to 3U/L is found, with a detection limit down to 0.07U/L. Additionally, the fluorescent assay developed here is utilized to assay the PPase activity in real biological samples and as well as to evaluate PPase inhibitor, illustrating the great potential for biological analysis. PMID:27093483

  14. 利用马铃薯淀粉生产的废水及废渣发酵制备蛋白饲料%The Preparation of Fermented Protein Feed from Waste Water and Wastes of Potate Starch Processing

    Institute of Scientific and Technical Information of China (English)

    陈辉; 李虹; 冯雷; 张露; 宋国勇

    2011-01-01

    Microorganism fermentation method was used to produce high value-added protein feed from waste-water and wastes of potato starch production. This can lower COD content in waste-water and comprehensive utilized the potato starch processing wastes. A yeast strain was screened in our laboratory, and the optimum preparation conditions of protein feed was determined as: 20% wastes in waste-water(W/V) , natural pH, 28℃, inoculation amount 10% , cultivation time 72 h. In the optimum condition, protein content of the protein feed production was 37.40% and waste-water COD decreased 72.29%.%以马铃薯淀粉生产的废水及废渣为原料,采用微生物发酵的方法,制备高附加值的蛋白饲料,同时降低废水的COD值,达到综合利用的目的。经试验筛选得到1株酵母,并确定了蛋白饲料的制各条件:废水中薯渣添加量20%、pH自然、温度28℃、接种量10%、发酵时间72h。在适宜条件下,制备的蛋白饲料蛋白含量达37.40%,废水COD降低72.29%。

  15. Prokaryotic expression, purification, and antiserum preparation of GAG and ENV proteins of human foamy virus%人泡沫病毒GAG、ENV蛋白的原核表达、抗血清制备及鉴定

    Institute of Scientific and Technical Information of China (English)

    卢飞; 孙燕; 李晶; 李治

    2012-01-01

    In order to detect the efficiency of human foamy virus infection on protein expression level, the recombinant prokaryotic expression vectors pET-32a-GAG and pET-32a-ENV were constructed successfully and transformed into E. coli BL21 Star (DE3), and the GAG, ENV fusion proteins were induced by IPTG and expressed at high level. Subsequently, the New Zealand rabbits were immunized with the fusion proteins for preparing the antiserums. Western Blot analysis showed that the antiserum could interact with GAG and ENV fusion proteins specifically. Based on the results from Western Blot and indirect immunofluotesent method, it is concluded that the antiserums were verified to interact with the natural GAG and ENV expressed from the viruses.%构建了HFV的GAG和ENV蛋白原核表达载体pET-32a-gag和pET-32a-env,转化E.coli BL21Star(DE3)后用IPTG诱导出高水平的GAG、ENV融合蛋白表达.以融合蛋白免疫新西兰兔制备了GAG和ENV抗血清.Western Blot检测表明,抗血清可以识别原核表达的GAG和ENV蛋白,说明抗血清具有较好的特异性.结合Western Blot和间接免疫荧光实验,表明抗血清可以检测到病毒表达的GAG和ENV蛋白.

  16. Cloning, Prokaryotic Expression of Echinococcus granulosus Heat Shock Protein 70 and Preparation of It's Antiserum%细粒棘球绦虫Hsp70基因的克隆、表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    赵莉; 薛晶; 石保新; 陈皓斐; 张文宝; 马正海; 张壮志; 张旭; 古努尔·吐尔逊; 米晓云; 金映红

    2012-01-01

    [Objective] The objective of the experiment is to express and purify E. granulosus (Eg) heat shock protein 70 (EgHsp70) in E. coli and prepare the antibody against E. granulosus. [ Methods] EgHsp70 gene was amplified by PCR and cloned into prokaryotic expression vector pMAL-p2x, and the recombinant plasmid was transformed into E. coil BL-21. The soluble expression conditions of fusion protein were optimized by induction with different concentrations, of IPTG different temperatures and cultivation times. The expressed fusion protein was purified by Mal-tag Magnetic Beads. To prepare the anti-serum, New Zealand white rabbits were immunized with purified EgHsp70 protein via hypodermic and volar. Western blot was used to determine the serum's specificity against EgHsp70 and native proteins. The serum titers were analyzed by ELISA. [Results] Full-length of EgHsp70 gene had an open reading frame of 765 bps encoding a protein mass of 68.6 kD. Restriction endonuclease analysis and DNA sequencing showed that EgHsp70 was cloned into the plasmid pMAL-p2x. Based on the optimization experiments, it was concluded that the best soluble expression conditions for the EgHsp70 protein are using 0.3 mmol· L-1 IPTG when bacterial cells growing to OD600 0.6 and induced for 4 h at 30℃. ELISA and Western blotting showed that the titers of the anti-serum were above 1 : 256 000, and the anti-serum could specifically bind with EgHsp70 protein and native proteins. [Conclusion] The EgHsp70 fusion protein was obtained by expressing in E.coli and purifying, and the antibody against EgHsp70 was prepared with the fusion protein immunized New Zealand white rabbits. This work will provide an antigen and detection antibody for further study on the EgHsp70 function. The protein is immunogenic and can be a vaccine candidate against Echinococcus infection.%[目的]克隆细粒棘球绦虫(Echinococcus granulosus,E.g)热休克蛋白家族基因Hsp70,在原核细胞中表达、纯化Hsp70蛋白并制

  17. Preparation and identification of water-soluble calcium-binding protein from grape (Vitis vinifera L.) seeds%葡萄籽中水溶性钙结合蛋白的分离和鉴定

    Institute of Scientific and Technical Information of China (English)

    吕晨艳; 赵广华

    2015-01-01

    grape seed protein by the ammonium sulfate sediment was approximately 3-fold larger than that by the traditional method, demonstrating that the ammonium sulfate sediment was a better way to isolate mineral-containing protein as compared to the traditional method. A high yield of calcium by the ammonium sulfate sediment could be derived from its mild condition, whereas acid and alkaline used in the alkali extraction and acid precipitation possibly inhibits the binding of calcium ions with grape seed protein. The following FT-IR (Fourier transform infrared spectroscopy) study showed that the prominent band of apo grape seed protein attributed to random coils turns (1 666 cm−1) was shifted to lower wave number (1 660 cm−1) with a marked decreased in intensity upon calcium binding with the protein and indicated that the binding of calcium to the protein stabilizes the secondary structure of WGSP by changing state of C=O. Moreover, the abundant amino acid residues were found in WGSP to be glutamic and aspartic acids, which accounted for about 26.7% and 9.0% of the total amino acid, respectively, and these amino acids might be beneficial for calcium binding. This study could provide a foundation for the preparation of mineral-containing protein in food industry. This method may have a potential use in food industry for isolation of mineral-containing protein from other sources.%为了开发植物源葡萄籽补钙制剂,该研究通过亚细胞定位试验表明,葡萄籽的胚乳中含有大量的钙元素。通过电泳分析发现,葡萄籽的水溶性蛋白包括2种主要成分,其中一种是11 S球蛋白(蛋白质B),也是最主要的钙结合蛋白,另一种是表观分子量为670 kDa的蛋白质A。在蛋白质组成相同的情况下,用传统的碱溶酸沉法来分离葡萄籽蛋白会导致大量的钙流失。但用30%~50%硫酸铵沉淀法得到的蛋白质得率是(22.5±0.02)g/kg,蛋白质中钙质量分数(3.47%)

  18. 不同干燥方法制备的星油藤分离蛋白功能性质的比较研究%Comparison of functional properties of Sacha Inchi protein isolates prepared by different drying methods

    Institute of Scientific and Technical Information of China (English)

    夏克东; 张骊; 李海旺; 田少君

    2015-01-01

    With Sacha Inchi ( Plukenetia volubilis L. ) cake as raw material, Sacha Inchi protein isolates were prepared by vacuum freeze-drying and spray drying methods. The influences of temperature, pH and mass concentration on the functional properties of the Sacha Inchi protein isolates obtained via the two drying methods were studied and compared. The results showed that oil absorption and solubility of the Sa-cha Inchi protein isolate reached the highest at 55℃;water retention reached the maximum at pH 7;vis-cosity decreased with the temperature increasing;the foamability, emulsifying ability and emulsion stabil-ity of Sacha Inchi protein isolate improved with the mass concentration of protein increasing. The solubili-ty, oil absorption, water retention and foamability of Sacha Inchi protein isolate obtained by vacuum freeze-drying method were better than those obtained by spray drying method;its emulsifying ability and emulsion stability obtained by vacuum freeze-drying method were higher than those obtained by spray drying method.%以星油藤饼为原料,分别采用真空冷冻干燥和喷雾干燥的方法制备星油藤分离蛋白。研究了温度、pH、质量浓度对两种干燥方法制备的星油藤分离蛋白功能性质的影响并进行比较。结果表明:两种方法制备的星油藤分离蛋白的溶解度和吸油性在55℃时最大;持水性在pH 7时最大;黏度随温度的升高而降低;起泡性和乳化活性、乳化稳定性均随蛋白质质量浓度的升高而增大。真空冷冻干燥样品的吸油性、持水性、溶解性、起泡性大于喷雾干燥的样品;其乳化活性与乳化稳定性高于喷雾干燥的样品。

  19. Comparison of functional properties of Sacha Inchi protein isolates prepared by different drying methods%不同干燥方法制备的星油藤分离蛋白功能性质的比较研究

    Institute of Scientific and Technical Information of China (English)

    夏克东; 张骊; 李海旺; 田少君

    2015-01-01

    以星油藤饼为原料,分别采用真空冷冻干燥和喷雾干燥的方法制备星油藤分离蛋白。研究了温度、pH、质量浓度对两种干燥方法制备的星油藤分离蛋白功能性质的影响并进行比较。结果表明:两种方法制备的星油藤分离蛋白的溶解度和吸油性在55℃时最大;持水性在pH 7时最大;黏度随温度的升高而降低;起泡性和乳化活性、乳化稳定性均随蛋白质质量浓度的升高而增大。真空冷冻干燥样品的吸油性、持水性、溶解性、起泡性大于喷雾干燥的样品;其乳化活性与乳化稳定性高于喷雾干燥的样品。%With Sacha Inchi ( Plukenetia volubilis L. ) cake as raw material, Sacha Inchi protein isolates were prepared by vacuum freeze-drying and spray drying methods. The influences of temperature, pH and mass concentration on the functional properties of the Sacha Inchi protein isolates obtained via the two drying methods were studied and compared. The results showed that oil absorption and solubility of the Sa-cha Inchi protein isolate reached the highest at 55℃;water retention reached the maximum at pH 7;vis-cosity decreased with the temperature increasing;the foamability, emulsifying ability and emulsion stabil-ity of Sacha Inchi protein isolate improved with the mass concentration of protein increasing. The solubili-ty, oil absorption, water retention and foamability of Sacha Inchi protein isolate obtained by vacuum freeze-drying method were better than those obtained by spray drying method;its emulsifying ability and emulsion stability obtained by vacuum freeze-drying method were higher than those obtained by spray drying method.

  20. Research progress on preparing functional polypeptide from whey protein hydrolysis%乳清蛋白水解制备功能性多肽的研究概况

    Institute of Scientific and Technical Information of China (English)

    李晓东; 蒋琛; 宋惠敏

    2014-01-01

    . In this experiment, whey protein was as raw material, hypocholestero-lemic peptides were separated and prepared from its hydrolysates. In this subject, by-product of cheese was full used, it not only reduced waste of resources and environmental pollution, but also provided a theoretical basis for the commercial development of hypocholesterolemic functional products. This paper reviews the whey protein source of functional polypeptides preparation, separation and purification technology and research status of active peptides.

  1. Expression and antibody preparation of nonstructural protein 1 for Japanese encephalitis virus from pigs%猪日本乙型脑炎病毒NS1基因的表达和抗体制备

    Institute of Scientific and Technical Information of China (English)

    沈红霞; 韩秀杰; 赵凡凡; 张保新; 余风艳; 王晓杜

    2013-01-01

    Japanese encephalitis virus (JEV) in breeding pigs,has caused reproductive disorders,such as orchitis,stillbirths,and mummified fetuses,and has produced encephalitis in piglets.The NS1 (nonstructural protein 1) gene is associated with viral RNA packaging and replication and with viral anti-host immunity.NS1 protein were expressed by prokaryotic expression system and polyclonal antibodies of NS1 were prepared.In this study,the cDNA of JEV was synthesized from a viral genome by reverse transcription-polymerase chain reaction (RT-PCR).The NS1 gene was cloned from cDNA by PCR and subcloned into pET-28(a) plasmid.The recombinant plasmid pET-28 (a)-JEV-NS1 was then transformed into Escherichia coli BL21 (DE3).Next,the recombinant JEV-NS1 protein (whose molecular weight is 46 kDa) was expressed by isopropyl-beta-D-thiogalactopyranoside (IPTG) induction.To improve the expression level of the recombinant JEV-NS1 protein,the 958-1 245 bp of the JEV-NS1 gene was truncated,and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for the analysis.Also,the protein was immunized into an Institute of Cancer Research (ICR) mouse; then the mouse anti-JEV-NS1 antiserum was prepared; the antiserum specificity were detected with western-blotting.Results showed that the truncated JEV-NS1 expression was greatly increased and the SDS-PAGE analysis confirmed this.In addition,purification production of the recombinant protein was 85% of the total protein content.The antiserum of NS1 can specifically recognized the production of JEV infected cells.This study will assist in JEV-NS1 functional research and exploration of JEV pathogenic mechanism.%猪Sus scrofa domestica日本乙型脑炎病毒(Japanese encephalitis virus,JEV)是引起母猪繁殖机能障碍的重要病原之一,其NS1蛋白参与病毒复制和组装、调节宿主免疫反应功能.提取猪日本乙型脑炎上海分离株的基因组RNA,反转录合成cDNA,扩增该病毒的NS1

  2. Difluoromethane preparation

    NARCIS (Netherlands)

    Wiersma, A.; Sandt, E.J.A.; Van Bekkum, H.; Makkee, M.; Moulijn, J.A.

    1996-01-01

    Abstract of NL 9401574 (A) The invention relates to a method for preparing difluoromethane, wherein dichlorodifluoromethane or monochlorodifluoromethane is brought into contact with hydrogen in the presence of palladium on activated carbon, wherein the loading of the palladium on the activated c

  3. Preparation of gluten free bread enriched with green mussel (Perna canaliculus) protein hydrolysates and characterization of peptides responsible for mussel flavour.

    Science.gov (United States)

    Vijaykrishnaraj, M; Roopa, B S; Prabhasankar, P

    2016-11-15

    Green mussel protein hydrolysates (GMPH) utilization for the enrichment of gluten-free bread followed by characterization of flavour peptides using chromatography and electronic nose techniques have been done. The degree of hydrolysis was carried out in each protease digest, and the higher degree of hydrolysis was observed in pepsin digestion. Gluten-free (GF) bread was formulated by using buckwheat flour (BWF), rice flour (RF) and chickpea flour (CPF) (70:20:10) and GMPH were added in the range of 0-20% in the GF bread for enrichment with GMPH. Radar plot of the electronic nose analysis showed that the sensors P30/2, T30/1 and T70/2 had a higher response to the GF bread and GMPH. Consequently, the peptide sequence was obtained manually by ESI-MS spectra of GMPH (KGYSSYICDK) and F-II (SSYCIVKICDK). Flavour quality was 97% discriminately comparable to the GMPH and F-II fractions. Mussel flavoured GF bread can be included in the celiac diet. PMID:27283688

  4. Purification and characterization of Moschatin, a novel type Ⅰ ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata),and preparation of its immunotoxin against human melanoma cells

    Institute of Scientific and Technical Information of China (English)

    CHAO TONG; HENG YU FAN; DA YUAN CHEN; XIANG FEN SONG; HEIDE SCHATTEN; QING YUAN SUN

    2003-01-01

    A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of~29 kD. It is a rRNA Nglycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

  5. Purification and characterization of Moschatin, a novel type I ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells.

    Science.gov (United States)

    Xia, Heng Chuan; Li, Feng; Li, Zhen; Zhang, Zu Chuan

    2003-10-01

    A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of approximately 29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

  6. A Study on Preparation and Identification of Mouse Monoclonal Antibody Against BMPR-ⅡProtein%小鼠BMPR-Ⅱ单克隆抗体的制备与鉴定的实验研究

    Institute of Scientific and Technical Information of China (English)

    周文婷; 杨岚兰; 黄欢; 陆勇; 张培湘; 王杨

    2012-01-01

      Objective To study the preparation and identification of mouse monoclonal antibody against BMPR-Ⅱ. Methods BalB/c mice were immunized with BMPR-Ⅱprotein. Hybridoma cell line that was capable of steadily se-creting monoclonal antibody against BMPR-Ⅱwas established through conventional hybridoma technique. After being cloned and cultured, the positive hybridoma cells were injected into the abdominal cavities of BalB/c mice to prepare the monoclonal antibody against BMPR-Ⅱ. Then they were indentified through indirect enzyme linked immunosorbent assay(ELISA), western blotting, immunohistochemistry and immunofluorescence technique. Results A hybridoma cell line that could steadily secrete BMPR-Ⅱmonoclonal antibody was obtained and named 3F6, with titer of ascites 105 and subtype being IgG1. The prepared antibodies were able to specifically bind to BMPR-II protein from various types of tissues and cells, confirmed by the detection of western blotting, immunohistochemistry and immunofluorescence tech-nique. Conclusion A hybridoma cell line that could steadily secret monoclonal antibody with biological activity against BMPR-Ⅱprotein was successfully prepared in this study and may be applied in various immunoassays.%  目的制备小鼠骨形成蛋白Ⅱ型受体(BMPR-Ⅱ)的单克隆抗体并鉴定其活性。方法以BMPR-Ⅱ为抗原免疫BalB/c小鼠,通过杂交瘤技术建立稳定分泌抗BMPR-Ⅱ单克隆抗体的杂交瘤细胞株,克隆化培养后接种于小鼠腹腔制备BMPR-Ⅱ的单克隆抗体,通过间接酶联免疫吸附试验、免疫印迹、免疫组织化学、免疫荧光技术对其进行鉴定。结果获得一株能稳定分泌抗BMPR-Ⅱ单克隆抗体的杂交瘤细胞,命名为3F6,其腹水效价为105,亚类为IgG1;通过免疫印迹、免疫组织化学、免疫荧光证实制备的BMPR-Ⅱ抗体可与组织和细胞中的BMPR-Ⅱ蛋白特异性结合。结论本实验成功制备了可稳定分泌具有生物活性

  7. 人乳头瘤病毒16型E2蛋白表达、纯化及抗血清制备%Expression and purification of HPV16 E2 protein and preparation of its antiserum

    Institute of Scientific and Technical Information of China (English)

    孙宇辉; 张沿君; 刘明明; 唐丽萍; 张光虹; 魏兰兰; 谷鸿喜; 商庆龙

    2013-01-01

    Objective To express and purity the human papillomavirus type 16 (HPV16) E2 protein in prokaryotic bacteria and prepare the antiserum of HPV16 E2.Methods After amplified by PCR,HPV16 E2 was inserted into pET21b vector.The recombinant pET21b-HPV16E2 vector was transfected into E.coli BL21 (DE3).Expression product was identified after induction.Through purification,denaturation and renaturation,soluble protein was obtained.With the HPV16 E2 protein,we immunized BALB/c mice and examined mouse IFN-γ,CD4+ T cells,CD8+ T cells,CD4/CD8 ratio and antiserum titer.Results Restriction digestion and DNA sequencing showed pET21b-HPV16E2 was constructed successfully.Relative molecular mass (Mr) of HPV16 E2 was 42 000 in SDS-PAGE and the specificity of the protein was confirmed with Western blotting.The antiserum could specifically bind with HPV16 E2 protein.In the immunized BALB/c mice,antiserum titre,CD4+ T cell count and CD4/CD8 ratio increased,while mouse IFN-γ did not change obviously.Conclusion Soluble HPV16 E2 protein was obtained successfully.The antiserum of high titer against HPV16 E2 was prepared in mice.%目的 表达人乳头瘤病毒16型(HPV16) E2蛋白,并制备小鼠抗HPV16 E2血清.方法 采用PCR技术扩增HPV16E2基因,构建入pET21b载体,重组表达载体pET21 b-HPV16E2经鉴定后转化大肠埃希菌BL21(DE3),诱导表达并鉴定表达产物.经纯化、变性和复性方法,制备可溶性HPV16 E2蛋白.免疫BALB/c小鼠制备抗血清,检测小鼠IFN-γ、CD4+T细胞、CD8+T细胞、CD4/CD8比值和抗血清滴度变化.结果 酶切和测序结果表明pET21b-HPV16 E2构建成功.表达蛋白相对分子质量(Mr)为42 000,Western blot法证明具有较高特异性.小鼠抗血清效价升高,CD4+T细胞数量和CD4/CD8比值升高,小鼠IFN-γ无升高.结论 成功制备可溶性HPV16 E2蛋白和小鼠抗HPV16 E2高效价的抗血清.

  8. 地木耳蛋白饮料的研制%Research on the Preparation of Nostoc commune-Protein Beverage

    Institute of Scientific and Technical Information of China (English)

    李贤煜; 李砧; 冯佳; 张猛; 谢树莲

    2011-01-01

    [ Objective ] The reference for the development and utilization of Nostoc commune resource was provided through the research on the prescription of Nostoc commune-protein beverage. [ Method ] The Nostoc commune being taken as main raw material and the peanut, walnut and others being taken as accessories, the best prescription of the beverage containing different materials was formed through reasonable adjustment of components based on the multi-factor orthogonal experiment combined with physical and chemical analysis, sensuous evaluation and microbiology test. [ Result ] The best prescription of the beverage containing the common Nostoc commune was that the dried algae was 20%;the peanut, 6%; the walnut, 6%; the sugar, 7% and the honey, 4% and the best prescription of the beverage containing the acid Nostoc commune was the dried algae, 20%; the peanut, 6%; the walnut, 6%; the sugar 6%; the honey, 5%; the acid pulp and juice, 3%. In the beverage the propylene glycol algiuate of 0.2% added as stabilizer was better than others. There was certain difference in the foaming and its stability among different species of Nostoc commune Vauch, but significant difference. [ Conclusion ] The beverage was pure natural functional protein one with special taste and nutrient value and suitable all of population with good market prospect.%[目的]研究地木耳蛋白饮料的配方,为地木耳资源的深层次开发利用提供参考.[方法]以地木耳为主要材料,并添加花生、核桃等辅料,通过合理调配.应用多因素正交试验,结合理化分析、感官评价、微生物检测等方法,得到该饮料成分的最佳配比.[结果]普通型地木耳蛋白饮料最佳配方:地木耳20%,花生6%,核桃6%,白砂糖7%,蜂蜜4%;酸性地木耳蛋白饮料最佳配方:地木耳20%,花生6%,核桃6%,白砂糖6%,蜂蜜5%,酸性果肉及果汁3%;选用0.2%藻酸丙二醇酯作为稳定剂,效果较好;不同品系地木耳蛋白饮料的起

  9. Expression, Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody Against NrfA%大肠杆菌NrfA蛋白表达、纯化及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    何婷婷; 龚钢明; 高然

    2012-01-01

    [ Objective] This study aimed to clone the E. Coli NrfA gene and construct the pET-28a( + )-NrfA prokaryotic expression vector for preparation of polyclonal antibody against E. Coli NrfA. [ Method] E. Coli NrfA gene was cloned from the E. Coli genome DNA by PCR and inserted into the vector pET-28a( + )to construct prokaryotic expression vector pET-28a( + )-NrfA. E. Coli NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by immunizing rabbit with routine method. The specificity and titer of polyclonal antibody were confirmed by ELISA and Western Blotting. [ Result] The constructed prokaryotic expression vector pET-28a( + )-NrfA was induced by IPTG, the recombinant NrfA protein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained after immunization and purification was about 1 =204 900. Western Blotting analysis indicated that the obtained polyclonal antibody against E. Coli NrfA protein had high titer and high specificity. [Conclusion] E. Coli NrfA gene was cloned and the prokaryotic expression vector pET-28a( + )-NrfA was constructed successfully, and the polyclonal antibody with high titer and high specificity was prepared, which had laid the foundation for the study of NrfA in different strains of bacteria.%[目的]克隆大肠杆菌NrfA基因,构建pET-28a(+ )-NrfA表达载体,制备相应的多克隆抗体并对其进行鉴定.[方法]以大肠杆菌基因组DNA为模板,PCR扩增得到NrfA基因编码区,构建pET-28a(+ )-NrfA表达载体;经IPTG诱导表达并纯化重组蛋白;再免疫新西兰雄兔,制备多克隆抗体;用ELISA方法检测抗体的效价,Western Blotting检测抗体的特异性.[结果]构建的表达载体pET-28a(+)-NrfA在大肠杆菌中诱导后可高效表达NrfA蛋白;免疫获得的多克隆抗体用ELISA检测,其效价为1∶204 900;经Western Blotting分析,抗体的特异性较好.[结论]成功克隆大肠杆菌的NrfA基

  10. 天津机场第二跑道工程道面混凝土抗冻性能研究%Research on Concrete Anti-freezing Performance of the Second Runway of Tianjin Airport

    Institute of Scientific and Technical Information of China (English)

    高志斌; 刘岩

    2011-01-01

    以天津机场第二跑道工程为背景,从配制参数、材料、试验条件等角度出发,开展机场道面混凝土抗冻性能影响因素的研究.基于此,运用混凝土损伤力学理论与材料疲劳理论,将损伤力学的损伤度概念应用于混凝土冻融疲劳破坏损伤中,结合天津机场道面混凝土多年的现场工程试验数据,建立了机场道面混凝土抗冻耐久性的数学预测模型,并将其应用到道面混凝土冬季施工配合比设计中,从而降低道面的耐久性劣化风险,提高机场道面结构安全性,保障跑道使用寿命和飞机飞行安全.%Based on construction of the second runway of Tianjin Airport, the research is carried out about the influence factors of the concrete anti-freezing performance of the airport pavement from the aspect of mix parameters, material and test condition. With the theory of damage mechanics and fatigue of materials, the damage degree of damage mechanics is applied to analyze fatigue damage of freezing-thawing of concrete. With several years of engineering test data of Tianjin Airport, a math-predicting model of the anti-freezing performance of the concrete of airport pavement is built and used in the design of concrete mix proportion of airport pavement for winter construction in order to reduce the durability degradation of concrete pavement, and improve the safety of the airport pavement structure and ensure the service life of the airport runway and the safety of aircraft flight.

  11. 准备配种期雌性水貂适宜日粮蛋白质水平的研究%Evaluation of Different Dietary Protein Levels on Preparative Mating Minks

    Institute of Scientific and Technical Information of China (English)

    蒋清奎; 张志强; 李光玉; 高秀华; 邢秀梅; 杨福合

    2012-01-01

    X The experiment was conducted to evaluate the regularity of digestibility and metabolism of diets with different levels in female minks on preparative mating period. 180 healthy female minks of one year and a half old were randomly assigned into six groups with 30 replicates and each replicate had 1 mink. The treatments were individually fed with 28. 59% (group I),32.31%(group Ⅱ),36. 21%(group Ⅲ),40. 35%(group Ⅳ) protein levels in fresh feed diets and with 32. 66% (group V ) ,40.47% (groupⅥ) protein levels in mixed feed diets. The period of trial lasted for 51 days,including 7 days preset period and 44 days test period. The results showed that the food intake of the fresh feed diets groups was higher than that of the mixed feed diets group,some had significantly difference(P<0. 05), On the item of the digestibility of dry mater, protein and fat,some had significantly difference (P<0. 05). The nitrogen intake, fecal nitrogen, urine nitrogen increased with the protein level in different groups. There was no significantly difference in nitrogen retention, the biology value of protein, net protein usage ratio. However, all of the three indexes tended to decrease when the dietary protein level reached 36. 21%. Of all the indexes, the mixed feed diets group were significantly or extremely lower than the fresh feed diets group(P<0.05 or P< 0.01). In conclusion, the minks had almost equally best nutrient digestibility and availability when the dietary protein level reached 32. 31% and 36. 21% , but in consideration of the feed expense and the particular characteristics of the preparative mating minks, 32. 31% protein level of diet would be the best choice in the preparative mating period, and the mixed feed diets were not recommended for this particular period.%本试验以准备配种期日粮蛋白质水平对水貂营养物质消化率及氮代谢的影响为研究目的.选择经产适龄母貂180只,随机分成6组,每组30个重复,每个重复1只水貂.6

  12. Preparation of Umami Peptides by Enzymatic Hydrolysis of Proteins from Aquatic Products%水产蛋白酶解制备鲜味肽

    Institute of Scientific and Technical Information of China (English)

    李莹; 黄开红; 周剑忠; 曾晓雄

    2012-01-01

    The aim of this study was to prepare umami peptides from the hydrolysis of a mixture of meat from 3 different species of aquatic products (silver carp, prawn and scallop) at a mass ratio of 1:1:1 with both flavourzyme and protamex at a mass ratio of 1:1. The hydrolysis process was optimized using response surface methodology based on a Box-Behnken experimental design. The effects of enzyme-to-substrate ([E]/[S]) ratio, hydrolysis temperature and hydrolysis time on degree of hydrolysis and sensory evaluations were explored. The optimal hydrolysis conditions were determined as follows: [E]/[S] ratio 7.5%0, substrate concentration 30 g/100 mL, natural pH, hydrolysis 56 ℃, and hydrolysis duration 5.9 h. Under these conditions, the degree of hydrolysis of aquatic products and the sensory evaluation score of the hydrolysate obtained were 56.32% and 6.8, respectively, close to the predicted values (55.17% and 6.9, respectively). The ultrafiltration of this hydrolysate resulted in 4 umami peptides. MaiUard reaction products of each umami peptide with molecular weight between 2.5 kD and 5 kD showed a strong umami and non-bitter taste and no fishy odor.%以3种不同类别的水产蛋A为原料,制备具有风味提升的短肽。通过比较酶解产物的感官特点,确定m鲢鱼:m对虾:m扇贝为1:1:1;采用风味蛋白酶与复合蛋白酶双酶水解,添加两酶质量比为1:1。采用Box—Behnken设计和响应面法(RSM)优化酶解水产蛋白的工艺,以水解度和感官评分为指标,探讨酶与底物比([E]/[S])、酶解温度和酶解时间对鲜味肽的感官影响。结果表明:水产蛋白制备鲜味肽的最佳工艺为【E】/[S]7.5‰、底物质量浓度30g/100mL、自然pH值、酶解温度56℃、酶解时间5.9h。验证实验表明,该条件下水产蛋白的水解度和鲜味肽的感官评分分别为56.32%和6.8,与模型的预测值(55.17%和6.9)基本

  13. 肿瘤抑制蛋白质P53单克隆抗体的制备及其免疫活性分析%Preparation and binding activity of monoclonal antibody against P53 protein

    Institute of Scientific and Technical Information of China (English)

    武军华; 魏文清; 贾培嫒; 赵宇; 王晨宇; 刘晶; 王玉霞

    2011-01-01

    OBJECTIVE To prepare monoclonal antibodies specially recognized P53 protein. METHODS Recombinant human wild-type P53 protein expressed in E. Coli. BL21 was used as antigen. Monoclonal antibodies were prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by affinity chromatography. Antibody titer was determined by enzyme linked immunosorbent assay ( ELJSA). The specific binding activity of antibody was detected by Westen blotting and immunohistochemistry. RESULTS Three antibodies, 1P15, 2P37 and 3P40, against P53 protein were prepared and purified by affinity chromatography. The purity of antibodies was higher than 90% shown by SDS-PAGE. The titers of antibodies were more than 1:6000 determined by ELJSA. All antibodies could recognize P53 protein by Western blotting, in which 3P40 had the sensitivity of 5 ng for P53. Two tumor cell lines with different p53 gene type (MDA-MB-231, p53 mutant;H1299, p53 null) were used to determine the binding activity of antibody 3P40 by Western blotting and immunohistochemistry. The result indicated that endogenous P53 protein in tumor cells could be identified by 3P40. CONCLUSION Three monoclonal antibodies against P53 are prepared. Antibody 3P40 is the most sensitive and specific one to P53.%目的 制备可特异性地识别肿瘤抑制蛋白质P53的单克隆抗体.方法 应用重组人野生型P53蛋白为免疫抗原,采用经典的细胞融合技术制备单克隆抗体.亲和层析纯化抗体蛋白;ELISA测定抗体滴度.纯化的抗体作用于含有不同p53基因型的MDA-MB-231[p53(mutant)]和[H1299,p53(null)]肿瘤细胞系,Western印迹、免疫组化和免疫荧光染色法检测抗体与内源性P53蛋白的结合特异性.结果 对筛选到的3株单克隆抗体1P15,2P37和3P40,进行了亲和层析技术纯化.SDS-PAGE结果显示,纯化后的3株抗体纯度均在90%以上;ELISA滴度测定结果显示,3株单抗的滴度均在1

  14. 番荔枝子去蛋白多糖制备及其体外降糖作用研究%Preparation of Protein-free Polysaccharide from Annonae Squamosae Semen and Its In Vitro Hypoglycemic Effect

    Institute of Scientific and Technical Information of China (English)

    邱海龙; 汤彬; 王玉; 李祥; 白刚刚; 陈建伟

    2013-01-01

    The study was aimed to research the influence of glucose consumption of HepG2 cell and insulin-resistance of HepG2 cell administrated with protein -free polysaccharide from A nnonae Squamosae Semen ( ASS ) . Crude polysaccharide from ASS was prepared by water extraction and alcohol precipitation method . Its protein was removed by sevag method . The content of its total sugar was measured by phenol-sulfuric acid method . Besides , the influences of glucose consumption of HepG2 cell and insulin-resistance of HepG2 cell administrated with different concentrations of protein-free polysaccharide were determined . The result showed that protein-free polysaccharide from ASS can slightly improve the glucose consumption of HepG2 cell , which was related to its concentration . The protein-free polysaccharide from ASS can obviously promote insulin-resis-tance of HepG2 cell . When the drug concentration was 0 . 08 mg•mL-1 , the effect is the best ( P < 0 . 01 ) . Be-sides , the protein-free polysaccharide from ASS had certain synergistic effect as physiological insulin . It was concluded that the protein-free polysaccharide from ASS had good in v itro hypoglycemic effect .%目的:研究番荔枝子去蛋白多糖对HepG2细胞糖消耗及胰岛素抵抗HepG2细胞糖消耗的影响。方法:水提醇沉法制备番荔枝子粗多糖,经过Sevag法除蛋白得番荔枝子去蛋白多糖,用苯酚-硫酸法检测其总糖含量。取对数生长期的HepG2细胞,分别给予不同浓度的番荔枝子去蛋白多糖,检测其对HepG2细胞葡萄糖消耗的影响;建立高胰岛素抵抗模型,同法测定其对细胞液中葡萄糖消耗的影响。结果:番荔枝子去蛋白多糖能轻度促进HepG2细胞的葡萄糖消耗,其作用与剂量呈正相关;番荔枝子去蛋白多糖能明显促进胰岛素抵抗HepG2细胞葡萄糖消耗作用,在浓度为0.08 mg·mL-1时,效果最佳(P <0.01)。同时,番荔枝子去蛋白多糖与生理胰

  15. Mix Study of C 50 Antifreeze Concrete of North Water Transfer Project%南水北调中线工程C50抗冻混凝土配合比试验研究

    Institute of Scientific and Technical Information of China (English)

    王利娟; 焦建国

    2011-01-01

    南水北调中线一期工程总干渠Ⅳ渠服役环境较为恶劣,混凝土冻融破坏问题突出.立足工程区域的地材特点,开展了原材料性能指标测试、混凝土试配以及抗冻性能检测等试验研究工作,配制出工作性能优异、耐久性能好的C 50F 200混凝土,并提出了相应的施工质量控制措施.%The service environment of main channel of midline first phase of North Water Transfer Project is serious, Freeze-thaw damage problem of concrete is prominent. Based on the material characters of local work site area, the performance index test of raw materials, concrete mix test and concrete antifreeze test are carried out. Through these test works, C 50 F 200 concrete with excellent work performance is obtained, and the corresponding measures of construction quality control are suggested.

  16. Preparation of antioxidant peptides of whey protein from the buffalo milk%水牛奶乳清蛋白制备抗氧化活性肽工艺的研究

    Institute of Scientific and Technical Information of China (English)

    闭秋华; 孙宁; 白文娟; 陈文硕; 王娇; 李全阳

    2012-01-01

    实验是以水牛奶为原料,分离纯化后得到乳清蛋白。利用碱性蛋白酶、中性蛋白酶、胰蛋白酶、木瓜蛋白酶和胃蛋白酶5种不同的蛋白酶对水牛奶乳清蛋白酶解以制备抗氧化活性多肽。酶筛选结果显示,中性蛋白酶是最适宜酶解水牛奶乳清蛋白制备抗氧化活性肽,其酶解液的还原能力和DPPH自由基清除率较其他4种酶高。探讨酶解反应时pH、温度、时间、酶浓度对酶解反应的水解度、酶解液的还原能力和DPPH自由基的清除率的影响,在单因素试验基础上,采用响应面法对酶解工艺进行优化。结果表明,中性蛋白酶酶解乳清蛋白的最佳工艺参数为:pH为7.4,温度为50.5℃,酶与底物浓度比为2.1%,酶解时间5.0h,此时2mg/mL酶解物的DPPH自由基清除率为32.58%。实测结果与预测值吻合效果良好。%The whey protein was separated from the buffalo milk. The whey protein of the buffalo milk was hydrolyzed with neutrase to prepare antioxidant peptide. Studying the effects of pH, temperature, time and enzyme-to-substrate ratio on degree of hydrolysis, DPPH radical-scavenging capacity and reducing power with neutrase. It was found that the hydrolysates with neutrase had the best effect of reducing ability and scavenging to DPPH free radical. Hydrolysis conditions for preparing protein hydrolysates from whey protein was used single factor experiments and response surface methodology (RSM) to optimize the enzymatic processes. An enzyme to substrate level of 2.1%, temperature of 50.5 %, a hydrolysis time of 5.0 h and the pH of 7.4 were found to be the optimum conditions to obtain a higher DPPH radical- scavenging capacity of hydrolysis, which the DPPH radical-scavenging capacity was up to 32.58%.Therefore, there was a good accordance between the predicted and observed values.

  17. 复合蛋白酶法制备玉米胚蛋白多肽及其抗氧化活性研究%Preparation of corn germ protein polypeptide by compound protease hydrolysis and its antioxidant activity

    Institute of Scientific and Technical Information of China (English)

    李艳娟; 李书国

    2015-01-01

    The corn germ protein polypeptides (CGPP)were prepared from protein of corn germ by com-pound enzyme (alkaline proteases:papain=1∶1 ).The reducibility of polypeptides was selected as a detec-ting index and the conditions of enzymatic hydrolysis were optimized by response-surface method designed by Box-Behnken on the basis of single factor experiment.The optimum enzymolysis conditions of the corn germ protein were determined:protease dosage 9 200 IU/g,substrate concentration 1 1% and hydrolysis time 188 min.The reducibility of polypeptides of corn germ protein was 0.229 under the optimal condi-tions.The polypeptide was added to the noodles to check its ability of oxidation resistance,anti-brown-ing,compared with the effect of ascorbic acid.The results showed that polypeptides of corn germ protein can effectively anti-browning in the storage of fresh noodles.When the additive amount was 0.9%,the effect of anti-browning was the same as ascorbic acid.TPA test results showed that adding 0.9% corn germ protein polypeptides into the noodles can improve its hardness and flexibility to a certain extent.%利用复合酶(碱性蛋白酶∶木瓜蛋白酶=1∶1)酶解玉米胚蛋白制备蛋白多肽,在单因素实验的基础上,采用Box-Behnken设计响应面法,以蛋白多肽的还原力为检测指标,优化酶解条件。确定玉米胚蛋白的最佳酶解条件为:加酶量9200 IU/g,底物浓度11%,酶解时间188 min,在此条件下测定的还原力为0.229。将玉米胚蛋白多肽添加到面条中,考察其在面条中的抗氧化防褐变能力,并与抗坏血酸的添加效果进行对比。结果表明,玉米胚蛋白多肽粉添加到面条中具有防褐变的作用,添加量为0.9%时对鲜面条褐变速率的抑制作用最强,与添加抗坏血酸的效果相当。此外质构仪分析(TPA)实验结果表明,在面条中添加0.9%的玉米胚蛋白多肽粉,对面条的硬度、弹性有一定程度的改善作用。

  18. Preparation of a poly(3'-azido-3'-deoxythymidine-co-propargyl methacrylate-co-pentaerythritol triacrylate) monolithic column by in situ polymerization and a click reaction for capillary liquid chromatography of small molecules and proteins.

    Science.gov (United States)

    Lin, Zian; Yu, Ruifang; Hu, Wenli; Zheng, Jiangnan; Tong, Ping; Zhao, Hongzhi; Cai, Zongwei

    2015-07-01

    Combining free radical polymerization with click chemistry via a copper-mediated azide/alkyne cycloaddition (CuAAC) reaction in a "one-pot" process, a facile approach was developed for the preparation of a poly(3'-azido-3'-deoxythymidine-co-propargyl methacrylate-co-pentaerythritol triacrylate) (AZT-co-PMA-co-PETA) monolithic column. The resulting poly(AZT-co-PMA-co-PETA) monolith showed a relatively homogeneous monolithic structure, good permeability and mechanical stability. Different ratios of monomers and porogens were used for optimizing the properties of a monolithic column. A series of alkylbenzenes, amides, anilines, and benzoic acids were used to evaluate the chromatographic properties of the polymer monolith in terms of hydrophobic, hydrophilic and cation-exchange interactions, and the results showed that the poly(AZT-co-PMA-co-PETA) monolith exhibited more flexible adjustment in chromatographic selectivity than that of the parent poly(PMA-co-PETA) and AZT-modified poly(PMA-co-PETA) monoliths. Column efficiencies for toluene, DMF, and formamide with 35,000-48,000 theoretical plates per m could be obtained at a linear velocity of 0.17 mm s(-1). The run-to-run, column-to-column, and batch-to-batch repeatabilities of the retention factors were less than 4.2%. In addition, the proposed monolith was also applied to efficient separation of sulfonamides, nucleobases and nucleosides, anesthetics and proteins for demonstrating its potential.

  19. Optimization of Preparation Process of Silver Carp Protein Hydrolysate by Double Enzyme Method%鲢鱼蛋白水解物的双酶法制备工艺优化

    Institute of Scientific and Technical Information of China (English)

    王岩

    2014-01-01

    Flavourzyme and alcalase are used to prepare silver carp protein hydrolysate,and response surface methodology is adopted to optimize the enzymatic hydrolysis process.The results show that the optimum hydrolysis conditions are:substrate concentration is 1∶2 (meat ∶water),the dosage of flavourzyme is 0.8%,the dosage of alcalase is 0.4%,the initial pH is 6.5,hydrolyzed for 4 h at 60 ℃,the hydrolysis degree is 65.2% under such conditions.%利用双酶法(Alcalase 碱性蛋白酶与Flavourzyme 复合风味蛋白酶)制备鲢鱼蛋白水解物,应用响应面分析法对酶水解工艺优化,最佳酶解条件为底物浓度为料水比1∶2,Alcalase 碱性蛋白酶用量为0.4%,Flavourzyme 复合风味蛋白酶用量为0.8%,水解温度60℃,水解pH 值6.5,水解时间4 h,此条件下水解度为65.2%。

  20. Preparation and scaling up of a low phenylalanine enzymatic hydrolysate of bovine whey proteins Preparação e escalonamento de um hidrolisado enzimático de proteínas do soro de leite bovino

    Directory of Open Access Journals (Sweden)

    Marilisa Guimarães Lara

    2005-12-01

    Full Text Available We describe the preparation of pancreatic enzymes hydrolysate of milk whey proteins containing low levels of aromatic amino acids. Pancreatin and trypsin/chymotrypsin (6.3% w/w protein when used to hydrolyze whey proteins for 27 h at 37±2 ºC, released 74% of the Phe, 100% of the Tyr and 100% of the Trp as free amino acids. Most of the free aromatic amino acids present in 2 kg hydrolysate were separated from the remaining peptides and other amino acids by gel filtration on a 15 liter Sephadex G-25 column eluted with 5% acetic acid at 60 liters h-1 at 25ºC. The product, recovered in 37% yield, contained 0.70 mmol Phe, 0.41 mmol Tyr, and Foi descrita a preparação de um hidrolisado de proteínas do soro de leite bovino com enzimas pancreáticas, contendo baixos níveis de aminoácidos aromáticos. Quando utilizadas pancreatina e tripsina/quimotripsina, por 27h a 37±2ºC, foram liberados 74% de Phe, 100% de Tyr e 100% de Trp como aminoácidos livres. A maioria dos aminoácidos aromáticos livres, presentes em dois quilos de hidrolisado (15 litros, foi separada dos peptídeos e outros aminoácidos remanescentes por filtração em coluna de gel de Sephadex G-25C eluída com ácido acético 5%, fluxo de 60 litros por hora a 25ºC. O produto, recuperado com 37% de rendimento, continha 0,70 mmol de Phe, 0,41 mmol de Tyr e <0,01 mmol de Trp/100 mmol de aminoácidos recuperados. A composição em aminoácidos do hidrolisado foi similar às proteínas do soro com as quais foi preparado. Após adição de aminoácidos aromáticos apropriados, ele pode ser usado como fonte de nitrogênio para pacientes com fenilcetonuria ou tirosinemia.

  1. Comparison of protein sample preparation methods of two-dimensional electrophoresis for Skeletonema costatum%中肋骨条藻蛋白质双向电泳样品制备方法的比较

    Institute of Scientific and Technical Information of China (English)

    王秀秀; 陈纪新; 黄邦钦

    2012-01-01

    Lysis buffer method and trichloroacetic acid (TCA)-acetone precipitation method both are the common methods in algal protein abstraction. We compared the 2-DE maps of these two protein extraction methods to find out the best protocol for Skeletonema costatum. As a result, the Lysis buffer-TCA-acetone method had better effect than the other two lysis buffer methods (lysis buffer-microcon &- lysis buffer-2 D clean-up kit) in removing the intracellular interferential factors, such as salt, nucleic acid, polyphenol and polysaccharide. The lysis buffer-TCA-acetone method and straight TCA-acetone precipitation method both represented clean background and clear protein dots in 2-DE maps while the later method showed better isoelectrofocusing results. We optimized the straight TCA-acetone precipitation methods. The TCA-acetone precipitation with 12 hours precipitation and a following ultrasonic cleaning process method was confirmed to be the appropiate sample preparation method for S. costatum for two-dimensional electrophoresis.%比较了裂解液法和直接三氯乙酸(TCA)丙酮沉淀法对中肋骨条藻Skeletonema costatum蛋白双向电泳的提取效果并优化了提取条件,结果表明:裂解液-TCA丙酮沉淀法在去除胞内干扰物质方面,较裂解液-超滤管法和裂解液试剂盒法的效果都好.裂解液TCA-丙酮沉淀法和直接TCA-丙酮沉淀法都能取得背景干净、蛋白点清晰的双向电泳图谱,但后者的双向电泳图谱聚焦更完全,在进一步优化条件后(即蛋白沉淀12h并增加超声波洗涤过程),可作为提取中肋骨条藻蛋白的最适方法.

  2. Prokaryotic Expression and Antiserum Preparation of the Coat Protein of Cymbidium Mosaic Virus%建兰花叶病毒CP基因的原核表达及抗血清制备

    Institute of Scientific and Technical Information of China (English)

    罗金水

    2009-01-01

    通过间接酶联免疫检测和电镜观察对从福建省漳州市采集的卡特兰病样进行检测,证明样品感染了建兰花叶病毒.设计一对特异性引物,扩增并克隆病毒分离物的外壳蛋白基因,随后将目的基因插入pET-29a(+)中构建相应的原核表达载体.目的蛋白经诱导表达及纯化后免疫家兔并获得了特异性抗血清.Westem blot检测结果表明.抗血清与诱导表达的CyMV外壳蛋白发生特异性反应.间接酶联免疫法检测结果表明,抗血清可检测病汁液的最低稀释度达1:51 200,最佳工作浓度为1:1000,病汁液灵敏度为0.39 mg/mL,而与TMV等11种同源或异源病毒均无明显的血清学交叉反应.%Cymbidium mosaic virus (CyMV) is one of the most important and worldwide viruses attacking orchids. This virus causes the symptoms of mosaic,chlorosis,necrosis and malformation in the orchids,and has a high economic impact to the orchid industry. Cattleya plants contracted with a disease were collected as samples from Zhangzhou,Fujian,and were identified to be infected with Cymbidium mosaic virus by using ID -ELISA and electronic microscopy assay. One pair of specific primers was designed for amplification of the coat protein(CP) gene from the samples infected with CyMV. The open reading frame encoding CP of CyMV isolate obtained from Zhangzhou,Fujian is 672 bp,encoding a 23.6 ku protein with 223 aa. The expected CP gene was then inserted into the pET-29a(+)vector for prokaryotic expression. And the aimed protein was purified and used to immune the rabbit for antiserum preparation. According the result of ID-ELISA analysis,specific rabbit anti-CyMV serum was prepared with a high titre of 1:51 200,a working concentration of 1:1 000 and sap sensitivity of 0.39 mg/mL. Western blot analysis confirmed that the antiserum reacted strongly and specifically to the CP of CyMV. There were no cross reactions between the antiserums and 11 species of homologous or heterologous

  3. Comparison of Functional Properties of Soybean Protein Isolates Prepared by Different Methods%不同方法制备的大豆分离蛋白功能性质的比较研究

    Institute of Scientific and Technical Information of China (English)

    郑二丽; 杨晓泉; 吴娜娜

    2012-01-01

    采用普通的碱溶酸沉方法、Samoto法、钙离子沉淀法制备出几种大豆分离蛋白,并对其产率、总脂含量及溶解性、乳化性等物化性质和功能性质进行系统比较。结果表明:钙离子沉淀法制备的大豆分离蛋白(Less-LPSPI)的产率是碱溶酸沉大豆分离蛋白(APP)产率的65%,但高于由Samoto法提取的7S与11S两种大豆分离蛋白产率之和;Less-LP SPI的总脂含量比APP降低了45%,由Samoto法提取的亲脂性蛋白(LP)中总脂含量达到7.48%,而7S与11S蛋白中脂含量分别为1.45%、2.36%。在功能性质上,LP的溶解性、乳化活性均比较差,7S、11S质量比1:2混合蛋白的溶解性最好,而Less-LP SPI在pH≤10时溶解性低于APP;乳化性方面,Less-LP SPI的乳化活性稍低于7S、11S质量比1:2的混合蛋白及APP,但乳化稳定性远高于后者。总体上,Less-LP SPI脂含量少、乳化稳定性好,方法简单,且可提高其货架期。%Several soybean protein isolates(SPI) were prepared by acid precipitation,Samoto method and calcium-precipitation method,respectively.The characteristics including protein yield,total lipid content,solubility and emulsification of these SPIs were investigated.Our results showed that the yield of calcium-precipitation protein(Less-LP SPI) was lower than that of acid precipitation protein(APP),but higher than 7S,11S extracted by Samoto fractionation procedure.The lipid content of Less-LP SPI was decreased by 45% when compared with that of APP.The lipid content of lipophilic protein(LP) obtained from Samoto fractionation was 7.48%,but the lipid contents in 7S and 11S were 1.45% and 2.36%,respectively.The Samoto LP-rich fraction had inferior solubility and emulsifying properties.Less-LP SPI had lower solubility(pH ≤ 10) and emulsifying activity than APP,while Less-LP SPI had superior emulsion stability.In summary,Less-LP SPI has lower lipid and better emulsion stability and therefore can improve the shelf

  4. Experimental Analysis on Mix Proportion Design and Mechanical Properties of Anti-freezing and Anti-permeability Concrete with Low Strength%低强度抗冻抗渗混凝土配合比设计及力学特性试验分析

    Institute of Scientific and Technical Information of China (English)

    黄丽彬

    2015-01-01

    In this paper, the mix proportion of low strength concrete was designed by the scien-tific method, and its performance of anti-freezing and anti-permeability were studied, and the effect of the air entrainment agent dosage and the optimal water cement ratio on the anti-freezing performance, the anti-permeability performance and the strength of concrete were synchronously analyzed. Through deep research we can find out that in cold environment, the requirements of the anti-permeability performance and the strength of concrete should be meet in the construc-tion project, by using of low strength concrete, as long as adding moderate amount of air en-trained agent and maintaining appropriate water cement ratio, the persistent anti-freezing per-formance of the low strength concrete could be enhanced.%通过科学设计低强度混凝土的配合比,研究分析其抗冻、抗渗功能,并同步分析引气剂掺量、最优水灰比例对混凝土抗冻性、抗渗性及强度的作用。通过深入研究发现,严寒环境下符合建筑项目混凝土抗渗、强度的要求,采用低强度混凝土,只要掺入适量的引气剂、保持合适的水灰比例,就能增强低强度混凝土的持久的抗冻性能。

  5. Racemic protein crystallography.

    Science.gov (United States)

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  6. Preparation of Garlic Oil Microcapsules by Soybean Protein Isolate-Chitosan Coacervation%大豆分离蛋白-壳聚糖复凝聚法制备大蒜油微胶囊的工艺研究

    Institute of Scientific and Technical Information of China (English)

    黄国清; 肖军霞; 仇宏伟

    2014-01-01

    研究以大豆分离蛋白和壳聚糖为复合壁材,以大蒜油为芯材,探讨了采用复凝聚法制备大蒜油微胶囊的最佳工艺。结果表明:大蒜油与复合壁材按质量比1∶2混合,在45℃,6000 r/min下乳化5 min后,调节乳状液的pH 值至6.5,于100 r/min搅拌反应10 min,再经18.75 U/g SPI的谷氨酰胺转氨酶固化1 h,大蒜油微胶囊的包埋效率和产率最高分别达到了69.20%和64.77%。在此条件下制备的大蒜油微胶囊具有典型的蒜香味,且大蒜油的刺激性气味有所降低。%The preparation of garlic oil microcapsules through complex coacervation by using soybean protein and chitosan as the complex wall material and garlic oil as the core material is studied.After the garlic oil is mixed with the complex wall material in the mass ratio of 1∶2 and emulsified at 6000 r/min and 45 ℃ for 5 min,the emulsion pH is adjusted to 6.5 and stirred at 100 r/min for 10 min.Then transglutaminase is added in the dosage of 18.75 U/g SPI to cross-link the microcap-sules for 1 h.Under such conditions,the microencapsulation efficiency and yield reach 69.20% and 64.77% respectively.The garlic oil prepared under the optimized conditions possesses the typical fla-vour of garlic oil with reduced pungent odor.

  7. Preparation of bovine whey protein antioxidant peptides using combined high pressure treatment and Alcalase digestion%超高压与Alcalase协同作用制备牛乳清蛋白抗氧化肽

    Institute of Scientific and Technical Information of China (English)

    盛小波; 木泰华

    2012-01-01

    为探讨超高压与碱性蛋白酶Alcalase协同作用下乳清蛋白抗氧化肽的制备,以牛乳清分离蛋白(WPI)为原料,采用Alcalase分别对100~600MPa的超高压处理中和超高压处理后的WPI进行水解,并采用邻苯三酚自氧化法对其水解产物的超氧阴离子自由基清除能力进行测定。结果表明,超高压与Alcalase协同作用显著地促进了WPI的水解,其水解产物的抗氧化活性也显著提高;分子量小于3ku的组分具有最强的超氧阴离子自由基清除能力,其半抑制浓度IC50值最小,为411.62μg/mL。因此,超高压与Alcalase协同作用于乳清蛋白可用于开发新型天然抗氧化剂。%To study the preparation of whey protein antioxidant peptides from whey protein isolate by the combination of high pressure treatment and Alcalase digestion,the proteolysis of WPI was conducted with Alcalase during or after the treatment at 100~600MPa.Pyrogallol autoxidation method was used to assess the superoxide anion radical scavenging ability.The results showed that the degree of hydrolysis for WPI was significantly enhanced by high pressure treatment,as well as the antioxidant activity of the hydrolysates.In addition,the fraction of molecular weight below 3ku had a maximum superoxide anion radical scavenging ability,with the half maximal inhibitory concentration(IC50) of 411.62μg/mL.Therefore,the combination of high pressure treatment and Alcalase digestion could be used to develop new natural antioxidants.

  8. Preparation and biological characteristics of the influenza virus matrix protein M1 monoclonal antibody%流感病毒M1单克隆抗体的制备及其生物学特征

    Institute of Scientific and Technical Information of China (English)

    陈帅帅; 丁建祖; 尤金彪; 沃恩康; 王怡婷; 曹毅; 郭霞; 杨新燕; 张儒轩

    2015-01-01

    mAb and HRP-M1 mAb could react with M1 protein and H1N1,H3N2 subtypes of influenza virus,indicating that they were mAb antibodies against the M1 protein.By using ELISA,the sensitivity of M1 mAb and HRP-M1 mAb detecting M1 antigen and influenza viruses were studied,and the results demonstrated that both of them could detect H1N1 and H3N2 subtypes of influenza A virus.In addition,the ability of HRP-M1 mAb to detect virus was higher than M1 mAb.The cell injected immune result showed that the M1 mAb could combine with the cells infected with H1N1 and H3N2 influenza virus.Conclusions The M1 mAb and HRP-M1 mAb that against M1 protein can be prepared.The antibodies can detect H1N1 and H3N2 subtypes of influenza A virus,while M1 protein and antibody can be applied to the research and development of the diagnosis and detect kit for influenza A virus.

  9. Characterization of a family of ice-active proteins from the Ryegrass, Lolium perenne.

    Science.gov (United States)

    Kumble, Krishnanand D; Demmer, Jerome; Fish, Steven; Hall, Claire; Corrales, Sofia; DeAth, Angela; Elton, Clare; Prestidge, Ross; Luxmanan, Selvanesan; Marshall, Craig J; Wharton, David A

    2008-12-01

    Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3. PMID:18835384

  10. Novel dimeric β-helical model of an ice nucleation protein with bridged active sites

    Directory of Open Access Journals (Sweden)

    Walker Virginia K

    2011-09-01

    Full Text Available Abstract Background Ice nucleation proteins (INPs allow water to freeze at high subzero temperatures. Due to their large size (>120 kDa, membrane association, and tendency to aggregate, an experimentally-determined tertiary structure of an INP has yet to be reported. How they function at the molecular level therefore remains unknown. Results Here we have predicted a novel β-helical fold for the INP produced by the bacterium Pseudomonas borealis. The protein uses internal serine and glutamine ladders for stabilization and is predicted to dimerize via the burying of a solvent-exposed tyrosine ladder to make an intimate hydrophobic contact along the dimerization interface. The manner in which PbINP dimerizes also allows for its multimerization, which could explain the aggregation-dependence of INP activity. Both sides of the PbINP structure have tandem arrays of amino acids that can organize waters into the ice-like clathrate structures seen on antifreeze proteins. Conclusions Dimerization dramatically increases the 'ice-active' surface area of the protein by doubling its width, increasing its length, and presenting identical ice-forming surfaces on both sides of the protein. We suggest that this allows sufficient anchored clathrate waters to align on the INP surface to nucleate freezing. As PbINP is highly similar to all known bacterial INPs, we predict its fold and mechanism of action will apply to these other INPs.

  11. Recombinant human brain myelin basic protein and its antibody preparation%重组人脑髓鞘碱性蛋白及其抗体的制备与研究

    Institute of Scientific and Technical Information of China (English)

    刘戟; 王若菡; 刘鱼; 陈俊杰

    2006-01-01

    BACKGROUND: Central nervous system (CNS) myelin is made up of 70% lipids and 30% proteins with human brain myelin basic protein (hMBP) constituting 1/3 of the proteins. MBP is a family of proteinswith four isoforms only in human brain. OBJECTIVE: To investigate the expression of MBP and its immunological function. DESIGN: Single sample study. SETTING: Department of Biochemistry and Molecule Biology, Huaxi College of Priclinical Medicine and Forensic Medicine, Sichuan University. MATERIALS: This experiment was conducted at Department of Bio chemistry and Molecule Biology, Huaxi College of Priclinical Medicine and Forensic Medicine, Sichuan University from August 2003 to March 2004. Relative molecular weight was 215000 hMBP cDNA clone pGEMP,prokaryotic expression recombinant vector pGEX-5T, T4 DNA Ligase,calf intestine alkaline phosphates, X-gal, IPTG, 123 Ladder (BRL), nitrocellu lose ,4-chloro-1-Naphthol;MBP and Anti- MBP ELISA kit. INTERVENTIONS: ①hBMP gene cDNA clone segment was digested with EcoR1 and BamH1; ②Construction and transformation of the recombinant expression vector p5TMP; ③ The growth and induced expression of the recombinant expression vector transformed bacteria; ④ Preparation of the antibody of recombinant hMBP. MAIN OUTCOME MEASURES: ① Detection and identification of the expressed protein product; ② Detection and identification of hMBP anti body. RESULTS: ① Screening and identification of recombinant MBP: The 4 999 bp pGEX-5T DNA and 557 bp MBP inserted fragment were obtained, indicating that the white colonies contained recombinant expression plasmid of exogenous DNA; ② A dense band disappeared from the control plasmid while a new special polypeptide band with apparent molecular weight 42 000 was detected in recombinant cell lysate; ③After 5 subcutaneous injections, antibody was obtained at 1:16 titer. The specificity of the antibody to MBP was confirmed.CONCLUSION: Compared with the original expression vector, the new

  12. Optimizing expression and antibody preparation of recombinant Streptococcus mutans surface protein%重组变异链球菌表面蛋白的可溶性表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    金洁; 樊明文; 李宇红

    2012-01-01

    Objective The soluble protein lecombinant Streptococcus mutans surface protein (rPAc) was expressed in Escherichia coli(E.coli) after the optimization of inducing conditions. The antisenim against rPAc was obtained by immunizing mice with the purified rPAc. Methods The soluble expression of rPAc in Exoli was further optimized by means of different culture conditions. Polyclonal antibody was made by immunizing mice with purified rPAc. Western blot and enzyme linked immunosorbent assay (ELISA) were carried out to identify the immunocompetence of the antibody. Results The highest soluble expression level of rPAc was obtained at Luria-Bertani (LB) medium (pH=7.2) when optical density (OD600cm) was 0.6 after being induced at 30℃ for 4 h and the concentration of isopropyl (S-D-l-thigalactopyranosideCIPTG) was l.0mmol'L-1. The titer of the mice antisenim against rPAc was about 1:6000 by ELISA analysis, and rPAc could be specifically recognized by Western blot analysis. Conclusion This study proved that rPAc can be effectively expressed as a soluble form in Exoli, and the high specific polyclonal antibody of rPAc was proved to be prepared, which shed light on further research of DNA prime-protein boost inoculation.%目的 探讨重组变异链球菌表面蛋白(rPAc)在大肠杆菌中可溶性表达的最佳诱导条件并制备其多克隆抗体.方法 通过改变pET20b (+)-AP/BL21 (DE3) plysS工程菌的培养条件,提高rPAc可溶性表达水平.用纯化的rPAc免疫BALB/c小鼠制备多克隆抗体,通过酶联免疫吸附测定(ELISA)和Western blot法鉴定抗体的免疫学活性.结果 pET20b(+)-AP/BL21 (DE3) plysS工程菌在Luria-Bertani (LB)培养基(pH值为7.2)上培养,至表示菌体密度的光密度值OD600nm=0.6时加入1.0 mmol·L-1异丙基-β-D-硫代吡喃半乳糖苷(IPTG),30℃诱导培养4h,rPAc的可溶性表达量最高.以纯化的rPAc免疫BALB/c小鼠,制备抗rPAc多克隆抗体,ELISA法测定抗体效价为1:6000,Western blot法鉴定

  13. Batter and method for preparing a pasta

    NARCIS (Netherlands)

    Wind, P.; Linden, van der E.

    2011-01-01

    This invention describes a batter that is suitable for preparing a pasta. The batter comprises water, a starch and a protein, whereby the weight ratio between the protein and the total amount of starch in the batter is represented by the symbol y and whereby the weight percentage of the total amount

  14. Pressure cryocooling protein crystals

    Science.gov (United States)

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  15. Preparation and Characterization of Monclonal Antibody against Protein Tyrosine Phosphatase PRL-3%酪氨酸磷酸酶PRL-3单抗的制备及特性鉴定

    Institute of Scientific and Technical Information of China (English)

    王莉; 杨艳丽; 宋子博; 廖世秀; 黄飞飞

    2009-01-01

    目的 制备并鉴定抗肝再生磷酸酶-3(Phosphatase of Regenerating Liver-3,PRL-3)单克隆抗体,为临床检测和以PRL-3为靶点的肿瘤治疗提供可能.方法 运用杂交瘤融合技术制备PRL-3单克隆抗体,通过Western blot检测和免疫沉淀鉴定其与原核及真核PRL-3蛋白的反应性;重组并诱导表达PRL-3的6个截短体,Western blot分析单抗结合的大致抗原表位.结果 共得到9株单抗,3株与PRL-1、PRL-2和PRL-3均反应,6株(9D8、9E2、9F4、11B2、4D3和4D10)只与PRL-3反应,其中4株特异性单抗可以与哺乳动物细胞表达的PRL-3反应;单抗9D8、9E2、9F4和11B2可以和PRL-3羧基末端(162-173位氨基酸)的短肽结合;4D3和4D10与中间部分(69~95位氨基酸)的短肽结合.结论 抗体9D8、9E2、9F4、11B2、4D3和4D10特异性强、亲和力高,为临床检测提供了可靠工具,并为进一步的功能研究奠定了基础.%Objective To prepare and identify specific Phosphatase of Regenerating Liver-3 (PRL-3) mon-oclonal antibodies for clinical detection and for further therapeutic intervention. Methods Hybridoma technology was used to prepare PRL-3 monocloual antibodies (MAbs), and the specificities of MAbs a-gainst PRL-3 were evaluated by Western blot and immunoprecipation. Six truncations of PRL-3 were cloned and expressed in prokaryotic cell for identifying the approximate epitope. The binding abilities of MAbs were analyzed by Western blot. Results Among 9 hybridoma clones obtained, 6 (9D8, 9E2,9F4, 11B2, 4D3 and 4D10)could specifically bind to PRL-3, and 4 could react to PRL-3 protein in eu-karyotic cells. Clone 9138,9E2,9F4 and 11B2. cloud bind to the COOH-terrninal of PRL-3, and clone 4D3 and 4D10 cloud bind to 69~95 amino acids. Conclusion MAbs 9D8, 9E2, 9F4, 11B2, 4D3 and 4D10 can react only to PRL-3, which provides potential applications in clinical diagnosis and in future study.

  16. Annealing condition influences thermal hysteresis of fungal type ice-binding proteins.

    Science.gov (United States)

    Xiao, Nan; Hanada, Yuichi; Seki, Haruhiko; Kondo, Hidemasa; Tsuda, Sakae; Hoshino, Tamotsu

    2014-02-01

    The Antarctic sea ice diatom Navicular glaciei produced ice-binding protein (NagIBP) that is similar to the antifreeze protein (TisAFP) from snow mold Typhula ishikariensis. In the thermal hysteresis range of NagIBP, ice growth was completely inhibited. At the freezing point, the ice grew in a burst to 6 direction perdicular to the c-axis of ice crystal. This burst pattern is similar to TisAFP and other hyperactive AFPs. The thermal hysteresis of NagIBP and TisAFP could be increased by decreasing a cooling rate to allow more time for the proteins to bind ice. This suggests the possible second binding of proteins occurs on the ice surface, which might increase the hysteresises to a sufficient level to prevent freezing of the brine pockets which habitat of N. glaciei. The secondary ice binding was described as that after AFP molecules bind onto the flat ice plane irreversibly, which was based on adsorption-inhibition mechanism model at the ice-water interface, convex ice front was formed and overgrew during normal TH measurement (no annealing) until uncontrolled growth at the nonequilibrium freezing point. The results suggested that NagIBP is a hyperactive AFP that is expressed for freezing avoidance.

  17. Optimizing Human Bile Preparation for Two-Dimensional Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hao-Tsai Cheng

    2016-01-01

    Full Text Available Aims. Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established. Methods and Material. The method involved acetone precipitation followed by protein extraction using commercially available 2D Clean-Up kit. The effectiveness was evaluated by 2-dimensional electrophoresis (2DE profiling quality, including number of protein spots and spot distribution. Results. The total protein of bile fluid in benign biliary disorders was 0.797 ± 0.465 μg/μL. The sample preparation method using acetone precipitation first followed by 2D Clean-Up kit protein extraction resulted in better quality of 2DE gel images in terms of resolution as compared with other sample preparation methods. Using this protocol, we obtained approximately 558 protein spots on the gel images and with better protein spots presentation of haptoglobin, serum albumin, serotransferrin, and transthyretin. Conclusions. Protein samples of bile prepared using acetone precipitation followed by 2D Clean-Up kit exhibited high protein resolution and significant protein profile. This optimized protein preparation protocol can effectively concentrate bile proteins, remove abundant proteins and debris, and yield clear presentation of nonabundant proteins and its isoforms on 2-dimensional electrophoresis gel images.

  18. Preparation of Sweet Orange Oil Microcapsule by Soybean Protein Isolate-Gum Arabic Complex Coacervation and its Characterization%SPI-GA复凝聚法制备甜橙油微胶囊及表征

    Institute of Scientific and Technical Information of China (English)

    肖军霞; 杨剑; 黄国清; 沈丽; 于海燕

    2012-01-01

    采用大豆分离蛋白(SPI)和阿拉伯胶(GA)为壁材,以甜橙油为模式芯材,通过复凝聚法结合真空冷冻干燥技术制备甜橙油微胶囊.考察了戊二醛和葡萄糖对甜橙油微胶囊包埋产率和包埋效率的影响.采用光学显微 镜和扫描电子显微镜对微胶囊粒径、微观结构进行表征.研究结果表明在固形物质量分数5%,芯材载量20%,SPI:GA=1时调节pH到4,室温下850 r/min搅拌30 min,加入1%的葡萄糖反应1h,真空冷冻干燥得到微胶囊.此时包埋产率和效率分别达到75.06%和93.5%.该方法制备的甜橙油微胶囊平均粒径是11.226 μm,胶囊表面光滑,颗粒均匀,色泽淡黄,香味愉悦.本研究结果说明SPI和GA具有良好的相容性,葡萄糖作为一种改良剂可显著改善微胶囊的性能.%The effect of glutaraldehyde and glucose on preparation of sweet orange oil microcapsule by the complex coacervation between soybean protein isolate (SPI) and gum Arabic (GA) was investigated and the microeapsules produced under optimized conditions were characterized by SEM in this paper. The results showed that the optimum glucose dosage and incubation time were 1% and 1 h respectively under the total solid concentration 5%, core load 20%, SPI/GA ratio 1:1, pH4.0, and stirring at 850 r/min for 30 min. Under these conditions, the microencapsulation yield and microencap-sulation efficiency reached 75.06% and 93.5% respectively. The microeapsules have a smooth outer surface and uniform size distribution of 11.226 μm in diameter. It is indicated that SPI is quite compatible with GA for flavor microencapsulation and glucose as a modifier can significantly improve the microencapsulation performance of the GA/SPI system.

  19. 双酶法制备葵花籽肽的工艺研究%Two-step Enzymatic Hydrolysis for Preparation of Peptides from Sunflower (Helianthus annuus L.) Protein Isolates

    Institute of Scientific and Technical Information of China (English)

    刘平伟; 刘会平; 陈苓

    2013-01-01

    利用双酶法水解葵花籽分离蛋白(SPI)制备葵花蛋白肽,探讨各因素对水解度(Dcgrcc of hydrolysis,DH)和肽含量的影响,目的在于确定葵花蛋白肽制备的最佳酶解条件.结果表明:最佳酶解条件为底物浓度5.0× 10-2 g/mL,温度为53.5℃,pH 8.5,按照[E/S]为1.0x 10-2 g/g加Alcalase 2.4 L,酶解104 min,调整pH为7.5,按照[E/S]为0.44×10-2 g/g添加Flavourzyme酶解120 min.此工艺得到水解液中肽含量为4.25 mg/mL,DH为30.97%,与单酶法相比,肽含量显著提高.%Two-step enzymatic hydrolysis reactions were investigated to prepare peptides from sunflower protein isolates. In order to maximize content of peptides and optimal degree of hydrolysis (DH), crucial hydrolysis parameters were optimized. Results showed that the maximum content of peptides of 4.249 mg/mL was obtained under the following conditions in the first enzymatic step: Alcalase 2.4 Ldosage 1.0 ×10-2g/g . hydrolysis time 104 min, temperaure 55 ℃, pH 8.5, and substrate concentration 5×10-2 g/mL. In the second enzymatic step, the optimum conditions were,: Flavourzyme dosage 0.44xl0"2 g/g, reaction time 120 min and pH 7.5. Unde the optimum condition, the DH was 30.97%. In addition, much higher peptide contents than using single enzyme method was found when using this two-step enzymatic method.

  20. Preparation of fish sauce with the hydrolysate from Tilapia protein and its antioxidative activity%罗非鱼蛋白酶解制取鱼露及其抗氧化研究

    Institute of Scientific and Technical Information of China (English)

    熊俊娟; 丁利君; 叶少芳

    2011-01-01

    目的:以罗非鱼为原料,采用生物酶解技术加工传统产品鱼露,提高产品质量,缩短加工时间。方法:采用枯草杆菌碱性蛋白酶(E1)、风味酶(E2)酶解罗非鱼蛋白,制取富含多肽的酶解物;并以酶解物为基本原料制备调味品鱼露。通过对自由基的清除作用,研究其抗氧化作用。结果:采用生物酶解技术生产罗非鱼鱼露,产品色泽好,风味浓,营养高,有良好的抗氧化性。结论:与传统技术生产鱼露比较,生产时间短,工艺条件容易控制,产品质量稳定,该研究为罗非鱼到深加工提供了理论基础。%Objective:To improve product quality,reduce processing time,the traditional fish sauce of tilapia was produced with biological enzyme technology.Methods:Tilapia protein was hydrolyzed with alkaline protease(E1) and flavor enzyme(E2) of Bacillus subtilis,and rich peptide hydrolysates was prepared;and fish sauce with the hydrolysates was processed.Its antioxidant effects were studied with scavenging rate to radicals.Results:The tilapia fish sauce with biological enzyme technology had good color,strong flavor,high nutrition,and good antioxidative activity.Conclusion:Comparing with the traditional fish sauce,the self-made fish sauce had short production time,and easy to control process conditions.The studies provide a theoretical foundation for deep processing of Tilapia.

  1. Comparison of Soy Protein Dope with Yeast Protein Dope on the Rheological Properties

    OpenAIRE

    Hayakawa, Isao; Chang, Hung Min; Katoh, Tatsuo

    1984-01-01

    Rheological properties of isolated soybean protein dopes were compared with those of yeart protein dopes in order to find out their application and processing. The elastic properties of soybean protein dope were better than those of yeast protein dope prepared with high protein concentrates because the viscoelastic absorption of soybean protein dope was smaller than that of yeast protein dope and the capacity of water holding was higher than that of yeast protein dope. On the other hand, yeas...

  2. 冰亲和吸附装置对胶原抗冻肽的分离纯化%Purification of antifreeze collagen peptides by ice affinity adsorption protocol

    Institute of Scientific and Technical Information of China (English)

    阮功成; 曹慧; 徐斐; 于劲松

    2015-01-01

    抗冻肽是一类可以非依数性降低体系冰点的多肽,在生命体内具有非常重要的生理作用.研究搭建了冰亲和吸附装置,并以微生物保护活性为指标,优化了其对猪皮胶原酶解复合物中胶原抗冻肽的吸附条件,同时对分离产物的性质进行了研究.结果表明:在胶原酶解复合物浓度为1 mg/mL,吸附时间为10h,吸附温度为-5℃,吸附次数为2次的条件下,所获得的胶原抗冻肽对微生物的低温保护活性最强.经过冰亲和吸附后,胶原抗冻肽的主要洗脱峰之间的分离效果明显增加,其胶原抗冻肽的分子质量主要分布小于1 000 Da、且富含甘氨酸、脯氨酸和羟脯氨酸.%Antifreeze collagen peptides is a family of peptides which can lowering the freezing point of the system non-colligatively.This plays an important role in protecting organisms from freezing injury and damage.In this study, we studied the ice affinity adsorption equipment, used micro-organisms protection as the index to optimize the condition of purifying pig skin collagen peptides by enzymatic hydrolysis.The optimum conditions were: crude mixture concentration of 1 mg/mL, at-5 ℃ for 10 hour, adsorption twice.After ice affinity adsorption, collagen peptides peaks had better separation effect, the molecular weight mainly distributed within the range of 1000 Da, and major collagen amino acids were Proline and Hydroxyproline.

  3. Proteomic comparison between maturation drying and prematurely imposed drying of Zea mays seeds reveals a potential role of maturation drying in preparing proteins for seed germination, seedling vigor, and pathogen resistance.

    Science.gov (United States)

    Wang, Wei-Qing; Ye, Jian-Qing; Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna I; Jensen, Ole Nørregaard; Møller, Ian Max; Song, Song-Quan

    2014-02-01

    We have studied the role(s) of maturation drying in the acquisition of germinability, seedling vigor and pathogen resistance by comparing the proteome changes in maize embryo and endosperm during mature and prematurely imposed drying. Prematurely imposed dried seeds at 40 days after pollination (DAP) germinated almost as well as mature seeds (at 65 DAP), but their seedling growth was slower and they were seriously infected by fungi. A total of 80 and 114 proteins were identified to change at least two-fold (p seed germinability. However, a relatively large number of proteins changed in the embryo (47 spots) and endosperm (76 spots) specifically during maturation drying. Among these proteins, storage proteins in the embryo and defense proteins in the endosperm may be particularly important for seedling vigor and resistance to fungal infection, respectively. PMID:24341390

  4. Preparation and preliminary application of monoclonal antibody against nucleocapsid protein of bovine parainfluenza virus type 3%牛副流感病毒3型NP单抗的制备及初步应用

    Institute of Scientific and Technical Information of China (English)

    吕闯; 朱远茂; 董秀梅; 蔡红; 于作; 高欲燃; 薛飞

    2011-01-01

    To prepare monoclonal antibody (Mab) against nucleocapsid protein (NP) of bovine parainfluenza virus type 3 (BPIV3), BALB/c mice were immunized with purified recombinant NP (Rnp) expressed by E. Coli and a hybridoma secreting Mab was screened from fusing the SP2/0 cells with the spleen cells of the immunized BALB/c mice by indirect ELISA coated with BPIV3. The titers of Mab in ascites were 2 x 106 and 1.28 x 105 as detected by Rnp and BPIV3 coated ELISA, respectively. The Mab was specifically reacted with BPIV3 identified by indirect ELISA, western blot, immunofluroescence assay. The specific tests indicated the Mab had no reaction with infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. The BPIV3 was detected in experimentally infected animals in immunohistochemical test with the Mab. Therefore, this Mab could be used to establish diagnosis method for BPIV3 and further study on the structure and function of NP.%为制备牛副流感病毒3型(BPIV3)核衣壳蛋白(NP)单克隆抗体(MAb),本研究利用原核表达并纯化的重组NP (rNP)免疫BALB/c小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合.采用以BPIV3为检测抗原的间接ELISA方法筛选阳性细胞克隆,经3次克隆纯化后获得1株稳定分泌抗NP特异性MAb的杂交瘤细胞株(5E5)并制备腹水,采用rNP及BPIV3包被的ELISA效价分别是2×106和1.28×105.间接ELISA、western blot、IFA试验表明该MAb具有良好的反应性和特异性.经抗体亚类鉴定该MAb亚类为IgGl/κ.特异性试验表明该MAb不与牛传染性鼻气管炎病毒、牛病毒性腹泻病毒反应.免疫组化试验表明该MAb可以检测BPIV3感染动物体内的病原.该MAb还可用于建立检测BPIV3病原及抗体的诊断方法,同时为研究NP的结构和功能提供了条件.

  5. Biosynthetic preparation of 35-S labelled methionine

    International Nuclear Information System (INIS)

    High specific activity methionine with sulfur-35 was prepared in our laboratory by growing Baker's yeast cells, in a medium containing 35S-sulfate. L-S35 methionine was prepared from the acid hydrolyzate of the proteins by chromatography on whatman paper. The specific activity was determined using o-phtaladehyde as a fluorophore to form a fluorescent complex. The specific activity was found to be usually greater than 800 Ci/mmol. (Author)

  6. 兔多杀性巴氏杆菌外膜蛋白A(OmpA)重组蛋白单克隆抗体的制备及潜在应用%Preparation and Potential Application of Monoclonal Antibody against Pasteurella multocida Outer Membrane Protein A (OmpA) Recombinant Protein

    Institute of Scientific and Technical Information of China (English)

    刘燕; 庞安娜; 韦强; 肖琛闻; 鲍国连; 季权安; 钱微

    2012-01-01

    The aim of this study was to prepare monoclonal antibody (McAb)against Pasteurella multocida. The DNA fragment encoding the mature domain of P. multocida outer membrane protein A (OmpA) was amplified from the genomic DNA and sub-cloned into pET28a (+) expression vector, 37.6 kD rOmpA fusion protein was expressed mainly as an insoluble protein, optimal sohibilization of the recombinant protein was obtained using 8 mol/L urea in lysis buffer. BALB/c mice (Mus musculus) were subcutaneously injected with 100 μg of P. multocida OmpA emulsified by equivolumminal freund's complete adjuvant at the age of 6-8 weeks. Thereafter they were boosted two times with 200 μg of P. multocida OmpA emulsified by Freund's incomplete adjuvant at intervals of three weeks. The spleen cells of BALB/ c mice immunized with recombinant Pm OmpA were collected and infused with SP2/ 0 cell. Sebsequently four hybridoma cell strains were obtained by indirect enzyme linked immunosorbent assay (ELISA). The ELISA titers of antibodies in culture supernatant were 1:128,1:128,1:256 and 1:128, respectively, and ascites titers were 1:6 400,1:6 400,1: 12 800 and 1:6 400, respectively. The McAbs did not cross-react with other gram-negative and gram-positive bacterial pathogens, including E. coli, Bordetella bmnchiseptka, Pseudomonas aeruginosa and slaphylococcus. High titer McAbs were secreted from the hybridoma cells after repeat freezing. The result of Western blotting assay showed that the four Mabs could react with Pm OmpA protein specifically. ELISA test revealed that the 2A2 McAb belonged to the subtype of IgG2b, with a concentration was 130 u-g/mL after protein A affinity purification. The purified 2A2 McAb was selected by Western blot and IFA assays. The result indicated that the McAb could react with the Pm isolate strain. The success of this study has built up a solid base for developing a novel diagnostic methodology to the Pasteurella multocida infection in rabbits.%制

  7. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... the vegetarian proteins, whether they have carbohydrate. Best Protein Choices The best choices are: Plant-based proteins ...

  8. 昆虫低温生物学: Ⅱ.冰核物质(冰核蛋白)和昆虫的耐冻性%Insect cryobiology: II. Ice nucleator agents (ice nucleator proteins) and insect antifreeze

    Institute of Scientific and Technical Information of China (English)

    李毅平; 龚和

    2000-01-01

    @@ 体系在低于熔点温度时才结冰的现象,叫过冷却(supercooling).体系开始结冰时的温度称为过冷却点(supercooling point, SCP).在适当的低温,体系内需存在一起始结冰的冰核,才能诱导冰晶产生,此物质称为冰核剂(ice nucleating agents, INA).昆虫体内各腔室充满组织液,各腔室(如消化系统和细胞内)因所含INA的冰核活性的不同,而使结冰的温度各异,所受低温伤害也不同.

  9. Fish protein hydrolysates

    Energy Technology Data Exchange (ETDEWEB)

    Mackie, I.M.

    1982-01-01

    Proteolytic enzymes now available in commercial quantities can be used to liquefy the fish and fish waste presently considered suitable for conversion to fish meal. The products obtained are readily dispersed or dissolved in water and have a high nutritional value. They have been satisfactorily used as substitutes for milk proteins in milk replacers for young animals. Further research is necessary on means of controlling the degree of hydrolysis to give protein preparations with acceptable functional properties as human food supplements. (Refs. 21).

  10. Ice Recrystallization in a Solution of a Cryoprotector and Its Inhibition by a Protein: Synchrotron X-Ray Diffraction Study.

    Science.gov (United States)

    Zakharov, Boris; Fisyuk, Alexander; Fitch, Andy; Watier, Yves; Kostyuchenko, Anastasia; Varshney, Dushyant; Sztucki, Michael; Boldyreva, Elena; Shalaev, Evgenyi

    2016-07-01

    Ice formation and recrystallization is a key phenomenon in freezing and freeze-drying of pharmaceuticals and biopharmaceuticals. In this investigation, high-resolution synchrotron X-ray diffraction is used to quantify the extent of disorder of ice crystals in binary aqueous solutions of a cryoprotectant (sorbitol) and a protein, bovine serum albumin. Ice crystals in more dilute (10 wt%) solutions have lower level of microstrain and larger crystal domain size than these in more concentrated (40 wt%) solutions. Warming the sorbitol-water mixtures from 100 to 228 K resulted in partial ice melting, with simultaneous reduction in the microstrain and increase in crystallite size, that is, recrystallization. In contrast to sorbitol solutions, ice crystals in the BSA solutions preserved both the microstrain and smaller crystallite size on partial melting, demonstrating that BSA inhibits ice recrystallization. The results are consistent with BSA partitioning into quasi-liquid layer on ice crystals but not with a direct protein-ice interaction and protein sorption on ice surface. The study shows for the first time that a common (i.e., not-antifreeze) protein can have a major impact on ice recrystallization and also presents synchrotron X-ray diffraction as a unique tool for quantification of crystallinity and disorder in frozen aqueous systems.

  11. 适于双向电泳分析的酵母胞外蛋白提取方法%Procedure to Prepare Samples for Two-dimensional Electrophoresis of Secreted Proteins from Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    杜维; 朴永哲; 黄玮; 谷月; 赵长新

    2015-01-01

    Saccraomyces cerevsiae FFC2144 was cultured in nitrogen base medium without protein. The secretory proteins of yeasts were extracted by ammonium sulfate precipitation, ultrafiltration and lyophilization-phenol extraction respectively. Extraction rates by 3 methods were calculated and the proteins were separated by two-dimensional electrophoresis. The proteins isolated were confirmed by MALDI-TOF-MS. The extraction rate by lyophilization-phenol method was 73.67% and 114 protein spots were obtained with the most protein spots and clearest electrophoretogram. Lyophilization-phenol method could be an ideal separation method for studying secretory proteomics.%采用了无蛋白酵母培养基培养FFC2144酵母细胞,用硫铵沉淀法、超滤法、冻干酚提等方法提取酵母胞外蛋白,计算3种方法的提取率并分别用双向电泳的方法对提取到的蛋白样品进行分离,同时运用质谱鉴定分离后蛋白.其中冻干-平衡酚法提取后得到114个蛋白点,提取率为73.67%,图谱识别的蛋白点最多图谱最清晰,是研究分泌类蛋白质组学理想的分离方法.

  12. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins

    DEFF Research Database (Denmark)

    Weber, Daniela; Davies, Michael J.; Grune, Tilman

    2015-01-01

    in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented...... different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used...

  13. 以低变性脱脂豆粉为原料生产大豆组织蛋白的研究%Study on the Preparation of the Soya Cell Protein With the Material of Low-denatured and Low-temperature Soybean Powder of the Degrease

    Institute of Scientific and Technical Information of China (English)

    韩玉洁; 张亚丽; 徐忠

    2000-01-01

    以低变性脱脂豆粉为原料生产大豆组织蛋白,详细讨论了原料的质量对生产工艺和产品质量的影响,指出了生产高质量的大豆组织蛋白的较适宜的工艺条件。%The preparation of the soya cell protein with the material of the low-denatured soybean powder of the degrease, study on the influence of quality of material on the production technology and the quality of the production. Point out that the optimal conditions of the technology for produce the soya cell protein of high quality.

  14. 蛋白质/多肽药物聚乳酸/乳酸-羟基乙酸共聚物微球研究进展%Recent advances in the preparation progress of protein/peptide drug loaded PLA/PLGA microspheres

    Institute of Scientific and Technical Information of China (English)

    徐风华; 张强

    2007-01-01

    Sustained release drug delivery from microparticles is an excellent alternative for daily protein/peptide drug administration protocol. Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) are the most commonly used polymer carriers in the development of protein/peptide microspheres. Basically there are three preparation methods for PLA/PLGA microspheres: the solvent extraction/evaporation based multiple emulsion (W/O/W emulsion) method, the phase separation method and the spray drying method. The stability of the protein/pipetide loaded, encapsulation efficiency, and the burst effect of the microspheres are key problems usually met in the preparation of microspheres. In this review the preparation techniques and progress in the development of protein/pipetide microspheres which aimed to stabilize protein/peptide structural integrity, keep the bioactivity of drugs, increase the encapsulation efficiency and improve the release profile were summarized and evaluated.%缓释微粒给药系统是蛋白质/多肽药物传输系统的一个重要研究方向,聚乳酸和乳酸-羟基乙酸共聚物是制备缓释微球最常用的载体材料.蛋白质/多肽药物聚乳酸/乳酸-羟基乙酸共聚物微球常用的制备方法包括溶剂萃取/挥发法(复乳法)、相分离法和喷雾干燥法.本文总结了微球制备中面临的难点如蛋白质/多肽药物稳定性、包封率、药物突释和药物吸附等问题,并综述了保持药物结构稳定性和生物活性、提高包封率、改善药物释放曲线等微球制备方法和进展.

  15. Neofunctionalization of zona pellucida proteins enhances freeze-prevention in the eggs of Antarctic notothenioids

    Science.gov (United States)

    Cao, Lixue; Huang, Qiao; Wu, Zhichao; Cao, Dong-Dong; Ma, Zhanling; Xu, Qianghua; Hu, Peng; Fu, Yanxia; Shen, Yu; Chan, Jiulin; Zhou, Cong-Zhao; Zhai, Wanying; Chen, Liangbiao

    2016-10-01

    The mechanisms by which the eggs of the Antarctic notothenioid fishes avoid freezing are not fully understood. Zona pellucida proteins (ZPs) are constituents of the chorion which forms a protective matrix surrounding the egg. Here we report occurrence of freezing temperature-related gene expansion and acquisition of unusual ice melting-promoting (IMP) activity in a family of Antarctic notothenioid ZPs (AnnotoZPs). Members of AnnotoZPs are shown to bind with ice and non-colligatively depress the melting point of a solution in a range of 0.26 to 0.65 °C at a moderate concentration. Eggs of zebrafishes expressing an AnnotoZP transgene show improved melting point depression and enhanced survival in freezing conditions. Mutational analyses in a representative AnnotoZP indicate the ZP domain and patches of acidic residues are essential structures for the IMP activity. AnnotoZPs, therefore, represent a group of macromolecules that prevent freezing by a unique ZP-ice interaction mechanism distinct from the known antifreeze proteins.

  16. International perspectives on coal preparation

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-12-31

    The report consists of the vugraphs from the presentations which covered the following topics: Summaries of the US Department of Energy`s coal preparation research programs; Preparation trends in Russia; South African coal preparation developments; Trends in hard coal preparation in Germany; Application of coal preparation technology to oil sands extraction; Developments in coal preparation in China; and Coal preparation in Australia.

  17. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    . The biophysical and structural investigations of PPIs consequently demand hybrid approaches, implementing orthogonal methods and strategies for global data analysis. Currently, impressive developments in hardware and software within several methodologies define a new era for the biostructural community. Data can......Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers...

  18. Angiotensin I-converting Enzyme Inhibitory Activity ofPeanut Protein Hydrolysates Prepared with Alcalase%花生蛋白碱性蛋白酶水解物对血管紧张素转化酶抑制作用的初步研究

    Institute of Scientific and Technical Information of China (English)

    刘焕; 黎观红; 施用晖; 乐国伟

    2005-01-01

    Peanut protein hydrolysates were prepared by enzymatic hydrolysis with Alcalase and Neutrase, and the angiotensin I-converting enzyme (ACE) inhibitory activities of the enzymatic hydrolysates were investigated at different hydrolysis times. The unhydrolyzed protein showed no inhibitory activity. Hydrolysates generated with Neutrase displayed very low ACE inhibitory activity, while those obtained with Alcalase exhibited high inhibitory activity. The highest ACE inhibitory activity with the IC50 value of 0.56 mg protein/mL was found in the hydrolysate obtained with Alcalase at 30 min of hydrolysis time. These results indicate that peanut protein is a good protein source of ACE inhibitory peptides when hydrolyzed with the protease Alcalase. The peanut protein hydrolysates prepared with Alcalase might be utilized for physiologically functional foods with antihypertensive activity.%分别以碱性蛋白酶Alcalase和中性蛋白酶Neutrase对花生分离蛋白进行水解,制备花生分离蛋白水解物,并测定不同水解时间所得产物对血管紧张素转化酶(ACE)的抑制活性.未水解的花生分离蛋白没有ACE抑制活性,用中性蛋白酶Neutrase水解所得的水解物显示弱ACE抑制活性.然而,碱性蛋白酶Alcalase水解物具有很强的ACE抑制活性,水解0.5 h时水解物活性最高,其半抑制浓度为(IC50)0.56 mg/mL.本研究表明,当用碱性蛋白酶Alcalase水解时,花生分离蛋白是生产ACE抑制肽的良好蛋白质来源,花生分离蛋白碱性蛋白酶Alcalase水解物可作为具有降压功能的功能食品添料.

  19. Preparation High Titer Anti-serum of Porcine Circovirus Type Ⅱ Capsid Protein by Hydrodynamics Gentic Immunization%水流动力学基因免疫制备猪Ⅱ型圆环病毒核衣壳蛋白高效价抗血清

    Institute of Scientific and Technical Information of China (English)

    樊宝良; 张瑾; 代红星; 黄培华

    2012-01-01

    为了建立一个简捷有效的抗血清的制备方法,本研究选用猪Ⅱ型圆环病毒核衣壳蛋白基因,使用水流动力学基因免疫的方法制备高效价抗血清的可行性.应用无内提取试剂盒制备猪Ⅱ型圆环病毒核衣壳蛋白基因真核表达载体pcDNA-Cap的无内毒素质粒.将该质粒使用水流动力学尾静脉注射法对小鼠(Mus musculus)进行基因免疫,重复免疫5次后采血收集血清;以原核表达获得的N末端去除了核定位序列的猪圆环病毒核衣壳蛋白表达产物作为抗原蛋白,以制备的小鼠血清作为一抗进行酶联免疫吸附试验(ELISA)和Western blot检测.结果显示,应用水流动力学尾静脉注射法获得抗血清稀释5000倍通过Western blot至少能够检测到10 ng的抗原蛋白,ELISA分析表明,其效价可达到1∶1000000,说明获得的抗血清具有很好的效价水平.这一研究为猪Ⅱ型圆环病毒相关研究用抗体的制备提供了一个简洁有效的方法,也为猪Ⅱ型圆环病毒的防治方法的建立提供了一个值得尝试的策略.%In order to establish a simple and efficient anti-serum preparation method for molecular biology research, in this research, porcine circovirus type II capsid protein gene was selected to research on the possiblity of preparing high titer anti-serum by hydrodynamics gentic immunization. Endotoxin free plasmid of pcDNA-Cap, with would express porcine circovirus type II capsid protein in the exkaryotic cell, was prepared using endotoxin free plasmid preparing kit and was delivered by hydrodynamics tail vein injection method for genetic immunization of mice (Mus musculus). After 5 times continuous immunization, the blood serum was collected. Using porcine circovirus type II capsid protein which has been deleted its nuclear laction signal sequence at its N-terminal and expressed in Escherichia coli as antigen, the prepared anti-serum was tested by enzymeliked immuno sorbent assay(ELISA) and

  20. The preparation method of the type 1 pneumococcal polysaccharide - protein binding biochemical and immunological characteristics of vaccine%不同方法制备的1型肺炎球菌多糖-蛋白结合疫苗生化及免疫学特性比较

    Institute of Scientific and Technical Information of China (English)

    戴吉平

    2014-01-01

    目的:比较不同方法制备的1型肺炎球菌多糖-蛋白结合疫苗生化及免疫学特性。方法采用胺还原法以及溴化氰活化法分别对结合物进行制备,并采取生化以及免疫学检测的方式对其进行检测。结果相对于胺还原法制造出来的结合物,利用溴化氰活化法具有较高的高分子结合物含量以及较高的结合率,与此同时,免疫小鼠产生的抗体提高的也十分明显。结论相对于胺还原法制造出来的结合物,利用溴化氰活化法对1型肺炎球菌多糖-蛋白结合疫苗进行制备要优。%Objective To compare the different methods of preparation of type 1 pneumococcal polysaccharide - protein conjugate vaccine biochemical and immunological properties. Methods amine reduction and cyanogen bromide activation method was used to conjugate prepared and take biochemical and immunological detection methods for its detection. The results relative to the amine reduction conjugates produced using cyanogen bromide activation method combined with high polymer content and higher binding rate, while the immunized mice produced antibodies increased very significantly. Conclusion reduction relative to the amine conjugates produced using cyanogen bromide activation method for type 1 pneumococcal polysaccharide - protein conjugate vaccine prepared to excellent.

  1. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    Science.gov (United States)

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  2. 碱溶酸沉法制备火麻仁蛋白工艺研究%Preparation of semen cannabis protein by alkali-extraction and acid-precipitation

    Institute of Scientific and Technical Information of China (English)

    汤茜

    2011-01-01

    Semen cannabis contains approximately 20%~25% protein including edestin, albumin, and complete proteins easy to be absorbed by human body. In this study, semen cannabis protein was separated by alkali-extraction and acid-precipitation using degreasing semen cannabis as raw material, The optimal technological conditions were obtained by orthogonal experiment as follows: extracting temperature 50℃, alkali-extraction time 0.5h,alkali-extraction pH .11.0, the ratio of solid to liquid 1:20 and acid-precipitation pH 5.0. Under these optimum extraction conditions, the extraction rate of semen cannabis protein reached 29.54%.%火麻仁中大约含20%~25%的蛋白质,主要为麻仁球蛋白和白蛋白,含人体所有必需氨基酸且容易消化的全价蛋白质.该试验以火麻仁为原料,对其进行脱脂处理后,利用碱溶酸沉法制备火麻仁分离蛋白并确定其最佳工艺条件.试验表明,提取温度50℃,碱提时间0.5h,pH 11.0,料液比1∶20,酸沉pH 5.0为最佳工艺条件,在此条件下蛋白质的提取率为29.54%.

  3. Materials Preparation Center

    Data.gov (United States)

    Federal Laboratory Consortium — MPC is recognized throughout the worldwide research community for its unique capabilities in purification, preparation, and characterization of: rare earth metals,...

  4. InterProScan Result: CK511306 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 00104 Antifreeze protein, type I Biological Process: homoiothermy (GO:0042309)|Molecular Function: ice binding (GO:0050825)|Biological Process: response to freezing (GO:0050826) ...

  5. InterProScan Result: BY918960 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 00104 Antifreeze protein, type I Biological Process: homoiothermy (GO:0042309)|Molecular Function: ice binding (GO:0050825)|Biological Process: response to freezing (GO:0050826) ...

  6. InterProScan Result: BY927519 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 00104 Antifreeze protein, type I Biological Process: homoiothermy (GO:0042309)|Molecular Function: ice binding (GO:0050825)|Biological Process: response to freezing (GO:0050826) ...

  7. InterProScan Result: BY928658 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 00104 Antifreeze protein, type I Biological Process: homoiothermy (GO:0042309)|Molecular Function: ice binding (GO:0050825)|Biological Process: response to freezing (GO:0050826) ...

  8. 抗流感病毒NS1蛋白抗体的制备及其生物特性研究%Preparation and biological characteristics study of multiclonal antibodies to influenza virus NS1 protein

    Institute of Scientific and Technical Information of China (English)

    王巧刚; 吴海波; 沃恩康; 王怡婷; 尤金彪; 郭潮潭

    2012-01-01

    目的 NS1蛋白在流感病毒复制和传播中起到重要的调节作用,其功能多样,如能调节宿主细胞的蛋白合成、诱导感染细胞的凋亡以及拮抗IFN作用等,此外还有许多功能至今尚未明了.因此,研制一个特异性高的抗NS1抗体,对NS1蛋白功能的进一步研究起到重要作用.方法 RT-PCR扩增流感病毒NS1基因,将其克隆进原核表达载体pET-28a(+)中.然后在大肠埃希菌BL21中表达,用切胶纯化方法获取NS1融合蛋白.用该蛋白免疫新西兰大白兔,制备效价较高的多克隆抗体,并测定其效价(间接ELISA法).用病毒感染细胞实验、Western印迹法和间接免疫荧光实验等方法,观察病毒复制过程中NS1蛋白的表达及细胞定位情况.结果 首先构建了能表达NS1蛋白的质粒,并经原核细胞表达获得了NS1蛋白.然后用该蛋白免疫大白兔,获得了效价较高(1∶256 000)的抗NS1蛋白抗体.实验发现,该抗体可以特异性结合非依赖亚型的甲型流感病毒NS1蛋白.病毒感染后6h即可以检测到NS1蛋白,随着病毒复制时间的延长NS1蛋白表达量变化不明显;24h后NS1蛋白主要分布于细胞核内并能定位于核仁中,也有部分位于细胞质.结论 本研究通过原核表达获得了NS1蛋白,并利用常规的免疫方法获得了效价较高的抗NS1多克隆抗体.所制备的多克隆抗体能够有效定位NS1蛋白的表达,并且能和甲型流感病毒各亚型结合,具有较高的特异性.该抗体不仅能用于NS1蛋白功能及其对宿主细胞影响的研究,而且可用于鉴别诊断猪或其他动物中的病毒感染还是接种疫苗后的反应,有较好的开发应用前景.%Objective NS1 protein of influenza virus plays an important regulatory role on the replication and dissemination.It has many functions,such as regulating host cell protein synthesis,inducing apoptosis of infection cell,as well as antagonistic action of interferon,but many functions are still

  9. Preparation and controlled release effect of soybean protein/multicarboxylic acids modified cotton fabric%大豆蛋白/复合羧酸改性棉织物的制备及其缓释效果

    Institute of Scientific and Technical Information of China (English)

    许云辉; 王晓明; 张晓丽

    2013-01-01

    To develop an eco-friendly and multifunctional cotton fabric,the modification treatment of cotton fabric was undertaken using citric acid,maleic acid and soybean protein so that the chemical bonding between soybean protein and cotton fabric was formed through the bridging function of multicarboxylic acids.Effects of treating conditions on the weighting percentage of cotton fabric were analyzed and the optimized process parameters were obtained.The characterization of infrared spectra and X-ray photoelectron spectrometry showed that the esterification crosslinking occurred between multicarboxylic acids and macromolecules of cotton fiber according to ring-anhydride mechanism,while the soybean protein was adhered to the cotton fabric with amido bond by the bridging function of multicarboxylic acids.The breaking strength of soybean protein modified cotton fabric slightly decreased,whereas the anti-ultraviolet-ray property of the modified cotton fabric after cactus extract treatment improved remarkably.Furthermore,the drug controlled release test showed that when soybean protein modified cotton fabric was used as the carrier for cactus extract,good controlled release effect was achieved.%为开发生态多功能棉织物,采用柠檬酸、马来酸、大豆蛋白对棉织物进行改性处理,通过复合羧酸的桥联作用,使大豆蛋白与棉织物形成化学键交联结合,研究处理条件对棉织物增重率的影响,并得出最优工艺参数.红外光谱和光电子能谱测试表明,复合羧酸与棉纤维大分子按环酐机制产生酯化交联,大豆蛋白借助复合羧酸的桥联作用以酰胺键共价结合在棉织物上.大豆蛋白改性棉织物的断裂强力略有下降,再经仙人掌提取物处理的大豆蛋白改性棉织物抗紫外线性能明显提高,药物缓释试验显示,大豆蛋白改性棉织物作为药物载体对仙人掌提取物的缓释效果良好.

  10. ZZ亲和肽-碱性磷酸酶融合蛋白的制备与初步应用%Preparation of fusion protein between ZZ affinity peptide and alkaline phosphatase and its primary application

    Institute of Scientific and Technical Information of China (English)

    鲍如梦; 赵爽佳; 薛敏; 杨洪鸣; 唐金宝

    2015-01-01

    目的:构建、表达ZZ亲和肽与碱性磷酸酶( AP)融合蛋白,并研究其生物学活性。方法:将ZZ亲和肽基因与AP基因重组,构建原核表达载体pEZZ-AP并利用大肠杆菌表达,金属离子亲和层析纯化目的蛋白,Western blot鉴定ZZ-AP生物学活性并初步应用于免疫细胞化学。结果:所构建重组质粒pEZZ-AP 经E.coli DH5α表达,SDS-PAGE结果显示目的蛋白相对分子量为62 kD,与理论值相近。 Western blot结果表明ZZ-AP融合蛋白既具有兔IgG抗体结合活性,又具有碱性磷酸酶活性,在细胞免疫组化应用中与AP标记的羊抗兔二抗显色模式与效果相似。结论:ZZ-AP融合蛋白构建成功,该融合蛋白具有兔IgG 抗体结合活性和碱性磷酸酶活性,细胞免疫组化应用结果表明该融合蛋白在免疫分析中具有潜在的应用价值。%Objective:To construct and express the fusion protein between ZZ affinity peptide and alkaline phosphatase and examine its biological activities.Methods:The alkaline phosphatase gene was cloned into pEZZ 18 vector containing ZZ peptide gene resulted in the pEZZ-AP recombinant vector.Then the vector was transformed and expressed in E.coli DH5α.And HisTrap affinity chromatography was employed to separate and purify the target protein.After analyzed by Western blot , ZZ-AP fusion protein was applied to immunocytochemistry as an alterative second antibody.Results:The result of SDS-PAGE showed that the fusion protein with a relative molecular weight was consistent with the theoretical values (62 kD).The fusion protein has rabbit IgG-binding ability and en-zymatic activity of alkaline phosphatase ,those were validated in Western blot;and it produced a good signal that was comparable in its staining pattern to that generated with goat anti-rabbit IgG-AP in immunocytochemistry.Conclusion: The ZZ-AP fusion protein was constructed successfully ,it has rabbit IgG-binding ability and enzymatic

  11. Expression and localization of an ice nucleating protein from a soil bacterium, Pseudomonas borealis.

    Science.gov (United States)

    Vanderveer, Tara L; Choi, Julie; Miao, Denian; Walker, Virginia K

    2014-08-01

    An ice nucleating protein (INP) coding region with 66% sequence identity to the INP of Pseudomonas syringae was previously cloned from P. borealis, a plant beneficial soil bacterium. Ice nucleating activity (INA) in the P. borealis DL7 strain was highest after transfer of cultures to temperatures just above freezing. The corresponding INP coding sequence (inaPb or ina) was used to construct recombinant plasmids, with recombinant expression visualized using a green fluorescent protein marker (gfp encoding GFP). Although the P. borealis strain was originally isolated by ice-affinity, bacterial cultures with membrane-associated INP-GFP did not adsorb to pre-formed ice. Employment of a shuttle vector allowed expression of ina-gfp in both Escherichia coli and Pseudomonas cells. At 27 °C, diffuse fluorescence appeared throughout the cells and was associated with low INA. However, after transfer of cultures to 4 °C, the protein localized to the poles coincident with high INA. Transformants with truncated INP sequences ligated to either gfp, or an antifreeze protein-gfp fusion showed that the repetitive ice-nucleation domain was not necessary for localization. Such localization is consistent with the flanking residues of the INP associating with a temperature-dependent secretion apparatus. A polar location would facilitate INP-INP interactions resulting in the formation of larger aggregates, serving to increase INA. Expression of INPs by P. borealis could function as an efficient atmospheric dispersal mechanism for these soil bacteria, which are less likely to use these proteins for nutrient procurement, as has been suggested for P. syringae. PMID:24930584

  12. 利用啤酒糟制备高品质饲料蛋白%Study on the preparation of high-quality feedstuffs protein by brewer's spent grain

    Institute of Scientific and Technical Information of China (English)

    蔡国林; 张麟; 陆健

    2015-01-01

    通过刀片粉碎和筛分技术对啤酒糟进行预处理,对筛下物进行球磨处理、固态酶解和有益微生物发酵,可以显著提高啤酒糟的饲用品质,制备高品质饲料蛋白.啤酒糟经刀片粉碎后过60目筛,筛下物粗蛋白含量达到38.8%,粗纤维含量降低到1.89%,实现蛋白-纤维的初步富集分离.对筛下物球磨后,经纤维素酶、木聚糖酶和蛋白酶混合固态酶解和产朊假丝酵母JX-09固态发酵之后,其粗蛋白含量达到45.17%,比啤酒糟原样提高了59.6%,多肽含量从0.68%提高到5.21%,蛋白体外消化率从53.7%提高到61.8%,水解氨基酸总量达到42.18%,比啤酒糟原样提高了60%,必需氨基酸总量达到17.3%,比啤酒糟原样提高了60%,粗纤维含量达到1.66%,比啤酒糟原样降低了89%.%After blade grinding and sieving pretreatment,the partial of brewer' s spent grain with high level of protein was dealt with ball milling,solid enzymatic digestion and fermentation with beneficial microorganism.The feeding quality of brewer' s spent grain has been significantly improved,resulting in the production of feed protein with high-quality.The contents of crude protein and crude fiber in the undersize brewer's spent grain were 38.8% and 1.89%,respectively after blade grinding and filtration through a 60 mesh sieve.Consequently,the preliminary enrichment and separation of protein and fiber had been achieved.Moreover,the enzymatic efficiency of undersize brewer' s spent grain could be further improved after milling of fiber bundle by ball milling.After the hydrolysis by cellulase,xylanase,protease and solid fermentation with Candida utilis JX-09,the crude protein content of brewer's spent grain was increased up to 45.17%,which was 59.6% higher than that of original sample.Polypeptides content was increased from 0.68% to 5.21%,while the in vitro digestibility of proteins was increased from 53.7% to 61.8%.Total amino acids

  13. 抗HBs Fab-IFNα融合蛋白的制备与初步鉴定%Preparation and preliminary identification of anti HBs Fab-IFNa fusion protein

    Institute of Scientific and Technical Information of China (English)

    陆慧琦; 宋杰; 叶伟民; 韩焕兴

    2011-01-01

    目的 构建抗HBsAg的pComb Fab-IFNα载体,原核表达具双重生物活性的抗HBs Fab-IFNα融合蛋白.方法 以pBAD-Fab和pBAD IFNα作为模板,分别扩增Fd、λ和IENα基因,经相应的限制性内切酶酶切后分3次克隆人pComBHss质粒,转化XL-1 Blue大肠埃希菌.限制性酶切和测序鉴定重组质粒,免疫蛋向印迹(Western blotting)和斑点印迹(Dot blotting)鉴定融合蛋白的表达及抗原结合活性.结果 重组载体的酶切、电泳及测序表明抗HBs Fab-IFNα基因克隆正确.表达产物经12%SDS-PAGE电泳、转印,Western blotting显示该融合蛋白分子量约为65kD,Dot blotting显示其与HBsAg具有结合能力.细胞病变抑制法测定IFNα生物学活性为7.8×104~5.1×105U/ml.结论 该原核系统成功表达了抗HBs Fab-IFNα融合蛋白,表明其既具有抗HBsAg结合能力,又具备IFNα的生物活性,为进一步的系统表达和应用研究提供了条件.%Objective To construct the anti HBsAg pComb Fab-IFNe vector, and to express the fusion protein consisting of IFN and anti-HBs Fab in prokaryon. Methods Using pBAD-IFN plasmid and pBAD-Fab plasmid as template, the anti-HBs Fd, and IFN were amplified separately with corresponding endonuclease sites by polymerase chain reaction (PCR). Each PCR product was digested with specific endonuclease and inserted into pComBHss vector, and then transformed into XL-1 Blue. The recombinant plasmid was isolated by miniprep for restriction analysis and sequencing. Fusion protein was identified by Western blotting and Dot blotting. Results The recombinant plasmid was confirmed by restriction electrophoresis and sequencing. Aliquots of human anti-HBs Fab-IFNα were concentrated and size fractionated by 12% SDS-PAGE and stained with Coomassie. The proteins were transferred to nitrocellulose, incubated with HRP- conjugated goat anti-human IgG Fab and Rah anti-human IFN. The molecular weight of the fusion protein was about 65kD. Anti HBs Fab-IFNa fusion

  14. 新鲜马铃薯渣同时制备膳食纤维和蛋白的研究%Attempt of Prepare Dietary Protein and Fiber from Fresh Potato Residue

    Institute of Scientific and Technical Information of China (English)

    吴海燕; 盖春慧; 钟振声

    2012-01-01

    Potato residue was the residue generated in the potato starch production process. The major ingredient is water, cell debris and residual starch granules. It contains a lot of starch and dietary fiber and small amounts of protein. It is a valuable resource. In this paper a-amylase and glucoamylase were used to deal with potato residue. And then separate the liquid portion and ferment it to edible protein, the solid part was bleached, modified to dietary fiber, and the potato residue was got full use. This study designed the process, selected the enzymes and optimized the process conditions. And the protein and dietary fiber's physical and chemical properties were determined.%马铃薯渣是马铃薯淀粉生产过程中产生的一种主要成分是水、细胞碎片和残余淀粉颗粒的副产物,含有大量的淀粉和膳食纤维以及少量蛋白质,实际上是一种宝贵的资源.本文采用α-淀粉酶和糖化酶处理马铃薯渣,分离出的液态部分再经发酵培养出可供食用的蛋白质,固体部分经漂白、改性后成为膳食纤维,薯渣得到全利用.设计了工艺流程,对酶的选择和工艺条件作了优化.测定了产物蛋白质和膳食纤维的理化指标.

  15. Optimization of Protein Preparation Suitable for 2-DE of Hevea brasiliensis C-serum%橡胶树胶乳 C-乳清双向电泳样品制备技术的建立及优化

    Institute of Scientific and Technical Information of China (English)

    戚继艳; 蓝基贤; 秦云霞; 龙翔宇

    2012-01-01

      在TCA/丙酮沉淀法的基础上,用乙醇和乙醚洗涤(称双乙法)沉淀获得橡胶树胶乳C-乳清蛋白样品,并在样品溶解时通过超声助溶进一步提高样品的溶解度.结果显示,利用改良后的样品制备方法(TCA/丙酮+双乙法+超声处理)所获得的C-乳清蛋白样品的溶解性大幅增加,蛋白得率高达8.47 mg/mL C-乳清,为传统TCA/丙酮法的1.6倍;同时降低了盐离子浓度,等电聚焦时升压顺利、聚焦完全,双向电泳的图谱质量明显改善,在银染2D胶上可获得1206个蛋白点,为传统TCA/丙酮法的2.7倍.本文对胶乳C-乳清蛋白样品制备方法进行深入优化,获得了重现性较好的高质量双向电泳蛋白图谱,促进了橡胶树蛋白质组学研究%  The C-serum is a major component of the latex and a key material for proteinomics of Hevea brasiliensis. Using the 2-D electrophoresis, there are some disadvantages with protein extraction from C-serum, such as unsatisfied solubility, higher ions concentrations and too many impurities. In this paper, the alcohol-ether method and sonication were used to further remove interfering substances and solubilized protein based on the traditional method of TCA/acetone. Comparing the traditional method of extraction, the results showed that the new method improved the solubility, ions concentration and purity of protein samples from C-serum. The optimized methods for extraction of protein will be extremely useful for the proteomic research of C-serum form H. brasiliensis latex.

  16. 抗重组 GST单克隆抗体的制备及其在融合蛋白纯化中的应用%Preparation of monoclonal antibody against recombinant GST and its application in purification of GST fusion protein

    Institute of Scientific and Technical Information of China (English)

    严馨蕊; 鲍永利; 董学斌; 柳忠辉

    2001-01-01

    目的制备抗重组 GST的单克隆抗体 (mAb), 并用来纯化重组 GST融合蛋白。方法用含重组 GST融合蛋白基因的 pGEX4T -1质粒转化 E.coli BL21, IPTG诱导 GST融合蛋白表达,亲和层析和凝胶过滤法分离表达的重组 GST融合蛋白。以此蛋白作为抗原,免疫 Balb/c小鼠,按传统的杂交瘤技术制备 mAb。将抗 GST mAb经 Protein A纯化后,与 Sepharose 4B偶联。结果经 3次亚克隆后,获得两株分泌抗 GST载体特异性 mAb的杂交瘤。采用该 mAb对两种不同的 GST融合蛋白进行亲和层析纯化后,经 SDS-PAGE鉴定达到了商品化 Glutathione-Resin的亲和层析纯化效果。结论用抗 GST蛋白特异性 mAb亲和层析纯化融合蛋白是一种经济、实用的方法,且可用于 Glutathione-Resin亲和层析纯化后的二次纯化。%Aim To prepare and characterize a monoclonal antibody against recombinant glutathione S-transferase(GST) for purifying GST fusion protein. Methods The GST-follistatin fusion protein was expressed by using a pGEX4T-1 expression vector in Escherichia coli BL21 and purified by glutathione-resin affinity column chromatography. Then female Balb/c mice were immunized with the GST-FS, The immunized splenocytes were fused with NS-1 hybridoma cells. Dreparation of the mAb was used by conventional hybridoma techniqal. The mAb purified by protein A, was culpled with Sepharose4B to purify further GST fusion protein by affinity chromatography. Results The SDS-PAGE showed that the GST fusion protein could be purified effctively by specific mAb affinity chromatography as same as by glutathione-resin affinity chromatography. Conclusion mAb affinity chromatography will be a ecnomical and useful method and it can be used for secondary purification of GST fusion protein following glutathione-resin affinity chromatography.

  17. PREPARATIVE SKIN PREPARATION AND SURGICAL WOUND INFECTION

    OpenAIRE

    Anjanappa; Arjun

    2015-01-01

    BACKGROUND AND OBJECTIVE: It is an established fact now that the normal skin of healthy human beings harbours a rich bacterial fl ora. Normally considered non - pathogenic , these organisms way be a potential source of infection of the surgical wound. Approximately 20% of the resident flora is beyond the reach of surgical scrubs and antiseptics. The goal of surgical preparation of the skin with antiseptics is to remove transient and pathogenic microorganism...

  18. A Protocol for Fast and Efficient Preparation of Genomic DNA and Total Proteins of Nosema bombycis%一种快速高效制备家蚕微孢子虫基因组DNA和总蛋白的方法

    Institute of Scientific and Technical Information of China (English)

    蔡顺风; 何欣怡; 何祥康; 邱海洪; 李光才; 何永强; 鲁兴萌

    2011-01-01

    家蚕微孢子虫孢子壁坚厚不易破碎,并对许多化学物质有很强的抵抗性,使用常规的核酸和蛋白抽提方法效果不够理想.研究建立玻璃珠