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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. 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.

  4. 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

    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...

  5. 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.

  6. 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.

  7. 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...

  8. 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.

  9. 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.

  10. Effects of polyhydroxy compounds on beetle antifreeze protein activity

    Science.gov (United States)

    Amornwittawat, Natapol; Wang, Sen; Banatlao, Joseph; Chung, Melody; Velasco, Efrain; Duman, John G.; Wen, Xin

    2016-01-01

    Antifreeze proteins (AFPs) noncolligatively depress the nonequilibrium freezing point of a solution and produce a difference between the melting and freezing points termed thermal hysteresis (TH). Some low-molecular-mass solutes can affect the TH values. The TH enhancement effects of selected polyhydroxy compounds including polyols and carbohydrates on an AFP from the beetle Dendroides canadensis were systematically investigated using differential scanning calorimetry (DSC). The number of hydroxyl groups dominates the molar enhancement effectiveness of polyhydroxy compounds having one to five hydroxyl groups. However, the above rule does not apply for polyhydroxy compounds having more than five hydroxyl groups. The most efficient polyhydroxy enhancer identified is trehalose. In a combination of enhancers the strongest enhancer plays the major role in determining the TH enhancement. Mechanistic insights into identification of highly efficient AFP enhancers are discussed. PMID:19038370

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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

    Antifreeze proteins (AFPs) are characterized by their ability to protect organisms from subfreezing temperatures by preventing tiny ice crystals in solution from growing as the solution is cooled below its freezing temperature. This inhibition of ice growth is called antifreeze activity, and in...... particular, certain insect AFPs show very high antifreeze activity. Recent studies have shown AFPs to be promising candidates as green and environmentally benign inhibitors for gas hydrate formation. Here we show that an insect antifreeze protein from the longhorn beetle, Rhagium mordax (RmAFP1), the most...... potent protein yet found for freezing inhibition, can inhibit methane hydrates as effectively as the synthetic polymeric inhibitor polyvinylpyrrolidone (PVP). In high pressure rocking cell experiments, onset hydrate nucleation temperatures and growth profiles showed repeatable results. RmAFP1 clearly...

  18. 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 ...

  19. 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 ...

  20. Why Does Insect Antifreeze Protein from Tenebrio molitor Produce Pyramidal Ice Crystallites?

    OpenAIRE

    Strom, Christina S.; Liu, Xiang Yang; Jia, Zongchao

    2005-01-01

    The antifreeze protein (AFP) reduces the growth rates of the ice crystal facets. In that process the ice morphology undergoes a modification. An AFP-induced surface pinning mechanism, through matching of periodic bond chains in two dimensions, enables two-dimensional regular ice-binding surfaces (IBSs) of the insect AFPs to engage a certain class of ice surfaces, called primary surfaces. They are kinetically stable surfaces with unambiguous and predetermined orientations. In this work, the or...

  1. 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...

  2. 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

  3. 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.

  4. 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

  5. Expression, purification, crystallization and preliminary crystallographic studies of Rhagium inquisitor antifreeze protein

    International Nuclear Information System (INIS)

    A novel hyperactive antifreeze protein from R. inquisitor (RiAFP) has been overexpressed, purified and crystallized. A complete native X-ray diffraction data set was recorded to 1.3 Å resolution. Antifreeze proteins (AFPs) are a specialized evolutionary adaptation of a variety of bacteria, fish, arthropods and other organisms to inhibit ice-crystal growth for survival in harsh subzero environments. The recently reported novel hyperactive AFP from Rhagium inquisitor (RiAFP) is the second distinct type of AFP in beetles and its structure could reveal important molecular insights into the evolution of AFPs. For this purpose, RiAFP was overexpressed in Escherichia coli, purified and crystallized at 293 K using a combination of 23% PEG 3350 and 0.2 M ammonium sulfate as a precipitant. X-ray diffraction data were collected to 1.3 Å resolution using a synchrotron-radiation source. The crystals belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = b = 46.46, c = 193.21 Å

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Ice nucleation in emulsified aqueous solutions of antifreeze protein type III and poly(vinyl alcohol).

    Science.gov (United States)

    Inada, Takaaki; Koyama, Toshie; Goto, Fumitoshi; Seto, Takafumi

    2011-06-23

    Antifreeze protein (AFP) III and poly(vinyl alcohol) (PVA) are known as anti-ice nucleating agents (anti-INAs), which inhibit heterogeneous ice nucleation. However, the effectiveness of these anti-INAs in inhibiting ice nucleation in water-in-oil (W/O) emulsions, in which homogeneous ice nucleation can be experimentally simulated, is unclear. In this study, the ice nucleation temperature in emulsified solutions of AFP III, PVA, and other nonanti-INA polymers was measured, and then the nucleation rate was analyzed based on classical nucleation theory. Results showed that ice nucleation was surface-initiated and, except for PVA solutions, probably caused heterogeneously by the emulsifier, SPAN 65, at the droplet surfaces. In this nucleation mode, AFP III had no significant effect on the ice nucleation rate. In contrast, PVA exhibited ice-nucleating activity only at the droplet surfaces, suggesting that the nucleation is due to the interaction between PVA and SPAN 65. PMID:21619040

  11. 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 ...

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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)

  19. 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.

  20. 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...

  1. 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.

  2. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules

    Science.gov (United States)

    Mitchell, Daniel E.; Congdon, Thomas; Rodger, Alison; Gibson, Matthew I.

    2015-10-01

    Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used ‘splat’ methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. A unique capsular polysaccharide structure from the psychrophilic marine bacterium Colwellia psychrerythraea 34H that mimics antifreeze (glyco)proteins.

    Science.gov (United States)

    Carillo, Sara; Casillo, Angela; Pieretti, Giuseppina; Parrilli, Ermenegilda; Sannino, Filomena; Bayer-Giraldi, Maddalena; Cosconati, Sandro; Novellino, Ettore; Ewert, Marcela; Deming, Jody W; Lanzetta, Rosa; Marino, Gennaro; Parrilli, Michelangelo; Randazzo, Antonio; Tutino, Maria L; Corsaro, M Michela

    2015-01-14

    The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a γ-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one- and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanics and dynamics calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments. PMID:25525681

  8. 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

  9. 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.

  10. 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.

  11. 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, are...... here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18...... equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is...

  12. 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

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

    Science.gov (United States)

    Buch, J L; Ramløv, H

    2016-06-01

    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 AFP could reliably be told apart from controls after only two minutes of recrystallisation. The goal of providing a fast, cheap and easy method for detecting antifreeze proteins in solution was met, and further development of the system can be followed at https://github.com/pechano/cryostage. PMID:27041219

  14. 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

  15. Evaluation of propanediol, ethylene glycol, sucrose and antifreeze proteins on the survival of slow-cooled mouse pronuclear and 4-cell embryos.

    Science.gov (United States)

    Shaw, J M; Ward, C; Trounson, A O

    1995-02-01

    Mouse pronuclear and 4-cell embryos were cryopreserved by slow cooling to -33 degrees C in 1.5 M 1,2-propanediol or 1.5 M ethylene glycol, with or without 0.1 M sucrose. Straws were thawed by immersion into a 37 degrees C water bath, immediately after their removal from liquid nitrogen (protocol 1), or after being held in air for 15 (protocol 2) or 30 s (protocol 3). Others were held in air until the ice melted (protocol 4). Embryos which formed blastocysts that hatched and attached to the Petri dish in vitro (plated) were considered viable. The thawing protocol did not significantly influence the viability of embryos frozen in propanediol with 0.1 M sucrose (52-72% of pronuclear and 69-97% of 4-cell embryos plated). In the other solutions tested, propanediol without sucrose and ethylene glycol with/without sucrose, only protocol 2 resulted in uniformly high development of both pronuclear (45-65% plating) and 4-cell embryos (70-97% plating). Thawing protocol 4 significantly reduced development, in particular for embryos frozen in ethylene glycol (0% 1-cell; 0-25% 4-cell plating). The difference between thawing protocols 2 and 4 was reduced by continuing slow cooling of ethylene glycol solutions to lower temperatures (-41 degrees C). Adding antifreeze proteins type I or III did not improve survival or development. Thus, although mouse pronuclear and 4-cell embryos can be frozen-thawed in either ethylene glycol or propanediol without significant loss of viability, an appropriate thawing protocol is essential for embryos frozen in ethylene glycol or propanediol-sucrose. PMID:7769070

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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. PMID:27059578

  1. 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.

  2. 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%\\}

  3. 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.

  4. Characterization of glycoprotein antifreeze biosynthesis in isolated hepatocytes from Pagothenia borchgrevinki

    International Nuclear Information System (INIS)

    Incorporation of 14C-leucine and 3H-alanine into TCA-precipitable protein. TCA-soluble protein, and antifreeze glycoproteins (AFGP) was measured in isolated hepatocytes from Pagothenia borchgrevinki Boulenger following acclimation to -1.5 degrees C and +4 degrees C. the rate of 3H-alanine incorporation into AFGP followed Michaelis-Menten kinetics with a Vmax of 4.8 nM X mg protein-1 X h-1 at -1.5 degrees C and 7.5 nM X mg protein-1 X h-1 at +4 degrees C. Km values were 27.9 microM and 41.7 microM at -1.5 degrees C and +4 degrees C, respectively. Incorporation of 14C-leucine into TCA-precipitable protein also showed Michaelis-Menten kinetics with a Vmax of 20 nM X mg protein-1 X hr-1 at 1.5 degrees C and 32.3 nM X mg protein-1 X hr-1 at +4 degrees C. Km values were 83.3 microM at -1.5 degrees C and 125 microM at +4 degrees C. AFGP synthesis was monitored over a 120-h period by radioimmunoassay in cultures of hepatocytes from cold acclimated fish (-1.5 degrees C) incubated at both -1.5 degrees C and +4 degrees C. The estimated Q10 for AFGP from these data is 3.23. Polyacrylamide gel electrophoresis of antifreeze glycoproteins produced by isolated hepatocytes showed that all four antifreeze fractions normally present in the serum of P. borchgrevinki are also synthesized by isolated hepatocytes. The two major conclusions from these experiments were that 1) P. brochgrevinki, unlike many northern fishes, does not show thermal acclimation, and 2) environmental factors responsible for modification of peptide antifreeze synthesis in northern fishes do not elicit changes in AFGP synthesis in P. borchgrevinki

  5. PREPARATION OF LIPOSOMES CONTAINING WHEY PROTEINS

    OpenAIRE

    A. Suha Yalçın; Murat Türkoğlu

    2010-01-01

    Aim: In recent years, it has been shown that whey and its components have a number of health-promoting effects. We aimed to isolate fractions containing whey proteins using chromatography and then to prepare antioxidant liposomes in order to obtain a gel suitable for cosmetic preparations.Methods: Fractionation of whey proteins was achieved by extraction, filtration and centrifugation followed by liquid chromatography. The antioxidant activities of the fractions was determined by their copper...

  6. 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

    protein from Rhagium mordax (RmAFP) and biodegradable synthetic kinetic inhibitor Luvicap Bio. A systematic capillary dispersion method was used, and this method enhanced the ability to detect the effect of various inhibitors on hydrate formation with small quantities. The presence of RmAFP and Luvicap...... Bio influence (inhibit) the hydrate formation phenomena significantly. Luvicap Bio (relative strength compared to buffer: 13.3 degrees C) is stronger than RmAFP (9.8 degrees C) as a nucleation inhibitor. However, the presence RmAFP not only delays hydrate nucleation but also reduces the amount of...... hydrate formed (20%-30%) after nucleation significantly. Unlike RmAFP, Luvicap Bio promoted the amount of hydrate formed after nucleation. The superior hydrate growth inhibition capability and predictable hydrate melting behavior compared to complex, heterogeneous hydrate melting with Luvicap Bio shows...

  7. PREPARATION OF LIPOSOMES CONTAINING WHEY PROTEINS

    Directory of Open Access Journals (Sweden)

    A. Suha Yalçın

    2010-01-01

    Full Text Available Aim: In recent years, it has been shown that whey and its components have a number of health-promoting effects. We aimed to isolate fractions containing whey proteins using chromatography and then to prepare antioxidant liposomes in order to obtain a gel suitable for cosmetic preparations.Methods: Fractionation of whey proteins was achieved by extraction, filtration and centrifugation followed by liquid chromatography. The antioxidant activities of the fractions was determined by their copper ion reducing capacity. Gel electrophoresis was used to analyze the proteins. Liposomes were made by the thin film hydration method.Results and Conclusion: Using Sephadex G-50 chromatography, two fractions were obtained. The first fraction contained major whey proteins, while the second fraction had small peptides. We have then determined the antioxidant activities of these fractions. The first fraction had the highest antioxidant activity. We prepared liposomes containing whey protein fractions and analyzed their sizes. Then, we investigated the liposome structures under a light microscope, electron microscope and atomic force microscope. Finally, we prepared a cosmetic formula from liposomes containing the whey fractions. We believe that preparing antioxidant liposomes containing whey proteins will be an important contribution to the cosmetic formulas for dermal applications.

  8. 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

  9. 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....

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Preparation and characterization of films from pea protein.

    Science.gov (United States)

    Viroben, G; Barbot, J; Mouloungui, Z; Guéguen, J

    2000-04-01

    The conditions for protein film preparation from an alkaline dispersion of a pea protein isolate were investigated in the presence of polyols as plasticizers. Mechanical and barrier properties of resulting films were studied as a function of protein dispersion conditions, protein and plasticizer concentrations and ratios, chain length of the plasticizer, and pH and composition of the alkaline medium. Neither the mode of protein hydration nor the pea isolate origin had a significant effect on the mechanical properties of pea protein films. However, increasing the plasticizer chain length induced slightly higher surface hydrophobicity but poor mechanical properties. Addition of monoglycerides to film-forming solution allowed a significant improvement of the films during aging. Both tensile strength and surface hydrophobicity increased when ammonium hydroxide was used as protein dispersing agent instead of sodium hydroxide. PMID:10775350

  17. Directly probing the antifreeze glycoprotein kinetics at the ice/solution interface

    Science.gov (United States)

    Zepeda, Salvador; Yokoyama, Etsuro; Furukawa, Yoshinor

    2009-03-01

    Antifreeze proteins (AFP) and glycoproteins (AFGP) help fish, plants, insects and bacteria survive sub-freezing environments. It is well known that these proteins function via some surface interaction, but the exact mechanism has eluded scientists. Aside from mutagenesis experiments directed towards examining the functional importance of specific residues, conclusions about the mechanism have been drawn from indirect studies or more precisely from studies that describe the proteins effects on the ice interface. Our work is aimed at directly studying the protein kinetics at the ice/solution interface. Fluorescent microscopy is used to determine interaction planes, surface concentrations as well as adsorption characteristics and the segregation constants, while fourier transform infra-red attenuated total reßectance (FTIR-ATR) is used to determine the protein structure vs. temperature in the liquid and solid states as well as the ice interface characteristics. All data show that AFGP do not function by the characteristic Gibbs-Thomson mechanism. While the surface coverage is similar for the AFPIII, segregation (amount in ice/amount in solution) is non-zero.

  18. Characterization of bromine-77-labeled proteins prepared using bromoperoxidase

    International Nuclear Information System (INIS)

    The halogenating enzyme bromoperoxidase, isolated from the red algae Bonnemaisonia hamifera and Penicillus capitatus, was used to catalyze the radiohalogenation of proteins at neutral pH. Human serum albumin and canine fibrinogen were halogenated as model compounds; the proteins were labeled with Br-77, produced by the 75As(α,2n)77Br reaction. For each enzyme, the essential reaction parameters (including the concentrations of hydrogen peroxide or of protein, the amount of enzyme used to catalyze the reaction, the pH of the reaction mixture, and the reaction time) were varied to obtain conditions that resulted in the highest yield of radiolabeled protein. The labeled proteins prepared with bromoperoxidase are stable with respect to loss of the radiolabel by hydrolysis and retain their biologic activity. The extension of this method to radiobromination of other types of compounds for imaging and receptor studies seems promising

  19. 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.

  20. Preparation of encapsulated proteins dissolved in low viscosity fluids

    International Nuclear Information System (INIS)

    The majority of proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. One potential approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques is to encapsulate them in a reverse micelle which is dissolved in a low viscosity fluid. Unfortunately, promising low viscosity fluids such as the short chain alkanes, supercritical carbon dioxide, and various halocarbon refrigerants all require the application of significant pressure to be kept liquefied at room temperature. Here we describe the design and use of a simple cost effective NMR tube suitable for the preparation of solutions of proteins encapsulated in reverse micelles dissolved in such fluids

  1. Antifreeze Polysaccharide Coating Study for De-icing Aircraft

    Science.gov (United States)

    Morita, Katsuaki; Sakaue, Hirotaka; Ando, Azuma; Matsuda, Yoshiyuki; Kawahara, Hidehisa

    2015-11-01

    Anti-icing or deicing of an aircraft is necessary for a safe flight operation. Mechanical processes, such as heating and deicer boot, are widely used. Deicing fluids, such as propyrene glycol and ethylene glycol, are used to coat the aircraft. However, these should be coated every time before the take-off, since the fluids come off from the aircraft while cruising. We study an antifreeze polysaccharide (AFPS) coating as a deicer for an aircraft. It is designed to coat on the aircraft without removal. Since an AFPS coating removes ice by reducing the interfacial energy, it would be an alternative way to prevent ice on the aircraft. We provide a temperature-controlled room, which can control its temperature under icing conditions (-8 and -4 °C). Ice adhesion tests are performed for AFPS coating and compared with a fundamental specimen without the coating.

  2. 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...

  3. 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)

  4. Antioxidative activity of protein hydrolysates prepared from alkaline-aided channel catfish protein isolates.

    Science.gov (United States)

    Theodore, Ann E; Raghavan, Sivakumar; Kristinsson, Hordur G

    2008-08-27

    Antioxidative activity of hydrolyzed protein prepared from alkali-solubilized catfish protein isolates was studied. The isolates were hydrolyzed to 5, 15, and 30% degree of hydrolysis using the protease enzyme, Protamex. Hydrolyzed protein was separated into hydrolysates and soluble supernatants, and both of these fractions were studied for their metal chelating ability, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and their ability to inhibit the formation of thiobarbituric acid reactive substances (TBARS) in washed tilapia muscle containing tilapia hemolysate. Both hydrolysates and supernatants were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that DPPH radical scavenging ability and reducing power of catfish protein hydrolysates decreased, whereas the ORAC value, metal chelating ability, and ability to inhibit TBARS increased, with an increase in the degree of hydrolysis. Hydrolysate samples showed higher DPPH radical scavenging ability and Fe(3+) reducing ability, and supernatant samples had higher metal chelating ability. In general, low molecular weight (MW) peptides had high ORAC values and high metal chelating ability, and high MW peptides had a higher reducing power (FRAP) and were more effective in scavenging DPPH radicals. In a washed muscle model system, the ability of catfish protein hydrolysates and their corresponding supernatants to inhibit the formation of TBARS increased with an increase in the degree of hydrolysis. PMID:18662014

  5. 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

  6. 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.

  7. Preparations and mechanical properties of low proteins radiation vulcanised natural rubber latex (RVNRL)

    International Nuclear Information System (INIS)

    Low proteins latex can be used in RVNRL preparation. Either Formulation Rll, uses single sensitiser or Formulation Rlll, uses a combination of sensitisers are useful in low proteins RVNRL preparation. However, these RVNRL formulations produce film vulcanisates of different mechanical properties and accelerated ageing resistance

  8. 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.

  9. 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)

  10. 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.

  11. 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...

  12. 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

  13. 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。

  14. Preparation, characterization and functional properties of flax seed protein isolate.

    Science.gov (United States)

    Kaushik, Pratibha; Dowling, Kim; McKnight, Stafford; Barrow, Colin J; Wang, Bo; Adhikari, Benu

    2016-04-15

    Flaxseed protein isolate (FPI) was extracted from flaxseeds, and its amino acid composition and functional properties (solubility, thermal stability, emulsifying properties and electrostatic charge density, water holding and fat absorption capacities) were determined. The highest purity of FPI (90.6%) was achieved by extraction at 60°C. FPI had a low lysine to arginine ratio of 0.25, which is desired in heart-healthy foods and infant formulas. The denaturation temperature of FPI was 105°C. FPI had the highest emulsion activity index (375.51 m(2)/g), highest emulsion stability index (179.5 h) and zeta potential (-67.4 mV) when compared to those of other commonly used proteins, such as sodium caseinate (SC), whey protein isolate (WPI), gelatin (Gel) and soy protein isolate (SPI). The average emulsion droplet size of emulsions stabilized by these proteins was in the order SCproteins. PMID:26616943

  15. 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.

  16. Biodegradability and groundwater pollutant potential of organic anti-freeze liquids used in borehole heat exchangers

    Energy Technology Data Exchange (ETDEWEB)

    Klotzbuecher, Thimo; Kappler, Andreas; Straub, Kristina L.; Haderlein, Stefan B. [Center for Applied Geosciences, Institute for Geosciences, Eberhard-Karls-University Tuebingen, Sigwartstrasse 10, D-72076 Tuebingen (Germany)

    2007-08-15

    Ground source heat pump systems are increasingly being used to exploit the energy content of shallow geothermal resources for space heating and cooling. In this study we evaluate the potential for groundwater contamination of the different organic anti-freeze compounds (ethylene glycol, propylene glycol and betaine) used in these pumps, based on a literature review of their biodegradability and the results of our own laboratory experiments on aquifer material. Ethylene and propylene glycol were found to be readily biodegradable under both oxic and anoxic conditions, without formation of toxic or persistent intermediates. Long-term groundwater contamination by the glycols is therefore not expected. Betaine is also expected to be readily biodegradable in oxic and anoxic groundwater. The potential formation of trimethylamine, an intermediate of anaerobic betaine degradation, is, however, regarded as critical due to its unpleasant odor even at very low concentrations. Additionally, betaine has the potential to complex metal ions and thus may mobilize toxic metals in groundwater. We therefore recommend that betaine not be used in borehole heat exchanger fluids. In addition to organic anti-freeze compounds such as glycols, borehole heat exchanger fluids also contain additives such as corrosion inhibitors or biocides. We demonstrate that potentially toxic additives in these fluids inhibit biodegradation of the organic anti-freeze compounds. In order to ensure environmental compatibility of borehole heat exchanger fluids, further research should be conducted on the impact of additives on subsurface microbiological activity and on groundwater quality. (author)

  17. 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.

  18. 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).

  19. 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...

  20. Preparation and evaluation of tara-modified proteins

    Science.gov (United States)

    Quebracho, a vegetable tannin, can be used to modify gelatin to produce a product that has been applied effectively as a filler in leather processing, as described in our previous report. In this ongoing study, another vegetable tannin tara is examined for its possible application in protein modifi...

  1. Thermophysical properties of starch and whey protein composite prepared in presence of organic acid and esters

    Science.gov (United States)

    Previously, we prepared starch and protein composite by reactive mixing in presence of various organic acids and found that use of these acid esters resulted in composites with good mechanical properties. In this study, concentration (% w/w) of acid citrates in the starch-protein composites were var...

  2. 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

  3. Preparation of denatured protein bone sterilized with gamma radiation

    International Nuclear Information System (INIS)

    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)

  4. Preparation and characterization of a thermoresponsive gigaporous medium for high-speed protein chromatography

    International Nuclear Information System (INIS)

    Highlights: • A high-speed thermoresponsive bioseparation medium was prepared in two steps. • Non-specific adsorption of proteins on thermoresponsive medium was greatly reduced. • Separation of proteins was achieved by only adjusting column temperature. • It was able to separate proteins at the mobile phase velocity up to 2167 cm h−1. - Abstract: A high-speed thermoresponsive medium was developed by grafting poly(N-isopropylacrylamide-co-butyl methacrylate) (P(NIPAM-co-BMA)) brushes onto gigaporous polystyrene (PS) microspheres via surface-initiated atom transfer radical polymerization (ATRP) technique, which has strong mechanical strength, good chemical stability and high mass transfer rate for biomacromolecules. The gigaporous structure, surface chemical composition, static protein adsorption, and thermoresponsive chromatographic properties of prepared medium (PS–P(NIPAM-co-BMA)) were characterized in detail. Results showed that the PS microspheres were successfully grafted with P(NIPAM-co-BMA) brushes and that the gigaporous structure was robustly maintained. After grafting, the nonspecific adsorption of proteins on PS microspheres was greatly reduced. A column packed with PS–P(NIPAM-co-BMA) exhibited low backpressure and significant thermo-responsibility. By simply changing the column temperature, it was able to separate three model proteins at the mobile phase velocity up to 2167 cm h−1. In conclusion, the thermoresponsive polymer brushes grafted gigaporous PS microspheres prepared by ATRP are very promising in ‘green’ high-speed preparative protein chromatography

  5. 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

  6. 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. PMID:24518312

  7. Preparation, characterization, and NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids

    International Nuclear Information System (INIS)

    Encapsulating a protein in a reverse micelle and dissolving it in a low-viscosity solvent can lower the rotational correlation time of a protein and thereby provides a novel strategy for studying proteins in a variety of contexts. The preparation of the sample is a key element in this approach and is guided by a number of competing parameters. Here we examine the applicability of several strategies for the preparation and characterization of encapsulated proteins dissolved in low viscosity fluids that are suitable for high performance NMR spectroscopy. Ubiquitin is used as a model system to explore various issues such as the homogeneity of the encapsulation, characterization of the hydrodynamic performance of reverse micelles containing protein molecules, and the effective pH of the water environment of the reverse micelle

  8. 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.

  9. 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

  10. 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.

  11. 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 (Pprotein complex. This method could be adopted for the production of stable iron enriched protein, an organic iron source. PMID:26593557

  12. Physicochemical characterization of protein-loaded pectin-chitosan nanoparticles prepared by polyelectrolyte complexation

    OpenAIRE

    Ahlin Grabnar, Pegi; Kristl, Julijana

    2015-01-01

    Recent advances in nanotechnology applied to proteins are directed towards safer and simpler methods of preparation, using naturally occurring polymers such as alginate, pectin and chitosan. In this study, pectin-chitosan nanoparticles (NPs) were designed by the mild process of polyelectrolyte complexation, which occurs at room temperature without using sonication or organic solvents. NPs with a mean diameter between 300 and 400 nm and 45 to 86% protein association efficiency were obtained by...

  13. 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

  14. Artificial receptor-functionalized nanoshell: facile preparation, fast separation and specific protein recognition

    International Nuclear Information System (INIS)

    This work combined molecular imprinting technology with superparamagnetic nanospheres as the core to prepare artificial receptor-functionalized magnetic nanoparticles for separation of homologous proteins. Using dopamine as a functional monomer, novel surface protein-imprinted superparamagnetic polydopamine (PDA) core-shell nanoparticles were successfully prepared in physiological conditions, which could maintain the natural structure of a protein template and achieved the development of molecularly imprinted polymers (MIPs) from one dimension to zero dimension for efficient recognition towards large biomolecules. The resultant nanoparticles could be used for convenient magnetic separation of homologous proteins with high specificity. The nanoparticles possessed good monodispersibility, uniform surface morphology and high saturation magnetization value. The bound amounts of template proteins measured by both indirect and direct methods were in good agreement. The maximum number of imprinted cavities on the surface of the bovine hemoglobin (Hb)-imprinted nanoshell was 2.21 x 1018 g-1, which well matched their maximum binding capacity toward bovine Hb. Both the simple method for preparation of MIPs and the magnetic nanospheres showed good application potential in fast separation, effective concentration and selective biosensing of large protein molecules.

  15. 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

  16. 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)

  17. 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

    Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG-gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling was...

  18. Preparative purification of antibodies with protein A-an alternative to conventional chromatography

    International Nuclear Information System (INIS)

    Protein A coated magnetic particles are used for the preparative purification of antibodies from up to 100 L cell culture supernatant. The comparison with conventional column and expanded bed chromatography results in similar yield and purity of the product but much faster separation times

  19. 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

  20. 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

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    Ace Baehaki

    2015-12-01

    Full Text Available 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–pikrilhidrazil, protein content, and molecular weight using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. The results showed that catfish protein hydrolysates prepared by papain enzyme has antioxidative activity. The highest degree of hydrolysis was 71.98% at enzyme concentration of 6%. Based on the DPPH scavenging method catfish protein hydrolysates has the antioxidative activity with the value 37.85-67.62%. The protein content of catfish protein hydrolysates were 20.86-54.47 mg/ml. The molecular weight of catfish protein hydrolyzates were 11.90-65.20 kDa.

  3. [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

  4. 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.

  5. Review of preparative and analytical procedures for the study of proteins in grape juice and wine.

    Science.gov (United States)

    Le Bourse, D; Jégou, S; Conreux, A; Villaume, S; Jeandet, P

    2010-05-14

    Proteins have a great influence on wine quality as they exhibit a various range of properties. In fact, they are involved among others in white wine turbidity, organoleptic characteristics and foam formation in sparkling wines. These compounds could also be of major interest for varietal differentiation, regarding wine authentication and traceability issues. To provide a better understanding of the role played by these biomolecules in wine processing and explore their potential applications, there is a manifest need for the quantification and characterization of each individual one in terms of sequence, structure and intrinsic and functional properties. We thus present an overview of preparative and analytical methods for the study of proteins in grape juices and wines, from routine techniques to dedicated methodologies. They include sample preparation with chromatographic methods for the purification and identification of proteins, quantification protocols and characterization procedures such as electrophoretic techniques, immunological methods, sequencing, mass spectrometry, physico-chemical and structural analyses, and so on. We expose advantages and limits of each technique and focus on the different but complementary information they can provide. Despite the past years advances in the field proteins identification, the elucidation of the full protein profile for grape juices and wines remains strenuous. Their interactions with other wine compounds make the challenge even harder. We therefore emphasize the requirement of the techniques to be refined and suggest the developments to be expected. PMID:20441863

  6. 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.

  7. Optimization of proteomic sample preparation procedures for comprehensive protein characterization of pathogenic systems

    Energy Technology Data Exchange (ETDEWEB)

    Brewer, Heather M.; Norbeck, Angela D.; Adkins, Joshua N.; Manes, Nathan P.; Ansong, Charles; Shi, Liang; Rikihisa, Yasuko; Kikuchi, Takane; Wong, Scott; Estep, Ryan D.; Heffron, Fred; Pasa-Tolic, Ljiljana; Smith, Richard D.

    2008-12-19

    The elucidation of critical functional pathways employed by pathogens and hosts during an infectious cycle is both challenging and central to our understanding of infectious diseases. In recent years, mass spectrometry-based proteomics has been used as a powerful tool to identify key pathogenesis-related proteins and pathways. Despite the analytical power of mass spectrometry-based technologies, samples must be appropriately prepared to characterize the functions of interest (e.g. host-response to a pathogen or a pathogen-response to a host). The preparation of these protein samples requires multiple decisions about what aspect of infection is being studied, and it may require the isolation of either host and/or pathogen cellular material.

  8. Single-input divergent flow IEF for preparative analysis of proteins

    Czech Academy of Sciences Publication Activity Database

    Šťastná, Miroslava; Šlais, Karel

    2008-01-01

    Roč. 29, č. 22 (2008), s. 4503-4507. ISSN 0173-0835 R&D Projects: GA AV ČR IAAX00310701; GA ČR GA203/06/1179 Institutional research plan: CEZ:AV0Z40310501 Keywords : continuous divergent flow * IEF * preparative protein analysis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.509, year: 2008

  9. 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%.

  10. Nanomaterials for efficiently lowering the freezing point of anti-freeze coolants.

    Science.gov (United States)

    Hong, Haiping; Zheng, Yingsong; Roy, Walter

    2007-09-01

    In this paper, we report, for the first time, the effect of the lowered freezing point in a 50% water/50% anti-freeze coolant (PAC) or 50% water/50% ethylene glycol (EG) solution by the addition of carbon nanotubes and other particles. The experimental results indicated that the nano materials are much more efficient (hundreds fold) in lowering the freezing point than the regular ionic materials (e.g., NaCl). The possible explanation for this interesting phenomenon is the colligative property of fluid and relative small size of nano material. It is quite certain that the carbon nanotubes and metal oxide nano particles could be a wonderful candidate for the nano coolant application because they could not only increase the thermal conductivity, but also efficiently lower the freezing point of traditional coolants. PMID:18019146

  11. 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

  12. 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。

  13. Preparation and Characterization of a Novel Chimeric Protein VEGI-CTT in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jiping Cai

    2008-08-01

    Full Text Available Vascular endothelial cell growth inhibitor (VEGI is a recently identified antiangiogenic cytokine that belongs to the TNF superfamily, and could effectively inhibit endothelial cell proliferation and angiogenesis. Synthetic peptide CTT (CTTHWGFTLC has been found to suppress invasion and migration of both tumor and endothelial cells by potent and selective inhibition of MMP-2 and MMP-9. To prepare chimeric protein VEGI-CTT for more potent antitumor therapy, the recombinant expression vector pET-VEGI-CTT was constructed. This fusion protein was expressed in inclusion bodies in E. coli BL21 (DE3, and was refolded and purified by immobilized metal affinity chromatography using His-tag. Purified VEGI-CTT protein was characterized by proliferation assays of the endothelial cells and casein degradation assay in vitro. The results demonstrated that chimeric protein VEGI-CTT had a potent activity of antiangiogenesis through inhibiting the proliferation of endothelial cells, and could effectively reduce the activity of MMP-2 and MMP-9. The preliminarily in vivo study demonstrated that chimeric protein VEGI-CTT had more potent antitumor activity than VEGI and/or CTT peptide against CA46 human lymphoma xenografts in nude mice. Thus, these facts that are derived from the present study suggest that the chimeric protein VEGI-CTT may be used for tumor therapy in the future.

  14. 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

  15. Properties and oxidative stability of emulsions prepared with myofibrillar protein and lard diacylglycerols.

    Science.gov (United States)

    Diao, Xiaoqin; Guan, Haining; Zhao, Xinxin; Chen, Qian; Kong, Baohua

    2016-05-01

    The objective of this study was to investigate the emulsifying properties and oxidative stability of emulsions prepared with porcine myofibrillar proteins (MPs) and different lipids, including lard, glycerolized lard (GL) and purified glycerolized lard (PGL). The GL and PGL emulsions had significantly higher emulsifying activity indices and emulsion stability indices than the lard emulsion (Pemulsion presented smaller droplet sizes, thus decreasing particle aggregation and improving emulsion stability. The static and dynamic rheological observations of the emulsions showed that the emulsions had pseudo-plastic behavior, and the PGL emulsion presented a larger viscosity and a higher storage modulus (G') and loss modulus (G'') compared with the other two emulsions (Pemulsions with PGL, GL and lard (Pemulsions prepared with MPs. PMID:26775153

  16. 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

  17. Preparation and Characterization of P(MAA-g-EG) Nanospheres for Protein Delivery Applications

    International Nuclear Information System (INIS)

    Novel complexation hydrogel nanospheres of poly(methacrylic acid-grafted-poly(ethylene glycol)) (P(MAA-g-EG)) were prepared by dispersion polymerization to be used for protein delivery applications. Polymerization was conducted in solvents such as deionized water, ethanol/water, sodium hydroxide, hydrochloric acid, and acetic acid solutions. When polymerizing in deionized water we produced nanospheres without agglomeration. Photon correlation spectroscopy studies revealed that the nanospheres possessed a narrow particle size distribution and the size was inversely proportional to the concentration of poly(ethylene glycol) incorporated in the monomer mixture. These nanospheres exhibited pH-sensitivity comparable to that encountered in hydrogel films with the same composition. The composition of the nanospheres was investigated by transmission Fourier transform infrared spectroscopy. The comparison between hydrogel films and nanospheres with the same monomer composition revealed that nanospheres possessed similar spectral characteristics than hydrogel films prepared by the same techniques. These nanospheres could be used for calcitonin release under physiological conditions

  18. 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.

  19. A fully automated plasma protein precipitation sample preparation method for LC-MS/MS bioanalysis.

    Science.gov (United States)

    Ma, Ji; Shi, Jianxia; Le, Hoa; Cho, Robert; Huang, Judy Chi-jou; Miao, Shichang; Wong, Bradley K

    2008-02-01

    This report describes the development and validation of a robust robotic system that fully integrates all peripheral devices needed for the automated preparation of plasma samples by protein precipitation. The liquid handling system consisted of a Tecan Freedom EVO 200 liquid handling platform equipped with an 8-channel liquid handling arm, two robotic plate-handling arms, and two plate shakers. Important additional components integrated into the platform were a robotic temperature-controlled centrifuge, a plate sealer, and a plate seal piercing station. These enabled unattended operation starting from a stock solution of the test compound, a set of test plasma samples and associated reagents. The stock solution of the test compound was used to prepare plasma calibration and quality control samples. Once calibration and quality control samples were prepared, precipitation of plasma proteins was achieved by addition of three volumes of acetonitrile. Integration of the peripheral devices allowed automated sequential completion of the centrifugation, plate sealing, piercing and supernatant transferral steps. The method produced a sealed, injection-ready 96-well plate of plasma extracts. Accuracy and precision of the automated system were satisfactory for the intended use: intra-day and the inter-day precision were excellent (C.V.<5%), while the intra-day and inter-day accuracies were acceptable (relative error<8%). The flexibility of the platform was sufficient to accommodate pharmacokinetic studies of different numbers of animals and time points. To the best of our knowledge, this represents the first complete automation of the protein precipitation method for plasma sample analysis. PMID:18226589

  20. 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

  1. 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.

  2. Research on raw material correlation and soy protein derivatives used for meat preparations in membrane

    OpenAIRE

    Constantin Moldovanu; Cornel Laslo

    2010-01-01

    Since the Romanian literature is pretty low quality data on the influence of hygienic quality ofraw materials and preparations of meat, in our research we intend to pursue these issues in pork andbeef quality I and II. Bacterial load of raw meat products brought in meat technology varies dependingon its quality. Thus, the total number is 44,640 germs / g beef I and 86,640 / g beef II. The number ofcoliform bacteria is 329 / g beef I and 420 / g beef II. Protein derivatives stands at a higher ...

  3. Modification of certain functional properties of protein preparations depending on the introduced technological treatment

    International Nuclear Information System (INIS)

    The paper shows the results of the work on the possibilities for the application of certain methods used for the improvement of quality of casein preparations. The presence of proteolytic and lipolytic enzymes of bacterial origin caused the undesirable changes of functional properties of high-protein products. Several technological treatments were applied, i.e. thermization of raw milk, thermization of casein grain, gamma-irradiation of casein and extrusion of casein. The microbiological quality of the product and the changes in viscosity of casein solutions during the storage, were evaluated. The high effectiveness of thermization and extruzion processes, was stated

  4. 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).

  5. 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.

  6. 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

  7. 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

  8. 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 ...

  9. 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.

  10. 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...

  11. 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.

  12. 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

  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. Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes.

    Science.gov (United States)

    Near, Thomas J; Dornburg, Alex; Kuhn, Kristen L; Eastman, Joseph T; Pennington, Jillian N; Patarnello, Tomaso; Zane, Lorenzo; Fernández, Daniel A; Jones, Christopher D

    2012-02-28

    The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6-5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity. PMID:22331888

  15. Solid nanotubes comprising alpha-Fe2O3 nanoparticles prepared from ferritin protein.

    Science.gov (United States)

    Qu, Xue; Kobayashi, Nao; Komatsu, Teruyuki

    2010-03-23

    Solid nanotubes comprising alpha-Fe2O3 nanoparticles were prepared from iron-storage protein ferritin. Their structure, magnetic properties, and photocatalytic activities were characterized. The initial ferritin nanotube precursors were fabricated using alternating layer-by-layer depositions of poly-L-arginine (PLA) and ferritin into a track-etched polycarbonate membrane (pore diameter, 400 nm) with subsequent dissolution of the template. The obtained uniform cylinders of (PLA/ferritin)3 (outer diameter, 410 +/- 14 nm) were calcinated at 500 degrees C under air, yielding reddish-brown iron oxide nanotubes. The one-dimensional hollow structure remained perfect, but its diameter, wall thickness, and maximum length were markedly diminished. Disappearance of the protein shell and the PLA layers were confirmed using IR and EDX spectroscopy. Subsequent SEM, TEM, and XPS measurements showed that the tubular walls comprise fine alpha-Fe2O3 nanoparticles with a 5 nm diameter. These alpha-Fe2O3 nanotubes demonstrated superparamagnetic properties with a blocking temperature of 37 K and efficient photocatalytic activity for degradation of 4-chlorophenol. PMID:20166700

  16. 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.

  17. Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location.

    Science.gov (United States)

    Liao, Shu Y; Lee, Myungwoon; Wang, Tuo; Sergeyev, Ivan V; Hong, Mei

    2016-03-01

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105-160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes. PMID:26873390

  18. 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...

  19. 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...

  20. 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...

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

    OpenAIRE

    Zheng Xiaojuan; Zhang Xin; Zhou Jiyong; Wu Yongping; Jiang Xuetao; Shi Lixue; Yin Wei; Wang Junhua

    2009-01-01

    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 viru...

  2. 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

  3. Effects of anti-freeze concentration in the engine coolant on the cavitation temperature of a water pump

    International Nuclear Information System (INIS)

    Improvements in engine-manufacturing technology have gradually increased the thermal efficiencies of engines as well as the burning temperature and pressure of fuels within the cylinders. Accordingly, greater heat dissipation are required. However, the volume of the radiators is constrained by the configuration of the engines, leading to excessive internal resistance in the engine-cooling system. Therefore, water pumps in engines are prone to cavitation, and air bubbles are likely to permeate into the anti-freeze, thereby severely reducing the performance, reliability and service life of the engines. Ethylene glycol (EG) is added to the radiator of some vehicles in cold areas to reduce the solidification point of the coolant and prevent freezing. This study probes the effects of the percentage of anti-freeze added to the cooling water in a water pump in an engine on the water-supply capability and cavitation temperature, whether air or burnt gas is present in the system. The results of this study have revealed that engines have a higher tolerance to air bubbles at lower rates of rotation. At a given fixed rotational speed, the tolerable cavitation temperature of an engine's water pump will fall slowly as the amount of air bubbles increases

  4. 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

  5. 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

  6. Preparation of HSA microspheres in a one-step thermal denaturation of protein aerosol carried in gas-medium

    International Nuclear Information System (INIS)

    A simple, one-step method of preparation of human albumin microspheres by thermal denaturation of protein aerosol in a gas medium is described. These microspheres were easily labelled with technetium-99m and iodine-131, and were characterized by short biological clearance and high lung uptake. (orig.)

  7. 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.

  8. 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.

  9. 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.

  10. Target protein separation and preparation by free-flow electrophoresis coupled with charge-to-mass ratio analysis.

    Science.gov (United States)

    Shen, Qiao-Yi; Guo, Chen-Gang; Yan, Jian; Zhang, Qiang; Xie, Hai-Yang; Jahan, Sharmin; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2015-06-01

    Herein, a novel strategy was developed to separate and prepare target protein from complex sample by free-flow electrophoresis (FFE), which mainly based on the charge-to-mass ratio (C/M) analysis of proteins. The C/M values of three model proteins, namely Cytochrome C (Cyt C), myoglobin (Mb) and bovine serum albumin (BSA) were analyzed under different pH and the separation of these proteins was predicted by CLC Protein Workbench software. Series of experiments were performed to validate the proposed method. The obtained data showed high accordance with our prediction. In addition, the chamber buffer (CB) of FFE system was optimized to improve the resolution of separation. Meanwhile, in order to evaluate the analytical performance of the proposed method, Cyt C was extracted from swine heart and further separated by FFE based on C/M analysis. Results showed that Cyt C was completely separated from the crude sample and a purity of 96.9% was achieved. The activity of prepared Cyt C was 98.3%, which indicate that the proposed method is promising in a wide variety of research areas where the native properties of proteins should be maintained for downstream analysis. PMID:25890440

  11. 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.

  12. “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 ...

  13. Preparation of some (1 goes to 6)-linked disaccharides, and their derivatives suitable for protein modification.

    Science.gov (United States)

    Lee, R T; Lee, Y C

    1982-02-16

    Synthetic methods for the preparation of per-O-acetylated, (1 goes to 6)-linked disaccharides containing either a D-galactose or a D-glucose residue at the reducing end are described. In these methods, 1,2,3,4-tetra-O-acetyl-6-O-trityl-beta-D-glucopyranose was first converted into 1,2,3,4-tetra-O-acetyl-beta-D-glucopyranose (1) by rapid treatment with 90% trifluoroacetic acid, followed by rapid isolation designed to minimize O-acyl migration. Disaccharides acid, followed by rapid isolation designed to minimize )-acyl migration. Disaccharides were formed by glycosylation of 1 or 1,2:3,4-di-O-isopropylidene-D-galactopyranose with per-O-acetylglycosyl halides. Isopropylidene groups in the resulting disaccharide, if present were removed, and the disaccharide was per-O-acetylated. Per-O-acetylated beta-Gal-(1 goes to 6)-Glc and beta-GlcNAc-(1 goes to 6)-Gal, and a mixture of per-O-acetylated alpha-Gal-(1 goes to 6)-Gal and beta-Gal-(1 goes to 6)-Gal (in the ratio of 3:7) were thus obtained. The per-O-acetylated Gal-(1 goes to 6)-Gal disaccharides were converted, by a reaction sequence previously reported, into (2,2-dimethoxyethyl)aminocarbonylmethyl 1-thio-beta-D-glycosides, which could then be coupled to proteins via reductive alkylation. For the anomeric mixture of per-O-acetylated Gal-(1 goes to 6(-Gal, conversion into the corresponding 1-thioglycoside permitted resolution of the isomers by chromatography on silica gel. When disaccharides, as borate complexes, were chromatographed on a column of a strong, anion-exchange resin, all of the (1 goes to 6)-linked disaccharides of neutral sugars tested (including melibiose) were eluted later than analogous disaccharides having other linkages, and also later than any neutral monosaccharides. PMID:7060054

  14. Preparation and in vivo evaluation of a novel stabilized linker for 211At labeling of protein

    International Nuclear Information System (INIS)

    Significant improvement of in vivo stability of 211At-labeled radioimmunoconjugates achieved upon employment of a recently reported new linker, succinimidyl N-2-(4-[211At]astatophenethyl)succinamate (SAPS), prompted additional studies of its chemistry. The 211At radiolabeling of succinimidyl N-2-(4-tributylstannylphenethyl)succinamate (1) was noted to decline after storage at -15oC for greater than 6 months. Compound 1 was found to degrade via a ring closure reaction with the formation of N-2-(4-tributylstannylphenethyl)succinimide (3), and a modified procedure for the preparation of 1 was developed. The N-methyl structural analog of 1, succinimidyl N-2-(4-tributylstannylphenethyl)-N-methyl succinamate (SPEMS), was synthesized to investigate the possibility of improving the stability of reagent-protein linkage chemistry. Radiolabeling of SPEMS with 211At generates succinimidyl N-2-(4-[211At]astatophenethyl)-N-methyl succinamate (Methyl-SAPS), with yields being consistent for greater than 1 year. Radiolabelings of 1 and SPEMS with 125I generated succinimidyl N-2-(4-[125I]iodophenethyl)succinamate (SIPS) and succinimidyl N-2-(4-[125I]iodophenethyl)-N-methyl succinamate (Methyl-SIPS), respectively, and showed no decline in yields. Methyl-SAPS, SAPS, Methyl-SIPS and SIPS were conjugated to Herceptin for a comparative assessment in LS-174T xenograft-bearing mice. The conjugates of Herceptin with Methyl-SAPS or Methyl-SIPS demonstrated immunoreactivity equivalent to if not superior to the SAPS and SIPS paired analogs. The in vivo studies also revealed that the N-methyl modification resulted in a superior statinated product

  15. Growth and metabolic responses in low-birth-weight infants fed human milk fortified with human milk protein or with a bovine milk protein preparation.

    Science.gov (United States)

    Moro, G E; Minoli, I; Fulconis, F; Clementi, M; Räihä, N C

    1991-08-01

    Unfortified human milk does not normally provide enough protein to secure maximal growth in low-body-weight (LBW) infants. Due to the practical difficulties in obtaining human milk protein (HMP), a bovine milk protein preparation (BMP) was designed by computer calculation to contain as close as possible the amino acid composition of the nutritionally available human milk proteins. Twenty-one AGA, LBW infants (BW of 1,180 to 1,600 g, GA of 27 to 33 weeks) were randomly assigned to be fed HM enriched either with HMP (9 infants) or BMP (12 infants). When full volume intake (170 ml/kg/day) was reached, the protein intakes were 3.6 +/- 0.5 and 3.3 +/- 0.3 g/kg/day, respectively, in the two diet groups. During the study period of 24 days, the infants achieved intrauterine or better weight gains: 32.9 +/- 3.3 g/day (17.7 +/- 1.9 g/kg/day) in the HMP group and 34.7 +/- 7.3 g/day (18.3 +/- 3.5 g/kg/day) in the BMP group. Serum urea nitrogen, acid-base status, and albumin values were normal and similar in both groups of infants. Plasma concentrations of total essential and total amino acids at the end of the study were 3,999 and 1,539 mumol/L and 3,899 and 1,422 mumol/L in the HMP and the BMP groups, respectively. The concentrations of all individual plasma amino acids were similar in both feeding groups. These results show that feeding human milk fortified with a modified bovine milk protein preparation produces satisfactory growth and a plasma amino acid profile similar to that found in LBW infants fed exclusively human milk protein at similar intakes. PMID:1941407

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

    Science.gov (United States)

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

    2015-04-27

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

  17. 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)

  18. 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.

  19. 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 ...

  20. 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

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

    International Nuclear Information System (INIS)

    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

  2. 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.

  3. Preparation of scaffolds from human hair proteins for tissue-engineering applications

    International Nuclear Information System (INIS)

    Human hair proteins were isolated and purified for the fabrication of tissue-engineering scaffolds. Their cellular compatibility was studied using NIH3T3 mice fibroblast cells. The proteins were characterized using FTIR spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis for molecular weights and two-dimensional polyacrylamide gel electrophoresis for their isoelectric points (pIs). The molecular weights of keratins were in the range of 40-60 kilo-Daltons (kDa) and of matrix proteins were in the range of 15-30 kDa. The pIs of keratins were found to be in the range of 4.5-5.3. Sponges of the proteins were formed by lyophilization. Scanning electron microscopy was performed to examine the surface. Swelling studies were carried out in phosphate buffer saline at physiological pH 7.4. The hydrophilic character of the protein surface was studied by determining an average contact angle, which came to be 370. The wells of tissue culture plates were coated with these proteins for studying the attachment and morphology of the cells. The protein detachment study was done to ensure the adsorption of proteins on the wells until the completion of the experiments. The cellular growth on a protein-coated surface showed three-dimensional 'bulged' morphology due to cell-cell and cell-matrix contacts. The sponges of human hair proteins supported more cells for a longer period than control. The morphology and cell proliferation studies exhibited by NIH3T3 cells on these proteins have shown their potential to be used as tissue-engineering scaffolds with better cell-cell contacts and leucine-aspartic acid-valine (LDV)-mediated cell-matrix interactions

  4. 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

  5. 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°.

  6. 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°

  7. 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)

  8. Preparation of 125I-protein A usable for up to 10 months in immunoassays

    International Nuclear Information System (INIS)

    Chloramine-T iodination of protein A from Staphylococcus aureus and gel electrophoretic purification of the iodination mixture results in a stable tracer of high specific and functional activity. Following repeated gel electrophoresis of the tracer only a single component was observed. The specific activity of the 125I-protein A was between 30 and 55 μCi/μg. The binding of 125I-protein A to rabbit immunoglobulin exceeded 90% and the tracer competed effectively with unlabelled protein A in binding to cells incubated with sera containing surface antibodies. Storage of the tracer for up to 46 weeks resulted in a moderate decrease in maximal binding to immunoglobulin (from 91% to 64%), in TCA precipitable radioactivity (from 97% to 80%) and an approx. 30% decrease in the ability to detect cell bound immunoglobulin. It is concluded that gel electrophoretic purification of 125I-protein A produces a tracer with a very long shelf life. (Auth.)

  9. 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

  10. 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.

  11. 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

  12. Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis

    OpenAIRE

    Aebersold, Ruedi H.; Teplow, David B.; Hood, Leroy E .; Kent, Stephen B. H.

    1986-01-01

    We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restric...

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

    OpenAIRE

    He, Wei; Tan, Yanan; Tian, Zhiqiang; Chen, Lingyun; Hu, Fuqiang; Wu, Wei

    2011-01-01

    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, β-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-dip...

  14. 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

  15. 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

  16. 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

  17. Preparation and characterization of water/oil/water emulsions stabilized by polyglycerol polyricinoleate and whey protein isolate.

    Science.gov (United States)

    Mun, Saehun; Choi, Yongdoo; Rho, Shin-Joung; Kang, Choon-Gil; Park, Chan-Ho; Kim, Yong-Ro

    2010-03-01

    In this study we tried to prepare stable water-in-oil-in-water (W/O/W) emulsions using polyglycerol polyricinoleate (PGPR) as a hydrophobic emulsifier and whey protein isolate (WPI) as a hydrophilic emulsifier. At first, water-in-oil (W/O) emulsions was prepared, and then 40 wt% of this W/O emulsion was homogenized with 60 wt% aqueous solution of different WPI contents (2, 4, and 6 wt% WPI) using a high-pressure homogenizer (14 and 22 MPa) to produce W/O/W emulsions. The mean size of final W/O/W droplets ranged from 3.3 to 9.9 microm in diameter depending on the concentrations of PGPR and WPI. It was shown that most of the W/O/W droplets were small (oil droplets (d > 20 microm) was also occasionally observed. W/O/W emulsions prepared at the homogenization pressure of 22 MPa had a larger mean droplet size than that prepared at 14 MPa, and showed a microstructure consisting of mainly approximately 6 to 7-microm droplets. When a water-soluble dye PTSA as a model ingredient was loaded in the inner water phase, all W/O/W emulsions showed a high encapsulation efficiency of the dye (>90%) in the inner water phase. Even after 2 wk of storage, >90% of the encapsulated dye still remained in the inner water phase; however, severe droplet aggregation was observed at relatively high PGPR and WPI concentrations. PMID:20492231

  18. 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...

  19. 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.

  20. 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%.

  1. 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....

  2. 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

  3. 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

  4. 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.; Meldal, M

    1998-01-01

    Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish...... the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluorescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluorescent Abz...... group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA-beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide...

  5. 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.

  6. 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)

  7. 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...

  8. Preparation of 125I-protein A usable for up to 10 months in immunoassays

    DEFF Research Database (Denmark)

    Dyrberg, T; Billestrup, Nils

    1984-01-01

    Chloramine-T iodination of protein A from Staphylococcus aureus and gel electrophoretic purification of the iodination mixture results in a stable tracer of high specific and functional activity. Following repeated gel electrophoresis of the tracer only a single component was observed. The specif...

  9. Partial Molecular Characterization of Arctium minus Aspartylendopeptidase and Preparation of Bioactive Peptides by Whey Protein Hydrolysis.

    Science.gov (United States)

    Cimino, Cecilia V; Colombo, María Laura; Liggieri, Constanza; Bruno, Mariela; Vairo-Cavalli, Sandra

    2015-08-01

    In this article, we report the cloning of an aspartic protease (AP) from flowers of Arctium minus (Hill) Bernh. (Asteraceae) along with the use of depigmented aqueous flower extracts, as a source of APs, for the hydrolysis of whey proteins. The isolated cDNA encoded a protein product with 509 amino acids called arctiumisin, with the characteristic primary structure organization of typical plant APs. Bovine whey protein hydrolysates, obtained employing the enzyme extracts of A. minus flowers, displayed inhibitory angiotensin-converting enzyme (ACE) and antioxidant activities. Hydrolysates after 3 and 5 h of reaction (degree of hydrolysis 2.4 and 5.6, respectively) and the associated peptide fraction with molecular weight below 3 kDa were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization/time of flight mass spectrometry, and reverse phase-high-performance liquid chromatography. The results obtained in this study demonstrate the viability of using proteases from A. minus to increase the antioxidant and inhibitory ACE capacity of whey proteins. PMID:25575270

  10. 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

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

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus; Christiansen, Helle; Schmidt, Steffen; Mollenhauer, Jan

    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 into...

  12. Design and characterization of controlled-release edible packaging films prepared with synergistic whey-protein polysaccharide complexes.

    Science.gov (United States)

    Liu, Fei; Jiang, Yanfeng; Du, Bingjian; Chai, Zhi; Jiao, Tong; Zhang, Chunyue; Ren, Fazheng; Leng, Xiaojing

    2013-06-19

    This paper describes an investigation into the properties of a doubly emulsified film incorporated with protein-polysaccharide microcapsules, which serves as a multifunctional food packaging film prepared using common edible materials in place of petroleum--based plastics. The relationships between the microstructural properties and controlled release features of a series of water-in-oil-in-water (W/O/W) microcapsulated edible films prepared in thermodynamically incompatible conditions were analyzed. The hydrophilic riboflavin (V(B2)) nano-droplets (13-50 nm) dispersed in α-tocopherol (V(E)) oil phase were embedded in whey protein-polysaccharide (WPs) microcapsules with a shell thickness of 20-56 nm. These microcapsules were then integrated in 103 μm thick WPs films. Different polysaccharides, including gum arabic (GA), low-methoxyl pectin (LMP), and κ-carrageenan (KCG), exhibited different in vitro synergistic effects on the ability of both films to effect enteric controlled release of both vitamins. GA, which showed a strong emulsifying ability, also showed better control of V(E) than other polysaccharides, and the highly charged KCG showed better control of V(B2) than GA did. PMID:23718814

  13. Preparation and characterization of PEM-coated alginate microgels for controlled release of protein

    International Nuclear Information System (INIS)

    In this study, calcium-alginate microgels coated with a polyelectrolyte multilayer (PEM) were fabricated as a controlled-release system. This system was constructed via an electrostatic droplet generation technique followed by a layer-by-layer (LbL) self-assembly technique. The electrostatic droplet generation technique was reported as an easy method of preparing microgels, due to their mild preparation conditions and ability to preserve the biological activity of the encapsulated drugs. With the LbL self-assembly technique, the PEM could be fabricated on the microgels attributed to the electrostatic attraction between positive-charged chitosan (Chi) and negative-charged dextran sulfate (Dex). The properties of the prepared microgels were investigated using dynamic laser scattering (DLS), scanning electron microscopy (SEM), x-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) spectrum and zeta potential analyzer. In vitro release study indicated that the initial burst release of the bovine serum albumin (BSA) from PEM-coated microgels was less compared to the uncoated microgels (19% versus 31% in 24 h). In addition, the sustained release of BSA from the PEM-coated microgels was recorded up to 1 month without any damage to BSA integrity. Thus, our results demonstrated that the PEM-coated microgels not only prolonged the release time, but also relieved the initial burst problem to some degree and preserved the biological activity of the encapsulated drugs. Moreover, the release rate of BSA could be regulated by controlling the number of deposited layers. In conclusion, this study presented an easy yet effective method for the controlled, sustained release of biological macromolecules. (paper)

  14. 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. PMID:26104337

  15. 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.

  16. 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...

  17. Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: Preparation and study of their structure

    Czech Academy of Sciences Publication Activity Database

    Mašek, J.; Bartheldyová, E.; Korvasová, Z.; Škrabalová, J.; Koudelka, Š.; Kulich, P.; Kratochvílová, Irena; Miller, A. D.; Ledvina, Miroslav; Raška, M.; Turánek, J.

    2011-01-01

    Roč. 408, č. 1 (2011), s. 95-104. ISSN 0003-2697 R&D Projects: GA ČR(CZ) GAP304/10/1951 Institutional research plan: CEZ:AV0Z10100520; CEZ:AV0Z40550506 Keywords : liposome * proteoliposome * detergent removal method * recombinant protein * metallochelatation * AF microscopy * TEM * dynamic light scattering Subject RIV: CE - Biochemistry Impact factor: 2.996, year: 2011

  18. Divergent flow isoelectric focusing: fast and efficient method for protein sample preparation in mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Mazanec, Karel; Bobálová, Janette; Šlais, Karel

    Zagreb : Croatian Society of Chemical Engineers, 2008 - (Šegudovič, N.). s. 80 ISBN 978-953-6894-36-9. [International Symposium on Separation Science /14./. 30.09.2008-03.10.2008, Primošten] R&D Projects: GA MŠk 1M0570; GA AV ČR IAAX00310701 Institutional research plan: CEZ:AV0Z40310501 Keywords : Isoelectric focusing * proteins * MALDI-TOF/TOF MS Subject RIV: CB - Analytical Chemistry, Separation

  19. 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.

  20. 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...

  1. 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

  2. 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.

  3. 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.

  4. Optimization of the Preparation of Fish Protein Anti-Obesity Hydrolysates Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Jinju Wang

    2013-02-01

    Full Text Available The enzymatic condition for producing the anti-obesity hydrolysates from fish water-soluble protein was optimized with the aid of response surface methodology, which also derived a statistical model for experimental validation. Compared with neutral protease, papain and protamex, the porcine pancreas lipase inhibitory rate of hydrolysates from fish water-soluble protein was higher with alkaline protease. Results showed that the model terms were significant, the terms of lack of fit were not significant, and the optimal conditions for the hydrolysis by alkaline protease were initial pH 11, temperature 39 °C, enzyme dosage 122 U/mL and 10 h of hydrolysis time. Under these conditions, the porcine pancreas lipase and the α-amylase inhibitory rate could reach 53.04% ± 1.32% and 20.03 ± 0.89%, while predicted value were 54.63% ± 1.75%, 21.22% ± 0.70%, respectively. In addition, Lineweaver-Burk plots showed noncompetitive inhibition. The Ki value calculated was 84.13 mg/mL. These results demonstrated that fish water-soluble protein could be used for obtaining anti-obesity hydrolysates.

  5. [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

  6. 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.

  7. 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

  8. 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)

  9. Factors affecting the oxidative stability of omega-3 emulsions prepared with milk proteins

    DEFF Research Database (Denmark)

    Horn, Anna Frisenfeldt; Nielsen, Nina Skall; Jacobsen, Charlotte

    Omega-3 fatty acids are prone to lipid oxidation due to their unsaturated nature. In oil-in-water emulsions, lipid oxidation is expected to be initiated at the oil-water interface. The properties of the emulsifier used and the structure at the interface are therefore expected to be of great...... importance for the resulting oxidation. This presentation will give an overview of parameters that are expected to change the properties and structure of milk protein components at the interface of 10% fish oil-in-water emulsions. Results from three different studies will be included. The first study...

  10. Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

    International Nuclear Information System (INIS)

    T. cruzi TcNDPK1 was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl2, 20% PEG 3350. Data were collected to 3.5 Å resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 Å. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. Preparation, characterization, and immunogenicity of meningococcal lipooligosaccharide-derived oligosaccharide-protein conjugates.

    Science.gov (United States)

    Gu, X X; Tsai, C M

    1993-05-01

    A method was developed for coupling carboxylic acid-containing oligosaccharides (OS) to proteins. An OS was isolated from Neisseria meningitidis group A strain A1 lipooligosaccharide (LOS). This LOS has no human glycolipid-like lacto-N-neotetraose structure and contains multiple immunotypes, including L8, found in group B and C strains. The carboxylic acid at 2-keto-3-deoxyoctulosonic acid of the OS was linked through adipic acid dihydrazide to tetanus toxoid. The molar ratio of the OS to tetanus toxoid in three conjugates ranged from 11:1 to 19:1. The antigenicity of the OS was conserved in these conjugates, as measured by an enzyme-linked immunosorbent assay (ELISA) and an inhibition ELISA with polyclonal and monoclonal antibodies to A1 LOS. These conjugates induced immunoglobulin G antibodies to A1 LOS in mice and rabbits. The immunogenicity of the conjugates in rabbits was enhanced by use of monophosphoryl lipid A plus trehalose dimycolate as an adjuvant. The resulting rabbit antisera cross-reacted with most of 12 prototype LOSs and with LOSs from two group B disease strains, 44/76 and BB431, in an ELISA and in Western blotting (immunoblotting), which revealed a 3.6-kDa reactive band in these LOSs. The rabbit antisera showed bactericidal activity against homologous strain A1 and heterologous strains 44/76 and BB431. These results indicate that conjugates derived from A1 LOS can induce antibodies against many LOS immunotypes from different organism serogroups, including group B. OS-protein conjugates derived from meningococcal LOSs may therefore be candidate vaccines to prevent meningitis caused by meningococci. PMID:8478076

  16. Preparation of ferrous chelate of hairtail (Trichiurus haumela) protein hydrolysate (Fe(II)-HPH) and its antibacterial activity

    Science.gov (United States)

    Lin, Huimin; Zhang, Bin; Yu, Tian; Deng, Shanggui

    The preparation of a ferrous chelate of hairtail (Trichiurus haumela) protein hydrolysate (Fe(II)-HPH) and its antibacterial activity were studied. The optimal conditions of hydrolysis by papain and ferrous chelation were obtained by single-factor experiments and orthogonal test, with the antibacterial activities as the index. In addition, the antibacterial activity of Fe(II)-HPH was evaluated using the Plackett-Burman design. The orthogonal test results showed that Fe(II)-HPH had an antibacterial activity of 98.3% under a temperature of 40 °C at pH 6.5 for an enzymolysis duration of eight hours in the presence of 20,000 U/g of enzyme. The Plackett-Burman design analysis showed that the three most significant factors (P < 0.05) influencing the antibacterial activity of Fe(II)-HPH were pH, the concentration (mg/mL), and presence of magnesium sulfate.

  17. 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.

  18. 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.

  19. 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.

  20. Enhanced inactivation of avian influenza virus at −20°C by disinfectants supplemented with calcium chloride or other antifreeze agents

    OpenAIRE

    Guan, Jiewen; Chan, Maria; Brooks, Brian W.; Rohonczy, Elizabeth

    2015-01-01

    Avian influenza outbreaks have occurred during winter months, and effective disinfection of poultry premises at freezing temperatures is needed. The commercial disinfectants Virkon and Accel, supplemented with an antifreeze agent [propylene glycol (PG), methanol (MeOH), or calcium chloride (CaCl2)], were evaluated for their effectiveness in killing avian influenza virus (AIV) at −20°C or 21°C. An AIV suspension was applied to stainless steel disks, air-dried, and covered with a disinfectant o...

  1. 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...

  2. A Unique Capsular Polysaccharide Structure from the Psychrophilic Marine Bacterium Colwellia psychrerythraea 34H That Mimics Antifreeze (Glyco)proteins

    OpenAIRE

    Carillo, Sara; Casillo, Angela; Pieretti, Giuseppina; Parrilli, Ermenegilda; Sannino, Filomena; Bayer-Giraldi, Maddalena; Cosconati, Sandro; Novellino, Ettore; Ewert, Marcela; Deming, Jody W.; Lanzetta, Rosa; Marino, Gennaro; Parrilli, Michelangelo; Randazzo, Antonio; Tutino, Maria Luisa

    2015-01-01

    The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a γ-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structu...

  3. 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...

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. Enhanced inactivation of avian influenza virus at -20°C by disinfectants supplemented with calcium chloride or other antifreeze agents.

    Science.gov (United States)

    Guan, Jiewen; Chan, Maria; Brooks, Brian W; Rohonczy, Elizabeth

    2015-10-01

    Avian influenza outbreaks have occurred during winter months, and effective disinfection of poultry premises at freezing temperatures is needed. The commercial disinfectants Virkon and Accel, supplemented with an antifreeze agent [propylene glycol (PG), methanol (MeOH), or calcium chloride (CaCl₂)], were evaluated for their effectiveness in killing avian influenza virus (AIV) at -20°C or 21°C. An AIV suspension was applied to stainless steel disks, air-dried, and covered with a disinfectant or antifreeze agent for 5 to 30 min. Virkon (2%) and Accel (6.25%) with 30% PG, 20% MeOH, or 20% CaCl₂ inactivated 6 log₁₀ AIV within 5 min at -20°C and 21°C. At these temperatures PG and MeOH alone did not kill AIV, but the 20% CaCl₂ solution alone inactivated 5 log10 AIV within 10 min. The results suggested that CaCl₂ is potentially useful to enhance the effectiveness of disinfection of poultry facilities after outbreaks of AIV infection in warm and cold seasons. PMID:26424918

  11. 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

  12. 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

  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

    -gel digestion of protein samples directly on the MALDI-MS metal probe. Removal of detergent and reagents as well as protein reduction and S-alkylation were performed prior to cutting of protein samples from the polyacrylamide gel slab. The general utility of this approach was demonstrated by on-probe digestion...... preparation protocol while being less labour intensive and more cost-effective due to minimal consumption of reagents, enzymes and consumables. Preliminary data obtained on a MALDI quadrupole-TOF tandem mass spectrometer demonstrated the utility of the on-probe digestion protocol for peptide mass mapping and...

  14. 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

  15. A Novel Strategy for the Preparation of Codon-Optimized Truncated Ulp1 and its Simplified Application to Cleavage the SUMO Fusion Protein.

    Science.gov (United States)

    Wang, Xiaohua; Liu, Haifeng; Liu, Yawei; Li, Yuting; Yan, Lei; Yuan, Xiaohuan; Zhang, Yufei; Wu, Yan; Liu, Jieting; Zhang, Chunlei; Chu, Yanhui

    2016-04-01

    Ubiquitin-like protease 1 (Ulp1) of Saccharomyces cerevisiae emerges as a fundamental tool to obtain the natural N-terminal target protein by cleavage of the small ubiquitin-related modifier (SUMO) fusion protein. However, the costly commercial Ulp1 and its complicated procedures limit its application in the preparation of the target protein with natural N-terminal sequence. Here, we describe the preparation of bioactive codon-optimized recombinant truncated Ulp1 (Leu403-Lys621) (rtUlp1) of S. cerevisiae in Escherichia coli using only one-step with Ni-NTA affinity chromatograph, and the application of rtUlp1 to cleave the SUMO fusion protein by simply mixing the purified rtUlp1, SUMO fusion protein and DL-Dithiothreitol in Tris-HCl buffer. The optimal expression level of non-fusion protein rtUlp1 accounts for approximately 50 % of the total cellular protein and 36 % of the soluble form by addition of isopropyl β-D-l-thiogalactopyranoside at a final concentration of 0.4 mM at 18 °C for 20 h. The purification of target protein rtUlp1 was conducted by Ni-NTA affinity chromatography. The final yield of rtUlp1 was 45 mg/l in flask fermentation with a purity up to 95 %. Furthermore, the high purity of rtUlp1 could effectively cleave the SUMO-tTβRII fusion protein (SUMO gene fused to truncated transforming growth factor-beta receptor type II gene) with the above simplified approach, and the specific activity of the rtUlp1 reached up to 2.8 × 10(4) U/mg, which is comparable to the commercial Ulp1. The preparation and application strategy of the rtUlp1 with commonly available laboratory resources in this study will be convenient to the cleavage of the SUMO fusion protein to obtain the natural N-terminal target protein, which can be implemented in difficult-to-express protein functional analysis. PMID:26960810

  16. Preparation, characterization, and safety evaluation of poly(lactide-co-glycolide) nanoparticles for protein delivery into macrophages.

    Science.gov (United States)

    Guedj, Anne-Sophie; Kell, Arnold J; Barnes, Michael; Stals, Sandra; Gonçalves, David; Girard, Denis; Lavigne, Carole

    2015-01-01

    Following infection, HIV establishes reservoirs within tissues that are inaccessible to optimal levels of antiviral drugs or within cells where HIV lies latent, thus escaping the action of anti-HIV drugs. Macrophages are a persistent reservoir for HIV and may contribute to the rebound viremia observed after antiretroviral treatment is stopped. In this study, we further investigate the potential of poly(lactic-co-glycolic) acid (PLGA)-based nanocarriers as a new strategy to enhance penetration of therapeutic molecules into macrophages. We have prepared stable PLGA nanoparticles (NPs) and evaluated their capacity to transport an active molecule into the human monocyte/macrophage cell line THP-1 using bovine serum albumin (BSA) as a proof-of-concept compound. Intracellular localization of fluorescent BSA molecules encapsulated into PLGA NPs was monitored in live cells using confocal microscopy, and cellular uptake was quantified by flow cytometry. In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects. The size of the PLGA NPs carrying BSA (PLGA-BSA) in culture medium containing 10% serum was ~126 nm in diameter, and they were negatively charged at their surface (zeta potential =-5.6 mV). Our confocal microscopy studies and flow cytometry data showed that these PLGA-BSA NPs are rapidly and efficiently taken up by THP-1 monocyte-derived macrophages (MDMs) at low doses. We found that PLGA-BSA NPs increased cellular uptake and internalization of the protein in vitro. PLGA NPs were not cytotoxic for THP-1 MDM cells, did not modulate neutrophil apoptosis in vitro, and did not show inflammatory effect in vivo in the murine air pouch model of acute inflammation. In contrast to BSA alone, BSA encapsulated into PLGA NPs increased leukocyte infiltration in vivo, suggesting the in vivo enhanced delivery and protection of the protein by the polymer nanocarrier. We demonstrated that PLGA

  17. 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.

  18. 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...

  19. Preparation and Characterization of All-Biomass Soy Protein Isolate-Based Films Enhanced by Epoxy Castor Oil Acid Sodium and Hydroxypropyl Cellulose

    OpenAIRE

    La Wang; Jianzhang Li; Shifeng Zhang; Junyou Shi

    2016-01-01

    All-biomass soy protein-based films were prepared using soy protein isolate (SPI), glycerol, hydroxypropyl cellulose (HPC) and epoxy castor oil acid sodium (ECOS). The effect of the incorporated HPC and ECOS on the properties of the SPI film was investigated. The experimental results showed that the tensile strength of the resultant films increased from 2.84 MPa (control) to 4.04 MPa and the elongation at break increased by 22.7% when the SPI was modified with 2% HPC and 10% ECOS. The increas...

  20. Incorporation of proteins and enzymes at different stages of the preparation of calcium phosphate coatings on a degradable substrate by a biomimetic methodology

    OpenAIRE

    Azevedo, Helena S.; Leonor, I. B.; C.M. Alves; Reis, R.L.

    2005-01-01

    In this work, the possibility of incorporating proteins into calcium phosphate (Ca-P) coatings, prepared on the surface of starch polymeric biomaterials by means of a biomimetic route, was investigated. The morphology, chemical composition and crystallinity of Ca-P coatings was assessed and related to the incorporation of the studied biomolecules. For that, bovine serum albumin (BSA) and aamylase were added in concentrations of 1 mg/ml to simulated body fluid (SBF) solutions, being both ad...

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

    Science.gov (United States)

    Choi, Yun-Sang; Kim, Hyun-Wook; Hwang, Ko-Eun; Song, Dong-Heon; Jeong, Tae-Jun; Seo, Kwang-Wook; Kim, Young-Boong; Kim, Cheon-Jei

    2015-01-01

    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.

  2. Rheological properties of oil-in-water emulsions prepared with oil and protein isolates from sesame (Sesamum Indicum

    Directory of Open Access Journals (Sweden)

    David Ramirez BREWER

    2016-01-01

    Full Text Available In this study, food emulsions of oil in water from sesame (Sesamum indicum protein isolates and their oil were formulated and standardised. The effect of the concentrations of sesame (Sesamum indicum protein isolates and base oil and the speed of the emulsification process for the food emulsion stability was studied. The protein isolates were achieved from the defatted sesame flour (DSF, obtaining a percentage of 80% ± 0.05% of protein. Emulsions were formulated through a factorial design 23. The rheological behaviour of sesame (Sesamum indicum protein isolates-stabilised emulsions and microstructural composition were investigated. Stable emulsions with suitable rheological properties and microstructure were formulated at a concentration of 10% sesame oil and different concentrations of protein isolates, between 1.5% and 2.5%, with the best droplet distribution characteristics being shown for the 2.5% sesame protein isolates. The emulsions showed a non-Newtonian fluid behaviour, adjusting the Sisko model.

  3. 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

  4. Preparation, characterization, and safety evaluation of poly(lactide-co-glycolide nanoparticles for protein delivery into macrophages

    Directory of Open Access Journals (Sweden)

    Guedj AS

    2015-09-01

    Full Text Available Anne-Sophie Guedj,1 Arnold J Kell,2 Michael Barnes,2 Sandra Stals,1 David Gonçalves,3 Denis Girard,3 Carole Lavigne11National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, 2National Research Council of Canada, Ottawa, ON, 3Laboratoire de recherche en inflammation et physiologie des granulocytes, Université du Québec, INRS-Institut Armand-Frappier, Laval, QC, CanadaAbstract: Following infection, HIV establishes reservoirs within tissues that are inaccessible to optimal levels of antiviral drugs or within cells where HIV lies latent, thus escaping the action of anti-HIV drugs. Macrophages are a persistent reservoir for HIV and may contribute to the rebound viremia observed after antiretroviral treatment is stopped. In this study, we further investigate the potential of poly(lactic-co-glycolic acid (PLGA-based nanocarriers as a new strategy to enhance penetration of therapeutic molecules into macrophages. We have prepared stable PLGA nanoparticles (NPs and evaluated their capacity to transport an active molecule into the human monocyte/macrophage cell line THP-1 using bovine serum albumin (BSA as a proof-of-concept compound. Intracellular localization of fluorescent BSA molecules encapsulated into PLGA NPs was monitored in live cells using confocal microscopy, and cellular uptake was quantified by flow cytometry. In vitro and in vivo toxicological studies were performed to further determine the safety profile of PLGA NPs including inflammatory effects. The size of the PLGA NPs carrying BSA (PLGA-BSA in culture medium containing 10% serum was ~126 nm in diameter, and they were negatively charged at their surface (zeta potential =-5.6 mV. Our confocal microscopy studies and flow cytometry data showed that these PLGA-BSA NPs are rapidly and efficiently taken up by THP-1 monocyte-derived macrophages (MDMs at low doses. We found that PLGA-BSA NPs increased cellular uptake and internalization of the protein in vitro. PLGA

  5. 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。

  6. 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.

  7. 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.

  8. Preparation of protein-loaded PLGA-PVP blend nanoparticles by nanoprecipitation method: entrapment, Initial burst and drug release kinetic studies

    Directory of Open Access Journals (Sweden)

    Shahryar Shakeri

    2015-07-01

    Full Text Available Objective(s:Despite of wide range applications of polymeric nanoparticles in protein delivery, there are some problems for the field of protein entrapment, initial burst and controlled release profile.   Materials and Methods: In this study, we investigated the influence of some changes in PLGA nanoparticles formulation to improve the initial and controlled release profile. Selected parameters were: pluronic F127, polysorbate 80 as surfactant, pH of inner aqueous phase, L/G ratio of PLGA polymer, volume of inner aqueous phase and addition of polyvinylpyrrolidone as an excipient. FITC-HSA was used as a model hydrophilic drug. The nanoparticles were prepared by nanoprecipitation.   Results:  Initial release of FITC-HSA from PLGA-tween 80 nanoparticles (opt-4, 61% was faster than control (PLGA-pluronic after 2.30 h of incubation. Results showed that decrease in pH of inner aqueous phase to pI of protein can decrease IBR but the release profile of protein is the same as control. Release profile with three phases including a initial burst b plateau and c final release phase was observed when we changed volume of inner aqueous phase and L/G ratio in formulation. Co-entrapment of HSA with PVP and pluronic reduced the IBR and controlled release profile in opt-19. Encapsulation efficiency was more than 97% and nanoparticles size and zeta potentials were mono-modal and -18.99 mV, respectively.   Conclusion:  In this research, we optimized a process for preparation of PLGA-PVP-pluronic nanoparticles of diameter less than 300 nm using nanoprecipitation method. This formulation showed a decreased initial burst and long lasting controlled release profile for FITC-HSA as a model drug for proteins.

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

    International Nuclear Information System (INIS)

    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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. Self-assembled luminescent CdSe-ZnS quantum dot bioconjugates prepared using engineered poly-histidine terminated proteins

    International Nuclear Information System (INIS)

    We report a simple and versatile approach for the conjugation of luminescent CdSe-ZnS core-shell quantum dots (QDs) to proteins through coordination of engineered C-terminal oligohistidine sequences. Several histidine tail containing proteins were self-assembled onto the QD surface using this method. A recombinant antibody specific for the high explosive 2,4,6-trinitrotoluene (TNT) was conjugated to QDs through a carboxy terminal histidine tail and the bioconjugate used to detect TNT by competitive immunoassay. TNT was detected over the range of 10 μg/ml down to 41 ng/ml using the scFv conjugated to QDs. These results open up the possibility to conjugate luminescent QDs to a whole range of proteins to form QD bioconjugates that can be effectively used in bio-oriented applications, such as sensing, imaging, immunoassay and other diagnostics

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

    International Nuclear Information System (INIS)

    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 P212121, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å

  18. Changes in the physical properties, solubility, and heat stability of milk protein concentrates prepared from partially acidified milk.

    Science.gov (United States)

    Eshpari, H; Tong, P S; Corredig, M

    2014-12-01

    A limiting factor in using milk protein concentrates (MPC) as a high-quality protein source for different food applications is their poor reconstitutability. Solubilization of colloidal calcium phosphate (CCP) from casein micelles during membrane filtration (e.g., through acidification) may affect the structural organization of these protein particles and consequently the rehydration and functional properties of the resulting MPC powder. The main objective of this study was to investigate the effects of acidification of milk by glucono-δ-lactone (GDL) before ultrafiltration (UF) on the composition, physical properties, solubility, and thermal stability (after reconstitution) of MPC powders. The MPC samples were manufactured in duplicate, either by UF (65% protein, MPC65) or by UF followed by diafiltration (80% protein, MPC80), using pasteurized skim milk, at either the native milk pH (~pH 6.6) or at pH 6.0 after addition of GDL, followed by spray drying. Samples of different treatments were reconstituted at 5% (wt/wt) protein to compare their solubility and thermal stability. Powders were tested in duplicate for basic composition, calcium content, reconstitutability, particle size, particle density, and microstructure. Acidification of milk did not have any significant effect on the proximate composition, particle size, particle density, or surface morphology of the MPC powders; however, the total calcium content of MPC80 decreased significantly with acidification (from 1.84 ± 0.03 to 1.59 ± 0.03 g/100 g of powder). Calcium-depleted MPC80 powders were also more soluble than the control powders. Diafiltered dispersions were significantly less heat stable (at 120°C) than UF samples when dissolved at 5% solids. The present work contributes to a better understanding of the differences in MPC commonly observed during processing. PMID:25459904

  19. 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. PMID:26263892

  20. Preparation and characterization of bio-nanocomposite films based on soy protein isolate and montmorillonite using melt extrusion

    Science.gov (United States)

    The non-biodegradable and non-renewable nature of plastic packaging has led to a renewed interest in packaging materials based on bio-nanocomposites (biopolymer matrix reinforced with nanoparticles such as layered silicates). Bio-nanocomposite films based on soy protein isolate (SPI) and montmorillo...

  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

    , nor any 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...

  2. 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

  3. Efficient preparation and metal specificity of the regulatory protein TroR from the human pathogen Treponema pallidum.

    Science.gov (United States)

    Liu, Yi; Li, Wei; Wei, Yaozhu; Jiang, Yindi; Tan, Xiangshi

    2013-10-01

    TroR is a putative metal-dependent regulatory protein that has been linked to the virulence of the human pathogen Treponema pallidum. It shares high homology with the well-known iron-dependent regulatory protein DtxR from Corynebacterium diphtheriae, as well as the manganese-dependent MntR from Bacillus subtilis. However, it has been uncertain whether manganese or zinc is the natural cofactor of TroR to date. Herein, we established an efficient method named "double-fusion tagging" to obtain soluble TroR for the first time. A series of studies, including ICP, CD, fluorescence, ITC, and electrophoresis mobility shift assay (EMSA), were performed to resolve the discrepancies in its metal-binding specificity. In addition, bioinformatic analysis as well as mutation studies were carried out to find the genetic relationships of TroR with its homology proteins. In conclusion, our findings indicate that TroR is a manganese-dependent rather than a zinc-dependent regulatory protein. PMID:23945957

  4. 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)

  5. 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 .

  6. 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

  7. 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...

  8. 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.

  9. 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...

  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 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.

  12. 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)

  13. 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.

  14. Multiple Acquisition of Magic Angle Spinning Solid-State NMR Experiments Using One Receiver: Application to Microcrystalline and Membrane Protein Preparations

    Science.gov (United States)

    Gopinath, T.; Veglia, Gianluigi

    2015-01-01

    Solid-State NMR spectroscopy of proteins is a notoriously low-throughput technique. Relatively low-sensitivity and poor resolution of protein samples require long acquisition times for multidimensional NMR experiments. To speed up data acquisition, we developed a family of experiments called Polarization Optimized Experiments (POE), in which we utilized the orphan spin operators that are discarded in classical multidimensional NMR experiments, recovering them to allow simultaneous acquisition of multiple 2D and 3D experiments, all while using conventional probes with spectrometers equipped with one receiver. POEs allow the concatenation of multiple 2D or 3D pulse sequences into a single experiment, thus potentially combining all of the aforementioned advances, boosting the capability of ssNMR spectrometers at least two-fold without the addition of any hardware. In this Perspective, we describe the first generation of POEs, such as dual acquisition MAS (or DUMAS) methods, and then illustrate the evolution of these experiments into MEIOSIS, a method that enables the simultaneous acquisition of multiple 2D and 3D spectra. Using these new pulse schemes for the solid-state NMR investigation of biopolymers makes it possible to obtain sequential resonance assignments, as well as distance restraints, in about half the experimental time. While designed for acquisition of heteronuclei, these new experiments can be easily implemented for proton detection and coupled with other recent advancements, such as dynamic polarization, to improve signal to noise. Finally, we illustrate the application of these methods to microcrystalline protein preparations as well as single and multi-span membrane proteins reconstituted in lipid membranes. PMID:25797011

  15. Preparation and crystallization of the stimulatory and inhibitory complexes of GTP cyclohydrolase I and its feedback regulatory protein GFRP.

    Science.gov (United States)

    Maita, N; Okada, K; Hirotsu, S; Hatakeyama, K; Hakoshima, T

    2001-08-01

    Mammalian GTP cyclohydrolase I is a decameric enzyme in the first and rate-limiting step in the biosynthesis of tetrahydrobiopterin, which is an essential cofactor for enzymes producing neurotransmitters such as catecholamines and for nitric oxide synthases. The enzyme is dually regulated by its feedback regulatory protein GFRP in the presence of its stimulatory effector phenylalanine and its inhibitory effector biopterin. Here, both the stimulatory and inhibitory complexes of rat GTP cyclohydrolase I bound to GFRP were crystallized by vapour diffusion. Diffraction data sets at resolutions of 3.0 and 2.64 A were collected for the stimulatory and inhibitory complexes, respectively. Each complex consists of two GTPCHI pentamer rings and two GFRP pentamer rings, with pseudo-52 point-group symmetry. PMID:11468403

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. Effect of surface microstructure and wettability on plasma protein adsorption to ZnO thin films prepared at different RF powers

    Energy Technology Data Exchange (ETDEWEB)

    Huang Zhanyun; Chen Min; Chen Dihu [State Key Laboratory of Optoelectronic Materials and Technologies, Sun Yat-Sen University, Guangzhou 510275 (China); Pan Shirong, E-mail: stscdh@mail.sysu.edu.c [Artificial Heart Lab, the 1st Affiliate Hospital of Sun Yat-Sen University, Guangzhou 510080 (China)

    2010-10-01

    In this paper, the adsorption behavior of plasma proteins on the surface of ZnO thin films prepared by radio frequency (RF) sputtering under different sputtering powers was studied. The microstructures and surface properties of the ZnO thin films were investigated by x-ray diffraction (XRD), scanning electron microscopy (SEM), UV-visible optical absorption spectroscopy and contact angle techniques. The results show that the ZnO thin films have better orientation of the (0 0 2) peak with increasing RF power, especially at around 160 W, and the optical band gap of the ZnO films varies from 3.2 to 3.4 eV. The contact angle test carried out by the sessile drop technique denoted a hydrophobic surface of the ZnO films, and the surface energy and adhesive work of the ZnO thin films decreased with increasing sputtering power. The amounts of human fibrinogen (HFG) and human serum albumin (HSA) adsorbing on the ZnO films and reference samples were determined by using enzyme-linked immunosorbent assay (ELISA). The results show that fewer plasma proteins and a smaller HFG/HSA ratio adsorb on the ZnO thin films' surface.

  4. 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%.

  5. 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。

  6. 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。

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Partition of synaptic membranes in aqueous two-phase systems at subzero temperatures by using anti-freeze solvent.

    Science.gov (United States)

    Johansson, G; Joelsson, M; Olde, B

    1990-11-16

    The freezing point of aqueous two-phase (liquid-liquid) systems containing water, dextran and poly(ethylene glycol) has been lowered by including glycerol. Biological membranes, obtained by fragmentation of a crude synaptosomal preparation from calf brain cortex, have been included in the two-phase systems. The effects of temperature and the concentration of glycerol on the partition of the membranes within the systems have been investigated. Considerable stabilisation of the membranes was noticed when they were partitioned at -10 degrees C compared with 0 degrees C. The influences of glycerol, ethylene glycol, N,N-dimethylformamide and tetrahydrofuran on the phase-forming properties of the systems and on enzyme activities are also presented. Possible use of the above systems for studies and separation of biological membranes are discussed. PMID:2245213

  12. 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.

  13. 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...

  14. 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

  15. 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

  16. 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

  17. 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

    International Nuclear Information System (INIS)

    Arsenic trioxide (arsenite, AsIII) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of AsIII on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of AsIII on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent AsIII-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of AsIII were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to AsIII 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 AsIII 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 AsIII-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 AsIII cytotoxicity between these cells. -- Highlights: ► Examination of effect of AsIII on primary cultured chorion (C) and amnion (A) cells. ► Dose-dependent AsIII-mediated cytotoxicity in C-cells, not in A

  18. 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

  19. 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.

  20. Preparation of uniformly labeled NMR samples in Escherichia coli under the tight control of the araBAD promoter: expression of an archaeal homolog of the RNase P Rpp29 protein.

    Science.gov (United States)

    Boomershine, William P; Raj, M L Stephen; Gopalan, Venkat; Foster, Mark P

    2003-04-01

    We report the first use of the tightly regulated araBAD promoter for generating uniformly labeled samples for NMR. The araBAD promoter provides a distinct advantage over that of the most commonly used protein overexpression systems in bacteria (e.g., in pET vectors: T7lac), in that it provides much tighter control over basal expression. However, use of araBAD-regulated expression for preparation of uniformly labeled protein samples for NMR is complicated by the fact that glucose (the most commonly used carbon source in defined minimal media) indirectly represses transcription, and thus, cannot be used. After experimenting with alternative media, we found that uniformly labeled NMR samples can be prepared using the highly regulated arabinose-inducible promoter and that suitable yields can be obtained in defined minimal media containing glycerol, not glucose, as the carbon source. PMID:12699688

  1. 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

  2. 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%.

  3. 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.

  4. 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。

  5. 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

  6. 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.

  7. 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%.

  8. Water-mediated influence of a crowded environment on internal vibrations of a protein molecule.

    Science.gov (United States)

    Kuffel, Anna; Zielkiewicz, Jan

    2016-02-01

    The influence of crowding on the protein inner dynamics is examined by putting a single protein molecule close to one or two neighboring protein molecules. The presence of additional molecules influences the amplitudes of protein fluctuations. Also, a weak dynamical coupling of collective velocities of surface atoms of proteins separated by a layer of water is detected. The possible mechanisms of these phenomena are described. The cross-correlation function of the collective velocities of surface atoms of two proteins was decomposed into the Fourier series. The amplitude spectrum displays a peak at low frequencies. Also, the results of principal component analysis suggest that the close presence of an additional protein molecule influences the high-amplitude, low-frequency modes in the most prominent way. This part of the spectrum covers biologically important protein motions. The neighbor-induced changes in the inner dynamics of the protein may be connected with the changes in the velocity power spectrum of interfacial water. The additional protein molecule changes the properties of solvation water and in this way it can influence the dynamics of the second protein. It is suggested that this phenomenon may be described, at first approximation, by a damped oscillator driven by an external random force. This model was successfully applied to conformationally rigid Choristoneura fumiferana antifreeze protein molecules. PMID:26805932

  9. 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

  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. 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。

  12. 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

  13. Purification and properties of a new ribosome-inactivating protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis.

    Science.gov (United States)

    Bolognesi, A; Barbieri, L; Carnicelli, D; Abbondanza, A; Cenini, P; Falasca, A I; Dinota, A; Stirpe, F

    1989-12-01

    A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1-8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an Mr of approx. 30,000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells. PMID:2597699

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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。

  20. 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.

  1. 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

  2. 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.

  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. 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

  6. 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

  7. 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

  8. 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。

  9. 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。

  10. 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.

  11. 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%.

  12. Preparation of whey protein hydrolysates using a single- and two-stage enzymatic membrane reactor and their immunological and antioxidant properties: characterization by multivariate data analysis.

    Science.gov (United States)

    Cheison, Seronei Chelulei; Wang, Zhang; Xu, Shi-Ying

    2007-05-16

    An initial 5% (w/v), followed thereafter with replacement aliquots of 3% (w/v), whey protein isolate (WPI) (ca. 86.98% Kjeldahl N x 6.38), was hydrolyzed using Protease N Amano G (IUB 3.4.24.28, Bacillus subtilis) in an enzymatic membrane reactor (EMR) fitted with either a 10 or 3 kDa nominal molecular weight cutoff (NMWCO) tangential flow filter (TFF) membrane. The hydrolysates were desalted by adsorption onto a styrene-based macroporous adsorption resin (MAR) and washed with deionized water to remove the alkali, and the peptides were desorbed with 25, 50, and 95% (v/v) ethyl alcohol. The desalted hydrolysates were analyzed for antibody binding, free radical scavenging, and molecular mass analysis as well as total and free amino acids (FAA). For the first time a quantity called IC50, the concentration of peptides causing 50% inhibition of the available antibody, is introduced to quantify inhibition enzyme-linked immunosorbent assay (ELISA) properties. Principal component analysis (PCA) was used for data reduction. The hydrolysate molecular mass provided the most prominent influence (PC1 = 57.35%), followed by inhibition ELISA (PC2 = 18.90%) and the antioxidant properties (PC3 = 10.43%). Ash was significantly reduced in the desalted fractions; the protein adsorption recoveries were high, whereas desorption with alcohol was prominently influenced by the hydrophobic/ hydrophilic amino acid balance. After hydrolysis, some hydrolysates showed increased ELISA reactivity compared with the native WPI. PMID:17432869

  13. 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.

  14. 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

  15. 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%。

  16. 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%)

  17. 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。

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. Infection by chikungunya virus modulates the expression of several proteins in Aedes aegypti salivary glands

    Directory of Open Access Journals (Sweden)

    Tchankouo-Nguetcheu Stephane

    2012-11-01

    Full Text Available Abstract Background Arthropod-borne viral infections cause several emerging and resurging infectious diseases. Among the diseases caused by arboviruses, chikungunya is responsible for a high level of severe human disease worldwide. The salivary glands of mosquitoes are the last barrier before pathogen transmission. Methods We undertook a proteomic approach to characterize the key virus/vector interactions and host protein modifications that occur in the salivary glands that could be responsible for viral transmission by using quantitative two-dimensional electrophoresis. Results We defined the protein modulations in the salivary glands of Aedes aegypti that were triggered 3 and 5 days after an oral infection (3 and 5 DPI with chikungunya virus (CHIKV. Gel profile comparisons showed that CHIKV at 3 DPI modulated the level of 13 proteins, and at 5 DPI 20 proteins. The amount of 10 putatively secreted proteins was regulated at both time points. These proteins were implicated in blood-feeding or in immunity, but many have no known function. CHIKV also modulated the quantity of proteins involved in several metabolic pathways and in cell signalling. Conclusion Our study constitutes the first analysis of the protein response of Aedes aegypti salivary glands infected with CHIKV. We found that the differentially regulated proteins in response to viral infection include structural proteins and enzymes for several metabolic pathways. Some may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by arboviruses. For example, proteins involved in blood-feeding such as the short D7, an adenosine deaminase and inosine-uridine preferring nucleoside hydrolase, may favour virus transmission by exerting an increased anti-inflammatory effect. This would allow the vector to bite without the bite being detected. Other proteins, like the anti-freeze protein, may support vector protection.

  8. Radioprotective preparation

    International Nuclear Information System (INIS)

    The invention is intended for radiation injuries prophylaxis in mammals. It has an well expressed radioprotective effect against acute gamma irradiation on cellular level as well as a prolonged action when applied up to 48 hours before the acute irradiation. The preparation is a coprecipitate of the natural tripeptide glutathione (reduced form) and polyvinyl pyrrolidone (pvp) in ratio 30-60/70-40. It is obtained by incubation method with subsequent lyophilization from water solution of the initial components. The molecular mass of the pvp is 20 till 360.103. 2 claims

  9. 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.

  10. 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.

  11. 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.

  12. 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%。

  13. 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

  14. 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

    DEFF Research Database (Denmark)

    Wang, Wei-Qing; Ye, Jian-Qing; Rogowska-Wrzesinska, Adelina;

    2014-01-01

    abundant in this group and may contribute to the acquisition of 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....

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Properties and biotechnological applications of ice-binding proteins in bacteria.

    Science.gov (United States)

    Cid, Fernanda P; Rilling, Joaquín I; Graether, Steffen P; Bravo, Leon A; Mora, María de La Luz; Jorquera, Milko A

    2016-06-01

    Ice-binding proteins (IBPs), such as antifreeze proteins (AFPs) and ice-nucleating proteins (INPs), have been described in diverse cold-adapted organisms, and their potential applications in biotechnology have been recognized in various fields. Currently, both IBPs are being applied to biotechnological processes, primarily in medicine and the food industry. However, our knowledge regarding the diversity of bacterial IBPs is limited; few studies have purified and characterized AFPs and INPs from bacteria. Phenotypically verified IBPs have been described in members belonging to Gammaproteobacteria, Actinobacteria and Flavobacteriia classes, whereas putative IBPs have been found in Gammaproteobacteria, Alphaproteobacteria and Bacilli classes. Thus, the main goal of this minireview is to summarize the current information on bacterial IBPs and their application in biotechnology, emphasizing the potential application in less explored fields such as agriculture. Investigations have suggested the use of INP-producing bacteria antagonists and AFPs-producing bacteria (or their AFPs) as a very attractive strategy to prevent frost damages in crops. UniProt database analyses of reported IBPs (phenotypically verified) and putative IBPs also show the limited information available on bacterial IBPs and indicate that major studies are required. PMID:27190285

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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...

  5. 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

  6. 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 ...

  7. 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

  8. 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)

  9. Mix proportion design of anti-freezing and anti-permeability low-strength concrete and its corresponding mechanical properties%低强度抗冻抗渗混凝土配合比设计及其力学特性

    Institute of Scientific and Technical Information of China (English)

    彭辉; 杨海宁; 刘绍林

    2012-01-01

    Through carefully choosing the mix proportion design of low-strength concrete,the anti-freezing,anti-perme- ability and mechanical properties of concrete were studied. The effects of optimal water-to-cement ratio, mixing amount of air-entraining agent on anti-freezing and anti-permeability characteristics of concrete were researched. Furthermore, the compressive strength of concrete under condition of saturated situation and natural curing were also studied respec- tively. The researches show that by using of mixing proper amount of air-entraining agent and choosing suitable water- to-cement ratio, the anti-freezing and anti-permeability of low-strength concrete can be obviously improved without re- ducing concrete strength and anti-permeability properties in severe cold areas in China.%通过对低强度混凝土配合比进行优化设计,并开展混凝土的抗冻、抗渗和力学等性能的试验研究。分别研究了最优水灰比、引气剂掺量等因素对混凝土抗冻性能的影响,并在上述条件下研究了其对强度和抗渗等性能的影响,以及饱和与自然养护条件下混凝土抗压强度变化特点。结果证明:在严寒地区,在满足工程结构混凝土强度和抗渗要求的情况下,即使配制低强度混凝土,通过掺适量引气剂和选用适当的水灰比能显著提高低强度混凝土在高寒干燥地区的抗冻耐久性。

  10. 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).

  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. Fundamentals of preparative and nonlinear chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Guiochon, Georges A [ORNL; Felinger, Attila [ORNL; Katti, Anita [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Shirazi, Dean G [unknown

    2006-02-01

    The second edition of Fundamentals of Preparative and Nonlinear Chromatography is devoted to the fundamentals of a new process of purification or extraction of chemicals or proteins widely used in the pharmaceutical industry and in preparative chromatography. This process permits the preparation of extremely pure compounds satisfying the requests of the US Food and Drug Administration. The book describes the fundamentals of thermodynamics, mass transfer kinetics, and flow through porous media that are relevant to chromatography. It presents the models used in chromatography and their solutions, discusses the applications made, describes the different processes used, their numerous applications, and the methods of optimization of the experimental conditions of this process.

  13. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    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. The...

  14. Thesis Preparation Manual

    OpenAIRE

    1999-01-01

    "This Thesis Preparation Manual has been written to provide you with format and procedure guidance for preparing and processing your thesis at the Naval Postgraduate School. It covers both unclassified and classified theses. All theses and thesis technical reports must be prepared in accordance with these guidelines. Please note that this manual is not written in the thesis format." form the Foreword

  15. 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.

  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. 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.

  18. Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (Prepared by reverse-phase evaporation) by Triton X-100 octyl glucoside, and sodium cholate

    International Nuclear Information System (INIS)

    The mechanisms governing the solubilization by Triton X-100, octyl glucoside, and sodium cholate of large unilamellar liposomes prepared by reverse-phase evaporation were investigated. The solubilization process is described by the three-stage model previously proposed for the detergents. In stage I, detergent monomers are incorporated into the phospholipid bilayers until they saturate the liposomes. At this point, i.e., stage II, mixed phospholipid-detergent micelles begin to form. By stage III, the lamellar to micellar transition is complete and all the phospholipids are present as mixed micelles. The turbidity of liposome preparations was systematically measured as a function of the amount of detergent added for a wide range of phospholipid concentrations. The results allowed a quantitative determination of the effective detergent to lipid molar ratios in the saturated liposomes. The monomer concentrations of the three detergents in the aqueous phase were also determined at the lamellar to micellar transitions. These transitions were also investigated by 31P NMR spectroscopy, and complete agreement was found with turbidity measurements. Freeze-fracture electron microscopy and permeability studies in the sublytic range of detergent concentrations indicated that during stage I of solubilization detergent partitioning between the aqueous phase and the lipid bilayer greatly affects the basic permeability of the liposomes without significantly changing the morphology of the preparations. A rough approximation of the partition coefficients was derived from the turbidity and permeability data. It is concluded that when performed systematically, turbidity measurements constitute a very convenient and powerful technique for the quantitative study of the liposome solubilization process by detergents

  19. Affinity purification of proteins binding to GST fusion proteins.

    Science.gov (United States)

    Swaffield, J C; Johnston, S A

    2001-05-01

    This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding. PMID:18265191

  20. 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%.

  1. 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

  2. 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,...

  3. Toddler test or procedure preparation

    Science.gov (United States)

    Preparing toddler for test/procedure; Test/procedure preparation - toddler; Preparing for a medical test or procedure - toddler ... Before the test, know that your child will probably cry. Even if you prepare, your child may feel some discomfort or ...

  4. Toddler test or procedure preparation

    Science.gov (United States)

    Preparing toddler for test/procedure; Test/procedure preparation - toddler; Preparing for a medical test or procedure - toddler ... about the procedure to 5 or 10 minutes. Toddlers have a short attention span. Any preparation should ...

  5. : Protein flexibility

    OpenAIRE

    Bornot, Aurélie; Offmann, Bernard; De Brevern, Alexandre

    2007-01-01

    Protein structures and protein structural models are great tools to reach protein function and provide very relevant information for drug design. Nevertheless, protein structures are not rigid entities. Cutting-edge bioinformatics methods tend to take into account the flexibility of these macromolecules. We present new approaches used to define protein structure flexibility.

  6. 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.

  7. 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.

  8. Total protein

    Science.gov (United States)

    The total protein test measures the total amount of two classes of proteins found in the fluid portion of your ... nutritional problems, kidney disease or liver disease . If total protein is abnormal, you will need to have more ...

  9. Preparation of tritiated TNT

    International Nuclear Information System (INIS)

    Tritiated TNT was prepared by catalytic exchange in tritiated aqueous medium. The tritiated water of high activity was prepared from micro amount of T2 and PdO/BaSO4. The crude product was purified by silica gel loaded paper chromatography. The radiochemical purity and specific activity are 90% and 54 mCi/mmol respectively

  10. Documents preparation and review

    International Nuclear Information System (INIS)

    Ignalina Safety Analysis Group takes active role in assisting regulatory body VATESI to prepare various regulatory documents and reviewing safety reports and other documentation presented by Ignalina NPP in the process of licensing of unit 1. The list of main documents prepared and reviewed is presented

  11. Polymers for Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Gianfranco Pasut

    2014-01-01

    Full Text Available Polyethylene glycol (PEG at the moment is considered the leading polymer for protein conjugation in view of its unique properties, as well as to its low toxicity in humans, qualities which have been confirmed by its extensive use in clinical practice. Other polymers that are safe, biodegradable and custom-designed have, nevertheless, also been investigated as potential candidates for protein conjugation. This review will focus on natural polymers and synthetic linear polymers that have been used for protein delivery and the results associated with their use. Genetic fusion approaches for the preparation of protein-polypeptide conjugates will be also reviewed and compared with the best known chemical conjugation ones.

  12. 人高致病性H5N1亚型禽流感病毒NS1蛋白多克隆抗体的制备及鉴定%Preparation and characterization the polyclonal antibody of the nonstructural protein of human highly pathogenic H5N1 avian influenza viruses

    Institute of Scientific and Technical Information of China (English)

    蒋培余; 黄惠莲; 周洪昌; 徐伯赢; 顾福萍; 闵丽姗; 钟婧; 戴利成

    2013-01-01

    目的 制备高灵敏度和高特异性的人高致病性H5N1亚型禽流感病毒NS1蛋白抗体并对其效价进行初步评估.方法 构建含有H5N1亚型禽流感病毒NS1序列的pET-28a(+)重组载体的大肠埃希菌BL21(DE3),诱导表达NS1蛋白,并经Ni-NTA色谱柱亲和层析纯化获得NS1重组蛋白,并进行SDS-PAGE和Western Blot鉴定.以纯化的蛋白为抗原免疫新西兰大白兔,获得兔抗NS1血清,亲和纯化获得多克隆抗体.应用ELISA和Western Blot检测纯化抗体的效价和特异性.结果 NS1融合蛋白得到高表达,且纯度>90%,用该融合蛋白免疫新西兰大白兔后得到的抗NS1多克隆抗体,效价达1∶80 000,并特异性识别H5N1亚型禽流感病毒NS1蛋白.结论 获得了NS1多克隆抗体,具有较好的效价和特异性.%Objective Of this study was to prepare high sensitivity and high specificity of highly pathogenic H5N1 subtype avian influenza virus NS1 protein antibody and a preliminary assessment of its potency.Methods Construct pET-28a (+) recombinant vector containing the H5N1 subtype of avian influenza virus NS1 sequences of E.coli BL21 (DE3),induced expression of NS1 protein,NS1 recombinant protein was obtained by Ni-NTA column purified by affinity chromatography,and SDS-PAGE and Western Blot analysis.Purified protein antigen to immunize New Zealand white rabbits,obtained rabbit anti-NS1 serum,affinity-purified polyclonal antibodies.Using ELISA and Western Blot analysis of purified antibody titer and specificity.Results NS1 fusion protein was highly expressed in a purity of greater than 90%,with the fusion protein was used to immunize New Zealand white rabbits anti-NS1 polyclonal antibody titer of 1:80000,and specific recognition of the H5N1 subtype of avian influenza virus NS1 protein.Conclusions NS1 polyclonal antibodies to NS1 recombinant protein purified antigen,with better potency and specificity,and to prepare the conditions for the development of the H5N1 subtype of avian

  13. 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...

  14. Preparation of wheat germ sports drinks of whey protein oligosaccharide%乳清蛋白-低聚糖型小麦胚运动饮料的研制

    Institute of Scientific and Technical Information of China (English)

    李涛; 陈雪勤; 雷雨

    2015-01-01

    以小麦胚、低聚麦芽糖、乳清蛋白、无机盐(NaCl、KCl)和柠檬酸、黄原胶为原料,制作新型运动饮料。其生产工艺为制取小麦胚汁、调配、均质、灌装、杀菌、冷却、静置、检验、成品。利用单因素试验和正交试验确定运动饮料的最优配方为小麦胚汁100g,黄原胶0.075g,乳清蛋白1.5g,无机盐1.8g,柠檬酸0.02g,低聚麦芽糖8g。该运动饮料呈乳白色,具有小麦胚特殊色泽,色泽均匀,清爽可口,既有小麦胚的清香味,又有乳清蛋白的香味,质地均匀一致,无沉淀,不分层,具有良好的流动性,对运动后的不适有良好的缓解作用。%Wheat germ ,oligo maltose ,whey protein ,inorganic salts (NaCl ,KCl) and citric acid ,xanthan gum were the raw materials to produce a new kind of sports drinks .The production process consisted of wheat germ juice extracting ,blending , homogenizing ,filling ,sterilizing ,cooling ,static ,inspection and finished product .Using single factor test and orthogonal test , the optimized formula were as:wheat germ juice 100 g ,xanthan gum 0 .075 g ,whey protein content 1 .5 g ,inorganic salt 1 .8 g ,citric acid 0 .02 g and oligo maltose 8 g .The sports drink was milk white ,with wheat germ special color ,uniform col‐or ,fresh and delicious ,both with wheat germ and whey protein scent ,muddy uniform ,non hierarchical ,non precipitation , with good fluidity .The sports drinks had a good mitigation for body after movement .

  15. Precise and automated microfluidic sample preparation.

    Energy Technology Data Exchange (ETDEWEB)

    Crocker, Robert W.; Patel, Kamlesh D.; Mosier, Bruce P.; Harnett, Cindy K.

    2004-07-01

    Autonomous bio-chemical agent detectors require sample preparation involving multiplex fluid control. We have developed a portable microfluidic pump array for metering sub-microliter volumes at flowrates of 1-100 {micro}L/min. Each pump is composed of an electrokinetic (EK) pump and high-voltage power supply with 15-Hz feedback from flow sensors. The combination of high pump fluid impedance and active control results in precise fluid metering with nanoliter accuracy. Automated sample preparation will be demonstrated by labeling proteins with fluorescamine and subsequent injection to a capillary gel electrophoresis (CGE) chip.

  16. PREPARATIVE SKIN PREPARATION AND SURGICAL WOUND INFECTION

    Directory of Open Access Journals (Sweden)

    Anjanappa

    2015-01-01

    Full Text Available 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 microorganisms on the skin surface and to reduce the resident flora to a low level. Povidone iodine (I odophors and chlorhexidine are most often used antiseptics for pre - operative skin preparation. OBJECTIVES : To evaluate the efficacy of povidone iodine alone and in combination with antiseptic agent containing alcoholic chlorhexidine in preoperative skin p reparation by taking swab culture. (2 To compare the rate of postoperative wound infection in both the groups. METHODS: One hundred patients (fifty in each group undergoing clean elective surgery with no focus of infection on the body were included in th e study. The pre - operative skin preparation in each group is done with the respective antiseptic regimen. In both the groups after application of antiseptics , sterile saline swab culture was taken immediately from site of incision. In cases which showed gr owth of organisms , the bacteria isolated were identified by their morphological and cultural characteristics. Grams staining , coagulase test and antibiotic sensitivity test were done wherever necessary and difference in colonization rates was determined as a measure of efficacy of antiseptic regimen. RESULTS: The results of the study showed that when compared to povidone iodine alone , using a combination of povidone iodine and alcoholic solution of chlorhexidine , the colonization rates of the site of incisi on were reduced significantly. As for the rate of post - operative wound infection , it is also proven that wound infections are also

  17. Interfacial Protein-Protein Associations

    OpenAIRE

    Langdon, Blake B.; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2013-01-01

    While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for ...

  18. Microfluidic Sample Preparation for Immunoassays

    Energy Technology Data Exchange (ETDEWEB)

    Visuri, S; Benett, W; Bettencourt, K; Chang, J; Fisher, K; Hamilton, J; Krulevitch, P; Park, C; Stockton, C; Tarte, L; Wang, A; Wilson, T

    2001-08-09

    Researchers at Lawrence Livermore National Laboratory are developing means to collect and identify fluid-based biological pathogens in the forms of proteins, viruses, and bacteria. to support detection instruments, they are developing a flexible fluidic sample preparation unit. The overall goal of this Microfluidic Module is to input a fluid sample, containing background particulates and potentially target compounds, and deliver a processed sample for detection. They are developing techniques for sample purification, mixing, and filtration that would be useful to many applications including immunologic and nucleic acid assays. Many of these fluidic functions are accomplished with acoustic radiation pressure or dielectrophoresis. They are integrating these technologies into packaged systems with pumps and valves to control fluid flow through the fluidic circuit.

  19. Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

    Science.gov (United States)

    Kim, Young Eun; Kim, Yu-Na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

    2015-05-01

    Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein-protein interactions and tools to manipulate receptor clustering on live cell surfaces.

  20. 乳清蛋白-麦芽糖糊精的Maillard反应复合物制备β-胡萝卜素纳米乳液%Preparation of β-carotene nanoemulsions stablilized by Maillard Reaction Products of whey protein isolate and maltodextrin

    Institute of Scientific and Technical Information of China (English)

    李燕; 刘成梅; 刘伟; 钟俊桢

    2013-01-01

    系统考察乳液制备参数对乳液粒径分布及稳定性的影响,同时以干热法制备乳清分离蛋白(Whey Protein Isolate,WPI)-麦芽糖糊精(Maltodextrin,MD)的Maillard反应复合物(Maillard Reaction Products,MRPs).以此为基础,制备WPI-MD MRPs稳定的β-胡萝卜、素纳米乳液,并进一步考察乳液的物理稳定性及β-胡萝卜、素的化学稳定性.结果表明,WPI-MD的MRPs能够显著降低纳米乳液的粒径,并提高纳米乳液的物理稳定性.同时,WPI-MD的MRPs可加速油相中β-胡萝卜素的降解,其机理有待进一步研究.%Conditons for preparing nanoemulsions were investigated systematically. The Maillard Reaction Products(MRPs) were prepared by dry heating which were further used to prepare β-carotene nanoemulsions. The physical stability of the obtained nanoemulsions and the β-carotene degregation kinetics were simultaneously studied. The result showed that nanoemulsions stabilized by MRPs with smaller fat globe size were much physically stable compared with the nanoemulsions stablized by native WPI. However,the β-carotene in MRPs stablized nanoemulsions degradated more rapidly which mechanism should be futher studied in future.

  1. 制备米糠蛋白降血压肽最佳用酶的筛选%Screening the Best Enzyme of the Preparation of Antihypertensive Peptides from Rice Bran Protein

    Institute of Scientific and Technical Information of China (English)

    于靖; 翟爱华

    2012-01-01

    试验以筛选制备米糠蛋白降压肽最佳用酶为目的。选用碱性蛋白酶、碱性蛋白酶Alcalase 2.4 L、中性蛋白酶、木瓜蛋白酶、胰蛋白酶、胰凝乳蛋白酶、蛋白酶K和双酶复合水解米糠蛋白,以血管紧张素转化酶(ACE)抑制率为主要指标,筛选出制备米糠蛋白降压肽的最佳用酶。结果表明,米糠降压肽ACE抑制率的大小与酶的种类及配比有关,筛选出碱性蛋白酶Alcalase为试验用酶,在酶解温度45℃,加酶量3 000 U.g-1,酶解pH8.5,米糠蛋白底物浓度3%酶解2 h时达到最大抑制率为71.1%。%The aim of this study was to select the best enzyme for rice bran protein.Rice bran protein was enzymolysised by alkali protease,Alcalase 2.4 L,neutral protease,papain,trysase,chymotrypsin,proteinase K and double enzyme composite to produce antihypertensive peptides,with ACE inhibiting rate as major indicator.The results indicated that ACE inhibiting rate of antihypertensive peptides in rice bran was related to the kind of enzyme and their ratio,and the best enzyme was alkali protease,the enzyme hydrolysis conditions were: hydrolysis temperature 45 ℃,the enzyme addition 3 000 U·g-1,pH8.5,substrate concentration 3%,hydrolysis time 2 h,its highest inhibiting rate reached 71.1%.

  2. Test Preparation: Your Role

    Science.gov (United States)

    ... could be asked to follow certain procedures to transport the specimen from home to the lab. Examples of some common laboratory tests that require advance preparation include: Glucose tolerance, fasting, and two-hour post-prandial blood ...

  3. Thyroid preparation overdose

    Science.gov (United States)

    Thyroid preparations are medicines used to treat thyroid gland disorders. Overdose occurs when someone takes more than the normal or recommended amount of this medicine. This can be by accident or ...

  4. Dukovany ASSET mission preparation

    International Nuclear Information System (INIS)

    We are in the final stages of the Dukovany ASSET mission 1996 preparation. I would like to present some of our recent experiences. Maybe they would be helpful to other plants, that host ASSET missions in future

  5. Preparation of ferritin national standard

    International Nuclear Information System (INIS)

    The raw material of ferritin is analyzed for preparation of ferritin national standard (NS) for immunoassay. No impure zone is found by polyacrylamide gel electrophoresis. The protein amount is 1.06 g/L and the immunoactivity is 1.015 g/L by RIA, with its dose-response curve being parallel to that of Human Ferritin 2nd International Standard coded 80/578(IS). This shows that the material used is suitable for preparation of ferritin NS. 1.25 mg of raw material diluted in 0.01 mol/L phosphate buffer solution (pH7.4) containing 1% human serum albumin is distributed into aliquot of 0.8 mL (2 μg per ampoule), lyophilized and assayed by RIA. The mean value of this NS is 1.962 μg per ampoule in terms of IS. Similar values are obtained for the ferritin contents of quality control serum samples at different content levels when either IS or NS is used as the assay calibrator. This indicates that there is no statistically significant difference between the serum sample values at the same levels

  6. Preparation and Characterization of Chitosan/soy Protein Isolate Packaging Composite Film%壳聚糖/大豆分离蛋白复合包装膜的制备与表征

    Institute of Scientific and Technical Information of China (English)

    刘幸幸; 王家俊; 刘海龙; 樊春艳

    2012-01-01

    采用壳聚糖(CS)和大豆分离蛋白(SPI)为基材,制备了可降解包装膜。用红外光谱、X射线衍射和扫描电镜对复合包装膜结构进行表征。对复合包装膜的拉伸性能和透光性能进行了测试和分析。结果表明:CS和SPI之间存在一定的相互作用;当SPI质量分数为10%时,复合包装膜的拉伸性能优于纯壳聚糖膜,透光性较纯壳聚糖膜略有下降。%The degradable packaging composite films made from chitosan(CS) and soy protein isolate(SPI) were developed. The structural features of CS/SPI composite films were investigated by mean of FTIR, XRD and SEM. The tensile properties and light transmission properties of the films were tested and analyzed. The results showed that certain interaction exists between CS and SPI; when the mass fraction of SPI is 10%, thepackaging composite film has higher tensile properties than that of chitosan film with slightly lower light transmittance than chitosan film.

  7. RHEOLOGY OF CHICKPEA PROTEIN CONCENTRATE DISPERSIONS

    OpenAIRE

    Aurelia Ionescu; Iuliana Aprodu; Gabriela Gurau; Iuliana Banu

    2011-01-01

    Chickpea proteins are used as ingredients in comminuted sausage products and many oriental textured foods. Rheological behaviour of chickpea protein concentrate was studied using a controlled stress rheometer. The protein dispersion prepared with phosphate buffer at pH 7.0 presented non-Newtonian shear thinning behaviour and rheological data well fitted to the Sisko, Carreau and Cross models. The viscoelastic properties of the chickpea protein suspensions were estimated by measuring the stora...

  8. Preparation and application of magnetic microsphere carriers

    Institute of Scientific and Technical Information of China (English)

    ZHANG Bo; XING Jianmin; LIU Huizhou

    2007-01-01

    Magnetic microsphere carriers have received considerable attention,primarily because of their wide applications in the fields of biomedicine and bioengineering.In this paper,preparation methods,surface modification and application of magnetic carriers are reviewed.Emphasis will be placed on recent biological and biomedical developments and trends such as enzyme immobilization,cell isolation,protein purification,target drugs and DNA separation.

  9. Preparation of food supplements from oilseed cakes

    OpenAIRE

    Sunil, L.; Appaiah, Prakruthi; Prasanth Kumar, P. K.; Gopala Krishna, A. G.

    2014-01-01

    Oilseed cakes have been in use for feed preparation. Being rich in proteins, antioxidants, fibers, vitamins and minerals, oilseed cakes have been considered ideal for food supplementation. These oilseed cakes can be processed and made more palatable and edible by suitable treatments and then incorporated as food supplements for human consumption. Rice bran pellets (RBP), stabilized rice bran (SRB), coconut cake (CC) and sesame cake (SC) were taken up for the study. These were mixed with disti...

  10. 发酵剂对双蛋白干酪理化特性及风味的影响%Physico-chemical Characteristics and Flavor of Dual-protein Cheese Prepared with Different Starters

    Institute of Scientific and Technical Information of China (English)

    李丽; 岳喜庆; 张莉; 张健; 杨贞耐

    2012-01-01

    Cheddar-type cheese was made by fermenting cow,s milk alone and in the presence of soybean milk with either aboratory-screened(consisting of L.lactis subsp.cremoris QH27-1 or L.lactis subsp.lactis XZ3303) and commercial fermentation starters and analyzed for physicochemical and sensory characteristics,texture and flavor composition.The results showed that the addition of soybean milk had no negative influence on the quality of Cheddar-type cheese.In addition,the Cheddar-type cheese fermented by L.lactis subsp.cremoris QH27-1 and L.lactis subsp.lactis XZ3303 revealed an improvement in quality.Hence,the laboratory-screened fermentation starter is applicable for the production of dual-protein cheese.%在牛乳和添加豆乳(质量分数10%)的牛乳中分别使用筛选发酵剂与商品发酵剂进行切达干酪生产,并对成熟干酪的理化成分、质地、风味成分和感官特性进行分析。结果表明,豆乳的添加对干酪的质地、风味和感官特性均无不良影响,而应用筛选发酵剂L.lactis subsp.cremoris QH27-1和L.lactis subsp.lactis XZ3303生产双蛋白切达干酪对干酪的品质有一定的改善作用,可将其应用于双蛋白干酪生产中。

  11. Preparation of Glycol-Fosfomycin Modified Zirconia Chromatography Stationary Phases and Its Application to the Separation of Proteins%乙二醇-磷霉素改性氧化锆色谱固定相的制备及其在蛋白质分离中的应用

    Institute of Scientific and Technical Information of China (English)

    张淑琼; 邹凤平; 李烃

    2009-01-01

    A novel glycol-fosfomycin modified zirconia (G-F-ZrO_2) stationary phase was prepared and characterized by elemental analysis, FT-IR and N_2 adsorption. Its chromatographic performance was evaluated using four standard proteins including lysozyme (Lys), ribonuclease-A (Rnase-A), α-chymotrypsin (α-Chy) and Cytochrome C (Cyt-C) as probes, and the effects of salt concentration, pH, salt type in eluents and temperature on retention behavior of proteins were investigated. The results indicate that the stationary phase behaves mainly as a hydrophobic interaction chromatographic packing with salt concentration in the mobile phase above 1.0 moh·L~(-1). At the same time, the retention mechanism between protein and G-F-ZrO_2 stationary phase was also discussed.%合成了分离蛋白质的乙二醇-磷霉素钠改性氧化锆高效液相色谱固定相,通过漫反射红外光谱、元素分析等分析方法对该固定相进行了表征.以溶菌酶、核糖核酸酶A、细胞色素C和糜蛋白酶四种标准碱性蛋白质为探针,系统地考察了固定相的疏水相互作用色谱性能.结果表明,乙二醇-磷霉素改性氧化锆固定相对蛋白质有一定的保留,表现出较高的分离选择性.

  12. 山羊PTHrP基因干扰重组腺病毒的制备与鉴定%Preparation and identification of recombinant adenoviruses carrying short hairpin RNA targeting parathyroid hormone related protein of goat

    Institute of Scientific and Technical Information of China (English)

    邢瑞芳; 郑惠玲; 刘雪梅; 闫林慧; 安俊辉; 杨振宇; 祝珍珍

    2011-01-01

    Parathyroid hormone related protein (PTHrP) has important biological functions in calcium metabolism. The aim of this study was to silence the expression of PTHrP by RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322/357) targeting two different sites of PTHrP mRNA. Then the oligonucleotides were inserted intoshuttle vector pENTR/CMV-GFP/U6. After detection of the interference efficiency by Western blotting, we chose pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST to produce recombinant vector pAD/PL-DEST/CMV-GFP/U6-322. The first generation recombinant adenovirus particles (AD-PTHrP-322) were produced and further amplified by transfecting HEK-293 cells. The titer of the recombinant adenovirus reached 2.0×l09 PFU/mL determined by TCID50 assays. The result of real-time quantitative PCR indicated that mRNA expression levels of gene were reduced 29.2%, 68.1% and 82.6% (P<0.05), respectively, when goat mammary gland epithelial cells were infected with AD-PTHrP-322 after24, 48 and 72 h, in which PTHrP. Western blotting also showed that the expression of PTHrP was reduced by infecting the cells with AD-PTHrP-322. AD-PTHrP-322 has been proved with significant interference effect on expression of PTHrP.%甲状旁腺激素相关蛋白(Parathyroid hormone related protein,PTHrP)具有广泛生物学功能,对调控钙代谢具有重要作用.采用RNA干扰和重组腺病毒技术,对山羊乳腺上皮细胞中PTHrP基因表达进行沉默,为进一步研究该基因在乳腺上皮细胞中的功能奠定基础.采用BLOCK-iT shRNA腺病毒干扰系统,将设计好的寡聚shRNA-322/357经退火复性后插入穿梭质粒pENTR/CMV-GFP/U6中.经Western blotting检测干扰

  13. Preparation and characterization of beta-carotene nanodispersions prepared by solvent displacement technique.

    Science.gov (United States)

    Chu, Boon-Seang; Ichikawa, Sosaku; Kanafusa, Sumiyo; Nakajima, Mitsutoshi

    2007-08-01

    This work demonstrated the preparation of protein-stabilized beta-carotene nanodispersions using the solvent displacement technique. The emulsifying performance of sodium caseinate (SC), whey protein concentrate (WPC), whey protein isolate (WPI), and a whey protein hydrolysate (WPH, 18% degree of hydrolysis) was compared in terms of particle size and zeta-potential of the nanodispersions. SC-stabilized nanodispersions exhibited a bimodal particle size distribution: large particles (stabilized by casein micelles) with a mean particle size of 171 nm and small particles (stabilized by casein submicelles) of 13 nm. This was confirmed with transmission electron microscopy analysis. Most of the beta-carotene precipitated (87.6%) was stabilized in the small particles. On the other hand, the nanodispersions stabilized by the whey proteins were polydispersed with larger mean particle sizes. The mean particle size of WPC and WPI was 1730 and 201 nm, respectively. The SC-stabilized nanodispersion was expected to be more stable as indicated by its higher absolute zeta-potential value (-31 mV) compared to that of WPC (-15 mV) and WPI (-16 mV). Partially hydrolyzed whey protein possessed improved emulsifying properties as shown by WPH-stabilized samples. It was interesting to note that increasing the SC concentration from 0.05 to 0.5 wt % increased the particle size of beta-carotene stabilized by casein micelles, while the reverse was true for those stabilized by SC submicelles. Microfluidization at 100 MPa of SC solution dissociated the casein micelles, resulting in a decrease in mean particle size of the casein micelle-stabilized particles when the SC solution was used to prepare nanodispersions. The results from this work showed that protein-stabilized beta-carotene nanodispersions could be prepared using the solvent displacement technique. PMID:17630759

  14. Surface preparation of niobium

    International Nuclear Information System (INIS)

    Any discussion of surface preparation for superconducting rf-surfaces is certainly connected with the question what is the best recipe for achieving high Q-values and high break-down fields. Since the break-down in a cavity is not understood so far and because several mechanisms play a role, it also is not possible to give one recipe which always works. Nevertheless in the past certain preparation techniques for niobium surfaces have been developed and certain rules for preparation can be applied. In the following the to-days state of the art will be described and it is attempted to give a short description of the surface in conjunction with the methods of surface treatments, which generally can be applied to niobium cavities. (orig./WTR)

  15. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  16. 大豆蛋白/κ-卡拉胶冷致凝胶的制备及控释特性%Preparation and Controlled Release Property of Soy Protein/κ-Carrageenan Cold-Set Gels

    Institute of Scientific and Technical Information of China (English)

    杨晓泉; 周小玲; 王晓园; 尹寿伟

    2011-01-01

    为构建具有控释特性的可食性输送载体,利用转谷氨酰胺酶交联大豆蛋白与κ-卡拉胶形成蛋白/多糖复合冷致凝胶,分析了复合凝胶的微结构与流变学性质,并将复合凝胶冷冻干燥压制成片剂,对其在模拟胃肠液中对核黄素的控释特性和释放动力学进行了分析.结果表明:复合凝胶呈现致密的网络结构,κ-卡拉胶的加入增强了复合凝胶的强度和硬度,荷载核黄素则弱化了复合凝胶的强度和硬度;在模拟消化道中,复合凝胶及片剂胃液释放1h的累积释放率低于17%,移入肠液后9h累积释放率为75%~100%,缓释效果良好;核黄素在模拟胃液和肠液中的释放机制不同,分别由Fick扩散和非Fick扩散控制.%In order to construct edible conveying carriers with excellent controlled release properties, soy protein/ K-carrageenah mixed cold-set gels were produced via the crosslinking of microbial transglutaminase ( MTGase). Then, the microstructure and rheological properties of the mixed gels were analyzed, and the tablets flaked by the gels via the freeze-drying were used as the controlled release carriers for riboflavin to investigate the release properties and the release dynamics in simulated gastrointestinal fluid. The results show that ( 1) the mixed gels are structurally in dense network; (2) K-carrageenan improves the strength and hardness of the mixed gels, while riboflavin weakens the gel properties; (3) in simulated gastrointestinal condition, the one-hour accumulative release rate of the gels and the tablets for riboflavin in simulated gastric fluid is lower than 17% but ranges from 75% to 100% after a moving to simulated intestinal fluid for 9h, which means that both the gels and the tablets have sustained release effect for riboflavin. In addition, it is found that the release mechanisms of riboflavin in simulated gastric and intestinal fluids are different from each other; Specifically, one is controlled

  17. 高蛋白浓缩牦牛骨汤煮制工艺及风味分析%Preparation method and taste analysis of enriched Yak bone soup with high protein

    Institute of Scientific and Technical Information of China (English)

    郑娅; 余群力; 张玉斌

    2012-01-01

    以牦牛骨为原料,采用Central Composite设计,通过测定浓缩骨汤中蛋白质含量,得出高蛋白浓缩牦牛骨汤最佳煮制工艺条件为:骨块径3cm、料液比3∶20、煮制时间4h,在此条件下煮制的牛骨汤,蛋白质含量较高,具有牛骨汤特有风味。通过GC/MS检测,高蛋白浓缩牦牛骨汤挥发性物质丰富,共74种,其特征香气主要来自醛类、酯类、醇类、酮类以及杂环类化合物,其中2,4-己二烯醛、反-2-庚烯醛、反式-2,4-癸二烯醛、反式-2-十二烯醛、反-2-己烯醛、苯并噻唑、4-丙烯基-2-甲氧基苯酚等物质都对高蛋白浓缩骨牛骨汤特征风味有贡献。高蛋白浓缩骨牛骨汤除了含有许多鲜牛骨所含有的特征性物质外,还产生了2,5-二甲基吡嗪、2,3-二甲基吡嗪、β-倍半水芹烯、2,3-辛二酮、1-辛烯-3-醇等形成骨汤风味的特色挥发性化合物。%Yak bone was raw material in this study,studied influence of optimization of concentrated bone soup of collagen protein content by Central composite design. It was concluded that the maximum response value corresponding to the optimal conditions which the bone block size was 3cm,liquid ratio was 3:20,and boiling time was 4h. Under the condition,the bone soup decocted best,volatile substances were found in bone soup rich in volatile components of 74 kinds by GC/MS testing. The characteristic aroma was from aldehydes, esters,alcohols, ketones and heterocyclic compounds, including 2,4-sorbaldehyde, heptyl-2-hepenal, heptyl- 2;4-decadiena;hepty2-ddecena-hepty-2-hexena-benzthiaze4-prpeny-2-meth~xyphen and so on which had contributed to concentrated bone soup characteristic flavour. In addition,contained many fresh bone that contains the characteristic substances,also produced a lot of material what the fresh bone did not contain. And it also produced 2,5 -dimethylpyrazine, 2,3 -dimethylpyrazine,13 -sesquiphellandrene, 2,3 - Octanedione, 1-octene-3-alcohol and so on,

  18. Preparation of americium amalgam

    International Nuclear Information System (INIS)

    Using the method of NGR-spectroscopy with the aid of 241Am isotope chemical state of transuranium elements in the volume and on the surface of amalgams is studied. Amalgam preparation was realized in a simplified electrolytic cell. It is shown that in the process of amalgam preparation the first order of reaction as to actinide is observed; americium is distributed gradually over the volume and it is partially sorbed by the surface of glass capillary. NGR spectrum of dry residue after mercury distillation at 200 deg C points to the presence of americium-mercury intermetal compounds

  19. Preparation of hydrophobic coatings

    Science.gov (United States)

    Branson, Eric D.; Shah, Pratik B.; Singh, Seema; Brinker, C. Jeffrey

    2009-02-03

    A method for preparing a hydrophobic coating by preparing a precursor sol comprising a metal alkoxide, a solvent, a basic catalyst, a fluoroalkyl compound and water, depositing the precursor sol as a film onto a surface, such as a substrate or a pipe, heating, the film and exposing the film to a hydrophobic silane compound to form a hydrophobic coating with a contact angle greater than approximately 150.degree.. The contact angle of the film can be controlled by exposure to ultraviolet radiation to reduce the contact angle and subsequent exposure to a hydrophobic silane compound to increase the contact angle.

  20. Protein Structure

    Science.gov (United States)

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  1. Preparing Global Citizens

    Science.gov (United States)

    Roberts, Dennis C.; Welch, Lucas; Al-Khanji, Khalid

    2013-01-01

    Global citizens are those who are aware of, demonstrate respect for, and are comfortable engaging across cultural boundaries. This article explores why preparing global citizens is important and how positive psychology can inform our understanding of those who engage comfortably in today's complicated world. Soliya's Connect program is described…

  2. Preparing for CLIC tests

    CERN Multimedia

    Laurent Guiraud

    1999-01-01

    The Canon 5 undergoes first brazing for preparation in the CLIC study at the CLIC Test Facility 2 (CTF2). This will test injection for a proposed linear collider that will further explore discoveries made at the LHC. Electric fields in the canon will boost electrons into the acceleration fields of the collider.

  3. Preparation of 1-bromoheptacosane

    International Nuclear Information System (INIS)

    Alkybromides are ones of the main organic precursors for fatty acids and alcohols labelling with Carbon 1-14. In this work the preparation of 1-bromoheptacosane by bromodescarboxylation of 1-octacosanoic acid is described. The synthesis yielded 80.5% of final product and more than 97% of chemical purity. Clean-up procedure modifications and spectral data bromoheptacosane are also reported

  4. Teaching Preparation Program (TPP).

    Science.gov (United States)

    California Univ., Los Angeles. Dept. of Geography.

    Because most graduate geography students will engage in professional teaching activities, the Teaching Preparation Program of UCLA's department of geography is viewed as an important part of graduate training. The program, co-directed by a graduate student and faculty member, is available to all graduate students on a voluntary basis and consists…

  5. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc;

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased...... concentration of tau protein in CSF from patients with relapsing-remitting MS and patients monosymptomatic at onset who progressed to MS, but interestingly no increased tau protein concentration in monosymptomatic ON. The concentration of tau protein was significantly correlated to Expanded Disability Status...

  6. Adhesives from modified soy protein

    Science.gov (United States)

    Sun, Susan; Wang, Donghai; Zhong, Zhikai; Yang, Guang

    2008-08-26

    The, present invention provides useful adhesive compositions having similar adhesive properties to conventional UF and PPF resins. The compositions generally include a protein portion and modifying ingredient portion selected from the group consisting of carboxyl-containing compounds, aldehyde-containing compounds, epoxy group-containing compounds, and mixtures thereof. The composition is preferably prepared at a pH level at or near the isoelectric point of the protein. In other preferred forms, the adhesive composition includes a protein portion and a carboxyl-containing group portion.

  7. Total Integrated Sample Preparation for Microfluidic Immunoassays in Complex Biological Matrices

    OpenAIRE

    Apori, Akwasi Asare

    2011-01-01

    A high-throughput protein analysis platform with integrated sample preparation is developed to address the identified technology gaps in biomarker validation, clinical and point-of-care diagnostics. The goals of the technology are to automate and integrate protein sample preparation with electrokinetic separations, implement immunoassays capable of processing raw biological fluids, and perform high-throughput protein assays targeted for disease diagnosis.Integration of multiple functions is ...

  8. 响应面法优化秘鲁鱿鱼(Dosidicus gigas)肌肉盐溶蛋白的提取和凝胶形成条件%Response Surface Optimization of Conditions for Extraction and Gel Preparation of Salt-soluble Proteins from Dosidicus gigas Muscle

    Institute of Scientific and Technical Information of China (English)

    金淼; 周逸; 徐亦及; 唐剑波; 杨文鸽; 张进杰

    2013-01-01

    The effect of extraction and gel preparation conditions of salt-soluble protein from Dosidicus gigas on its gel characteristics were discussed.Using respond surface methodology,the effects of KCl concentration,pH and lowtemperature heating time on the water holding capacity(WHC) and texture characteristics of salt-soluble protein gel from squid meat were studied.Result:(1) The effect of KCl concentration on the WHC of gel was extremely significant; the effect of KCl concentration and low-temperature heating time on the hardness of gel was significant ; both the effect of pH on the WHC and low-temperature heating time on the stickiness of gel were significant.(2) Response surface and contour were graphed with the WHC,hardness and stickiness of gel as the response value,the optimal extraction and gel preparation conditions of salt-soluble proteins from Dosidicus gigas muscle were 0.16mol· L-1 KCl,pH7.10 and 55min of low-temperature heating time,respectively.Experimentally,these conditions produced a salt-soluble protein gel with a WHC of 88.00%,hardness of 510.00 g,stickiness of-11.00 g,confirming the model prediction.%探讨了秘鲁鱿鱼肉盐溶蛋白的提取及凝胶形成条件对其凝胶特性的影响.采用响应面分析法,研究KCl浓度、pH值和低温加热时间对鱿鱼肉盐溶蛋白凝胶保水性和质构特性的影响.结果显示:(1)提取液KCl浓度对保水性影响极其显著,对硬度影响显著;提取液pH值对保水性影响显著;低温加热时间对硬度和粘性影响显著;(2)在分析各因素显著性及其交互作用的基础上,确定鱿鱼肉盐溶蛋白提取和凝胶形成的适宜条件为:KCl浓度0.16 mol·L-1、pH 7.10、40℃加热55min,此时凝胶保水性、硬度、粘性分别达到88.00%、510.00g、-ll.00g,与模型预测值相符.

  9. Interplay of phase II enzymes and transporters in futile cycling: influence of multidrug resistance-associated protein 2-mediated excretion of estradiol 17beta-D-glucuronide and its 3-sulfate metabolite on net sulfation in perfused TR(-) and Wistar rat liver preparations.

    Science.gov (United States)

    Sun, Huadong; Zeng, Ying-Ying; Pang, K Sandy

    2010-05-01

    The hepatic disposition of estradiol 17beta-D-glucuronide (E(2)17G), a substrate of the organic anion-transporting polypeptides Oatp1a1, Oatp1a4, and Oatp1b2, was investigated in Wistar and TR(-) [multidrug resistance-associated protein (Mrp) 2-mutant] rats to elucidate how absence of Mrp2, the major excretory transporter for both E(2)17G and its 3-sulfate metabolite (E(2)3S17G), affected the net sulfation. With absence of Mrp2, lower microsomal desulfation activity and higher Mrp3 but unchanged immunoreactive protein expression of other transporters (Oatps and Mrp4) and estrogen sulfotransferase were found in TR(-) rats. In recirculating, perfused liver preparations, the rapid decay of E(2)17G and sluggish appearance of low levels of E(2)3S17G in perfusate for Wistar livers were replaced by a protracted, biexponential decay of E(2)17G and greater accumulation of E(2)3S17G, whose levels reached plateaus upon the almost complete obliteration of biliary excretion of E(2)17G and E(2)3S17G in the TR(-) liver. Much higher amounts of E(2)17G (28x) and E(2)3S17G (11x) in liver and reduced net sulfation (40 +/- 6 from 77 +/- 6% dose, P Mrp2 function led to decreased net sulfation of E(2)17G by raising the intracellular concentration of the metabolite, E(2)3S17G, which readily refurnished E(2)17G via desulfation. PMID:20124397

  10. Isolation of monodisperse nanodisc-reconstituted membrane proteins using free flow electrophoresis

    DEFF Research Database (Denmark)

    Justesen, Bo Højen; Laursen, Tomas; Weber, Gerhard;

    2013-01-01

    Free flow electrophoresis is used for rapid and high-recovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e.......g., fluorescent tags. Preparative separation of membrane protein loaded nanodiscs from empty nanodiscs and protein aggregates results in monodisperse nanodisc preparations ideal for structural and functional characterization using biophysical methods. © 2013 American Chemical Society....

  11. 结核分枝杆菌调节蛋白RelA的原核表达及多克隆抗体制备%Preparation of polyclonal antibody and prokaryotic expression of recombinant protein RelA from Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    伊正君; 付玉荣; 许福亮; 高昆山; 李猛; 李建花

    2012-01-01

    AIM: To prepare polyclonal antibodies a-gainst RelA protein of Mycobacterium tuberculosis. METHODS : RelA gene segment was inserted into pET-32a ( + ) and the recombinant protein RelA was expressed in E. coli under IPTG induction. The protein was purified and identifed by SDS-PAGE and Western blot. Polyclonal antibody to RelA was got by immunizing rabbits with the protein. Quality and quantity of the antibody was identified. RESULTS: RelA gene segment was successfully inserted into pET-32a ( + ) and recombinant protein RelA was obtained. The polyclonal antibody to RelA had a good specificity, and the titer reached more than 1:6 400. CONCLUSION: RelA recombinant protein and rabbit anti-RelA polyclonal antibody with high specificity were obtained, which provided good tools for further studying functional characterization of RelA.%目的:克隆编码结核分枝杆菌调节基因RelA并在大肠杆菌中表达,纯化后制备兔抗RelA的抗体.方法:利用PCR从结核分枝杆菌H37Rv株中扩增RelA基因,构建重组表达质粒pET-32a(+ )-RelA;以重组质粒转化大肠杆菌BL21( DE3),筛选阳性重组菌株,IPTG诱导目的蛋白表达,在变性条件下对目的蛋白进行镍离子亲和层析纯化,通过SDS-PAGE和Westem blot鉴定目的蛋白的表达及反应原性;以表达的RelA蛋白免疫家兔,制备抗RelA的多克隆抗体并进行效价及特异性鉴定.结果:扩增了RelA基因,克隆于表达载体pET-32a(+)中,PCR筛选和酶切鉴定获得阳性克隆,测序证实正确.经诱导在大肠杆菌中表达出相对分子质量(Mr)为120 000的目的蛋白;纯化的RelA免疫家兔后,能有效地刺激特异性抗体的产生,抗血清的效价达到1:6 400以上,且具有良好的特异性.结论:已成功构建RelA基因的原核表达载体,并在大肠杆菌中获得高效表达;制备出兔抗RelA抗体,效价及特异性均良好,为进一步研究RelA蛋白在结核病中的致病机制奠定了基础.

  12. New properties of immunotropic preparation from porcine skin.

    Science.gov (United States)

    Belova, O V; Arion, V Y; Zimina, I V; Lukandina, T A; Krotova, S B; Sysoeva, O B; Tret'yakov, V A

    2007-09-01

    We studied new immunological and physicochemical properties of K-activin, immunotropic preparation from porcine skin isolated by the acetone method. The preparation restored the sensitivity of background rosette-forming cells in the spleen of thymectomized mice to the inhibitory effect of azathioprine in vivo and practically normalized serum thymic activity reduced in thymectomized mice. The molecular weight of proteins present in K-activin and previously detected by SDS-PAAG electrophoresis was determined by MALDI mass spectrometry PMID:18457058

  13. Preparation of UFsub(4)

    International Nuclear Information System (INIS)

    The Uranium tetrafluoride (UF4) has been prepared via a single step reduction of uranium hexafluoride (UF6) with hydrogen and fluorine gases in the cold wall reactor. The tap density and shape of dense UF4 power are largely dependent on the nozzles used, either concentric nozzle or the nozzle separated. The round shape of dense UF4 power are obtained by adding of hydrogen gas and UF6-F2 gaseous mixture through the nozzle separated. Feeding rate, ratio of three gases and preheating temperature have also a marked effect upon the its density and particle size. The specification of the UF4 product obtained using the nozzle separated in the cold wall reactor are well met on the preparation of uranium metal using UF4 and magnesium powder. (Author)

  14. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2009-01-01

    The Human Resources Department is organizing a preparation for retirement seminar, which will take place on the afternoons of the 11, 13, 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members below 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to availability of places. Registration: In view of the number of people concerned and the limited capacity of the main auditorium, you are requested to register in advance via ...

  15. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2009-01-01

    The Human Resources Department is organizing a preparation for retirement seminar, which will take place on the afternoons of the 11, 13, 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members below 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to the availability of places. Registration: In view of the number of people concerned and the limited capacity of the Main Auditorium, you are requested to register in advance ...

  16. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2009-01-01

    The Human Resources Department is organizing a preparation for retirement seminar, which will take place in the afternoons of 11, 13, 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members below 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to the availability of places. Registration: In view of the number of people concerned and the limited capacity of the Main Auditorium, you are requested to register in advance via Ind...

  17. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2009-01-01

    The Human Resources Department is organizing a preparation for retirement seminar, which will take place on the afternoons of the 11, 13, 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members under the age of 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to the availability of places. Registration: In view of the number of people concerned and the limited capacity of the Main Auditorium, you are requested to register ...

  18. Holographic characterization of protein aggregates

    Science.gov (United States)

    Wang, Chen; Zhong, Xiao; Ruffner, David; Stutt, Alexandra; Philips, Laura; Ward, Michael; Grier, David

    Holographic characterization directly measures the size distribution of subvisible protein aggregates in suspension and offers insights into their morphology. Based on holographic video microscopy, this analytical technique records and interprets holograms of individual aggregates in protein solutions as they flow down a microfluidic channel, without requiring labeling or other exceptional sample preparation. The hologram of an individual protein aggregate is analyzed in real time with the Lorenz-Mie theory of light scattering to measure that aggregate's size and optical properties. Detecting, counting and characterizing subvisible aggregates proceeds fast enough for time-resolved studies, and lends itself to tracking trends in protein aggregation arising from changing environmental factors. No other analytical technique provides such a wealth of particle-resolved characterization data in situ. Holographic characterization promises accelerated development of therapeutic protein formulations, improved process control during manufacturing, and streamlined quality assurance during storage and at the point of use. Mrsec and MRI program of the NSF, Spheryx Inc.

  19. Characterization of alkali-modified soy protein concentrate

    Directory of Open Access Journals (Sweden)

    Barać Miroljub B.

    2005-01-01

    Full Text Available To study the influence of the preparation mode, including mild alkali modification, of soy protein concentrate on soluble protein content and composition, some of its nutritive and functional properties were investigated. Soy protein concentrate prepared by aqueous alcohol leaching was modified in mild alkaline solutions (pH 8.0 at 40, 50 and 60° C for 60 minutes and compared with two principal types of commercial soy protein concentrate. Soluble protein content, composition and properties of soy protein concentrate, as well as their potential use are essentially determined by the preparation mode. Limited mild alkali hydrolysis increased protein solubility by 40-71%, while emulsion stability was increased by 18-56%. Major storage soybean proteins exhibited different stability to alcohol denaturation and mild alkali modification. The most susceptible were acidic -A3 - and -A5- subunits of glycinin.

  20. Sperm preparation for fertilization

    OpenAIRE

    Gadella, B.M.

    2014-01-01

    Description This book contains 19 chapters that discuss theoretical and applied andrology for domestic, zoo and wild animals. Topics include semen and its constituents; sperm production and harvest; determinants of sperm morphology; sperm preparation for fertilization; practical aspects of semen cryopreservation; evaluation of semen in the andrology laboratory; genetic aspects of male reproduction; emerging techniques and future development of semen evaluation and handling and applied androlo...

  1. PREPARATION OF PLUTONIUM HALIDES

    Science.gov (United States)

    Davidson, N.R.; Katz, J.J.

    1958-11-01

    A process ls presented for the preparation of plutonium trihalides. Plutonium oxide or a compound which may be readily converted to plutonlum oxide, for example, a plutonium hydroxide or plutonlum oxalate is contacted with a suitable halogenating agent. Speciflc agents mentioned are carbon tetrachloride, carbon tetrabromide, sulfur dioxide, and phosphorus pentachloride. The reaction is carried out under superatmospberic pressure at about 300 icient laborato C.

  2. Preparation for painting

    OpenAIRE

    2015-01-01

    "The aesthetic qualities of a painted work of art are determined by composition, the colours used and the application method. Equally important are the support and its preparation. Preparatory layers are fundamental to the creative process and to the perception of the work: they influence the final surface texture, the paints' luminosity and the durability of a painting. For centuries painters were well aware of these facts and took great care in this stage of the process." "These papers were...

  3. TORIS Data Preparation Guidelines

    Energy Technology Data Exchange (ETDEWEB)

    Guinn, H.; Remson, D.

    1999-03-11

    The objective of this manual is to present guidelines and procedures for the preparation of new data for the Tertiary Oil Recovery Information System (TORIS) data base. TORIS is an analytical system currently maintained by the Department of Energy's (DOE) Bartlesville Project Office. It uses an extensive field- and reservoir-level data base to evaluate the technical and economic recovery potential of specific crude oil reservoirs.

  4. Overview of Site Preparation

    International Nuclear Information System (INIS)

    The preparation of Cadarache as the host of ITER is organised at a double level: Europe, since the beginning of the candidature in 2001, is coordinating the so-called European ITER Site Studies; France, as the host country, has put in place a dedicated structure at a decisional level (close to the government), and operational level in the PACA region with two entities: The Agency Iter France (AIF), inside the CEA, interlocutor of international and European entities, in charge of site preparation and fund recollection; An accompanying prefectoral mission, in charge mainly of road adaptation and the international school. The paper will cover all the aspects related to the preparation of the implementation of ITER: Technical aspects: the progress of site preparation itself, its servicing (water supply, electrical supply, Internet...), the road adaptation between the large harbour of Fos-sur-mer and Cadarache, etc. will be detailed. Regulatory procedures: in the framework of the delegation that the ITER partners gave to the CEA/AIF on 14th September 2005, two main large files are in progress: The public debate, organised by an independent authority, informs the population of the challenges and impacts of ITER in Provence; The safety documents: the writing of the preliminary safety report, which will be submitted to the Nuclear Safety Authority and the files submitted to the public during the public enquiries are ongoing. Socioeconomic aspects: the welcome of ITER staff and their families is operational, via a dedicated Welcome Office; the location of an international school in Manosque leads now to its pre-figuration. The overall organisation will be described, as well as all planning forecast for the coming years, leading to the start of construction. (author)

  5. Preparation of americium amalgam

    International Nuclear Information System (INIS)

    The authors describe a method for the electrochemical preparation of an americium amalgam from americium dioxide and americium 241 and 243 for use in determining the physicochemical properties of the alloy. Moessbauer spectra were made using neptunium dioxide, in the neptunium 237 form, as an absorber. Results show that electrolysis produces a homogeneous amalgam that gives an unoxidized product on vacuum distillation at 200 degrees C

  6. The Preparation of Graphene

    Institute of Scientific and Technical Information of China (English)

    Chen Yanyan

    2015-01-01

    Graphene has unique structure and possesses excellent physical and chemical properties, and it has received a great deal of attention in related research fields. The quality, quantity and application of graphene are related to its preparation methods. At present the bottleneck of graphene research is that both high-quality and large quantity of graphene could not be obtained simultaneously and the reason is that the basic mechanism of graphene formation has mot been wel understood.

  7. Preparation to exceptional operations

    International Nuclear Information System (INIS)

    Preparation to special maintenance operations requires a specific approach according to the considered intervention type. Replacement of vapor generators is representative of a kind of intervention where technics is generally only an adaptation to the power plant context of processes already in application in construction, and where methodology, planning and organization have an important role because of the variety and the quantity of taskworks to be done, the involved manpower, the dosimetry and time lag requirements

  8. Preparing for ERP Implementation

    OpenAIRE

    Karkio, Antti

    2014-01-01

    This Thesis develops a proposal for Operations and the local management in the case company how to prepare for ERP implementation and ensure successful implementation. ERP implementation can be considered successful when the ERP system has been in effective use after a certain period of time after implementation. The objective of this Thesis is to create recommendations and action points how to ensure the efficient implementation of ERP. The Thesis is focused on the pre-implementation ph...

  9. Preparation of chitosan gel

    Directory of Open Access Journals (Sweden)

    Lagerge S.

    2012-06-01

    Full Text Available Aerogel conditioning of the chitosan makes it possible to prepare porous solids of significant specific surface. The increase in the chitosan concentration or the degree of acetylation decreases the specific surface of the synthesized chitosan gel. Whereas drying with supercritical CO2 more effectively makes it possible to preserve the volume of the spheres of gel and to have a more significant specific surface in comparison with evaporative drying.

  10. Protein politics

    OpenAIRE

    Vijver, Marike

    2005-01-01

    This study is part of the program of the interdisciplinary research group Profetas (protein foods, environment, technology and society). Profetas consists of technological, environmental and socio-economic research projects on protein food systems which result in the development of scenarios and strategies for guiding a shift towards a more plant protein based diet. The different research projects focus on the goal of identifying viable options for a more sustainable food system. Profetas aro...

  11. Principles of protein-protein interactions.

    OpenAIRE

    Jones, S; Thornton, J. M.

    1996-01-01

    This review examines protein complexes in the Brookhaven Protein Databank to gain a better understanding of the principles governing the interactions involved in protein-protein recognition. The factors that influence the formation of protein-protein complexes are explored in four different types of protein-protein complexes--homodimeric proteins, heterodimeric proteins, enzyme-inhibitor complexes, and antibody-protein complexes. The comparison between the complexes highlights differences tha...

  12. Nanochemistry of Protein-Based Delivery Agents.

    Science.gov (United States)

    Rajendran, Subin R C K; Udenigwe, Chibuike C; Yada, Rickey Y

    2016-01-01

    The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior. PMID:27489854

  13. Preparation and purification of mouse monoclonal antibody against NMDAR1 by ProteinA - Sepharose CL - 4B affinity chromatography%应用Protein A亲和层析法制备及纯化R1JHL单克隆抗体

    Institute of Scientific and Technical Information of China (English)

    李桂新; 孙长凯; 程桂芝; 罗建红; 李芳; 徐淑君

    2008-01-01

    [目的]制备、纯化N-甲基D-门冬氨酸受体(NMDAR,NR)主亚基RIJHL单克隆抗体.[方法]NR1杂交瘤细胞腹腔注射BalB/C小鼠制备抗体,Protein A-Sepharose CL-4B亲和层析纯化单抗,Western-blot鉴定该单抗特异性.[结果]纯化后单抗识别大鼠脑组织膜蛋白中约115 kD大小的单一条带.[结论]制备、纯化了具有高度特异性的抗NR1单克隆抗体,为进一步研究NMDA受体提供了工具.

  14. Preparation of polyclonal antiserum against recombinant NSP2 protein of PRRSV HH08 strain and study on biological functions of the polyclonal antiserum%猪繁殖与呼吸综合征病毒HH08株NSP2蛋白多克隆抗体的制备及其生物学功能的研究

    Institute of Scientific and Technical Information of China (English)

    马玲; 李广兴; 洪琴; 任玉东; 任晓峰

    2013-01-01

    利用RT-PCR和SOE PCR技术扩增得到猪繁殖与呼吸综合征病毒(PRRSV) HH08株NSP2全长基因,经抗原性和亲水性分析,将NSP2部分序列成功亚克隆于pET-30a(+)和PVAX1栽体中.将阳性重组质粒pET30a-NSP2转化E.coli Rosetta(DE3)感受态细胞,经诱导表达获得107 ku的重组NSP2蛋白.Western-blot检测表明,重组蛋白能够与PRRSV参考阳性血清反应.以纯化的重组NSP2蛋白免疫新西兰白兔制备多克隆抗体,其ELISA效价达到1∶1015以上;Western-blot试验表明,其具有良好的反应性和特异性.间接免疫荧光试验显示,用抗NSP2多克隆抗体可以检测到PVAX-NSP2转染BHK 21细胞所表达的NSP2蛋白,且与经典型和高致病型PRRSV毒株均有很好的特异性反应.病毒感染抑制试验表明,多抗血清对PRRSV经典和高致病性毒株的抑制率可达到68%和53%.上述研究结果为PRRSV检测及NSP2蛋白功能的深入研究奠定了基础.%In this study,the complete PRRSV HH08 NSP2 gene was cloned and the partial gene sub-cloned into prokaryotic expression vector pET-30a( + ) and eukaryotic expression vector PVAX1 after hy-drophilicity plot and antigenic index analysis. E. coli Rosetta (DE3) was transformed with the recombinant plasmid pET30a-NSP2. The recombinant NSP2 protein that molecular weight is 107 ku was expressed. It could be recognized by specific PRRSV antisera in western blot. Then the purified recombinant NSP2 protein as antigen was used to immunize rabbit for preparation of anti-NSP2 polyclonal antibody. An indirect ELISA assays showed that the titer of anti-NSP2 polyclonal antibody was 1 : 1015 , and it had highly reactivity and specialty in Western-blot. Also.IFA test demonstrated that this polyclonal antibody could react with the BHK-21 cells which can express PRRSV NSP2 protein and the Marc-145 cells infected with PRRSV. Both attenuated and highly pathogenic PRRSV strains could be inhibited by the anti-NSP2 polyclonal antibody and the

  15. Preparation of poly(guanidinium ionic liquid)s materials and evaluation of their recognition properties for protein%聚胍基离子液体材料制备及其对蛋白质识别性能评价

    Institute of Scientific and Technical Information of China (English)

    郭艳玲; 顾雨辰; 邓启良

    2016-01-01

    Protein phosphorylation is one of the most important post-translational modifications in life activities,which has a close relationship to the precise regulation of life activities. Thus, preparation of materials capable of selective enrichment of phosphoproteins is significantly important to phosphoproteome research. In this research,we prepared poly( guanidinium ionic liquid)s materials by using a synthesized guanidinium ionic liquid functional monomer. Poly ( guanidinium ionic liquid )s materials were characterized by Fourier transform infrared spec-trometer( FT-IR ), scanning electron microscope ( SEM ) and thermo gravimetric analyzer ( TGA). The results indicated that microspheres had a mean size of 200 nm in diameter. Usingβ-casein as a model phosphoprotein,the recognition properties of the obtained materials for phosphoprotein were evaluated. The adsorption results showed that microspheres not only had a high adsorption capacity for phosphorylation proteins(599. 1 mg/g for β-casein)and a rapid balance in 1 h,but also displayed high selectivity to phosphorylation proteins.%磷酸化修饰是蛋白质翻译后修饰中最为重要的修饰之一,蛋白质的磷酸化修饰几乎参与生命活动的每一个环节。因此,制备对磷酸化蛋白具有选择性识别性能的材料在磷酸化蛋白质组学中具有重要意义。本实验首先合成胍基离子液体功能单体,通过沉淀聚合法合成聚胍基离子液体材料。通过傅里叶红外光谱( FT-IR)、扫描电子显微镜( SEM)、热重分析仪( TGA)考察了材料的结构、形貌、热稳定性。结果显示所制备材料为粒径约200 nm 的球形颗粒。并以标准磷酸化蛋白(β-酪蛋白)为模型蛋白质,考察了聚胍基离子液体材料的识别性能。研究结果表明:材料对磷酸化蛋白具有较高吸附容量(对β-酪蛋白的最大吸附量达到599.1 mg/g)、较快的吸附速度(1 h内达平衡),而且对磷

  16. Preparation and identification of monoclonal antibody against liver fatty acid binding protein%肝脏型脂肪酸结合蛋白的重组表达及其单克隆抗体的制备和鉴定

    Institute of Scientific and Technical Information of China (English)

    宋巍; 杨海波; 陈兰英

    2014-01-01

    目的:制备抗肝脏型脂肪酸结合蛋白(LFABP)的单克隆抗体(mAb)并进行亚型和特异性鉴定。方法:以重组的LFABP蛋白为免疫原,经过原核表达和纯化后,免疫BALB/c小鼠,获得分泌鼠抗人LFABP蛋白mAb的杂交瘤细胞株,通过ELISA和Western blot方法检测其特异性。结果:纯化的LFABP重组蛋白免疫小鼠后经过筛选得到2株稳定分泌抗人LFABP的mAb杂交瘤细胞株,分别命名为3E6和5B7,其亚型分别为IgG1和IgG2a,抗体经纯化后浓度达到2 mg/mL,效价达到1∶10000以上。Western blot分析结果表明可与细胞中表达的内源LFABP发生特异性免疫反应。结论:成功制备了鼠抗人LFABP的mAb,为进一步研究LFABP的生物学特性,并为相关疾病的治疗奠定了基础。%OBJECTIVE:To prepare anti-liver-type fatty acid binding protein (LFABP) monoclonal antibody (mAb) and identified subtype and specificity. METHODS:The LFABP recombinant protein was used to immunize BALB/c mice,spleen cells from immunized mice were fused with myeloma cell Sp2/0. After HAT selective culture and indirect ELISA screening,we obtained hybridoma cell line secreting mouse mAb against human LFABP. The specificity was verified by ELISA and Western blotting. RESULTS:After using purified recombinant protein immunized mice and screening,we obtained two hybridoma cell lines secreting the mAb against human LFABP,named 3E6 and 5B7,both were of the IgG1 subtype. The antibody was purified,reaching a concentration of 2 mg/mL,and titer of 1∶10 000 above. Western blotting showed that mAb had a specific reaction with the LFABP endogenous expression in the cells. CONCLUSION:We successfully prepared mice mAb against human LFABP ,which could not only provide an important foundation to further studies of the biological characteristics of LFABP,mechanism involved in regulation of fatty acid metabolism and drug interaction,but also provide experimental evidence for the treatment

  17. Radio decontamination of the biological preparation iron-protein

    International Nuclear Information System (INIS)

    Is showed the gamma radiation application for to diminish the bio burden of the experimental lots possessing an elevated bio burden. The gamma radiation no change the quality parameter of product. Is no possible to apply others sterilization methods why changed composition of this medicine Instituto Nacional de Endocrinologia

  18. 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, and...... their addition to foods is therefore limited by the development of unpleasant off-flavors. Hence, efficient strategies are necessary to protect the lipids and thereby make fish oil-enriched food products successful in the marketplace. In an attempt to increase the oxidative stability of fish oil...... oxidatively stable product. Thus, a better understanding of factors influencing lipid oxidation in delivery emulsions themselves is therefore needed to understand the differences observed between food systems. In oil-in-water emulsions, lipid oxidation is expected to be initiated at the oil-water interface...

  19. Acidic preparations of platelet concentrates release bone morphogenetic protein-2.

    OpenAIRE

    Wahlström, Ola; Linder, Cecilia; Kalén, Anders; Magnusson, Per

    2008-01-01

    BACKGROUND AND PURPOSE: Growth factors released from platelets have potent effects on fracture and wound healing. The acidic tide of wound healing, i.e. the pH within wounds and fractures, changes from acidic pH to neutral and alkaline pH as the healing process progresses. We investigated the influence of pH on lysed platelet concentrates regarding the release of growth factors. MATERIAL AND METHODS: Platelet concentrates free of leukocyte components were lysed and incubated in buffers with p...

  20. 100% Organic Livestock Feeds – preparing for 2005

    OpenAIRE

    Hancock, Jake; Weller, Richard; McCalman, Heather

    2003-01-01

    A project entitled “100% Organic Livestock Feeds – Preparing for 2005” looking at the implications for organic farmers of the removal of the current derogation to use a percentage of non-organic livestock feeds. 1) Researching the required volume of feed stuff, in particular protein crops, and potential for feed production within Wales. 2) Researching the suitability of alternative protein sources, and evaluating livestock systems regarding the potential for a reduction in energy and pr...

  1. Optimizing Human Bile Preparation for Two-Dimensional Gel Electrophoresis

    OpenAIRE

    Hao-Tsai Cheng; Sen-Yung Hsieh; Chang-Mu Sung; Betty Chien-Jung Pai; Nai-Jen Liu; Carl PC Chen

    2016-01-01

    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 distribut...

  2. Progress and Application of Plastein Reaction in Food Proteins

    Institute of Scientific and Technical Information of China (English)

    ZHOU Zunlai; FENG Zhibiao

    2006-01-01

    Plastein reaction is considered a reversal of the usual protein hydrolysis by proteinase, which was applied to prepare a higher-molecular, protein-like substance. It can improve biological value and functional properties of food proteins, meliorate flavor of protein hydrolysates and, especially, provide a way to synthesize new sources of proteins. Although the mechanism(s) of the plastein reaction is not clarified, it will have great values in food industry with the development of technologies in enzymology and microbiology.

  3. Incorporation of cellular proteins into enveloped virus particles

    OpenAIRE

    Hammarstedt, Maria

    2006-01-01

    This thesis work aimed to investigate the assembly and budding of enveloped virus particles with focus on the fate of cellular proteins, present in or near the plasma membrane (PM) where the budding occurs. It was previously shown that compact viruses, like alphaviruses, with a covering outer protein coat, did not contain any cellular proteins in the envelope. However, cellular proteins were found in purified retroviral preparations and these proteins were thought to be spec...

  4. Preparing for Emergency Situations

    Science.gov (United States)

    Asproth, Viveca; Amcoff Nyström, Christina

    2010-11-01

    Disaster relief can be seen as a dynamic multi actor process with actors both joining and leaving the relief work during the help and rescue phase after the disaster has occurred. Actors may be governmental agencies, non profit voluntary organisations or spontaneous helpers comprised of individual citizens or temporal groups of citizens. Hence, they will vary widely in agility, competence, resources, and endurance. To prepare for for disasters a net based Agora with simulation of emergency situations for mutual preparation, training, and organisational learning is suggested. Such an Agora will ensure future security by: -Rising awareness and preparedness of potential disaster responders by help of the components and resources in the netAgora environment; -Improving cooperation and coordination between responders; -Improving competence and performance of organisations involved in security issues; -Bridging cultural differences between responders from different organizations and different backgrounds. The developed models are intended to reflect intelligent anticipatory systems for human operator anticipation of future consequences. As a way to catch what should be included in this netbased Agora and to join the split pictures that is present, Team Syntegrity could be a helpful tool. The purpose of Team Syntegrity is to stimulate collaboration and incite cross fertilization and creativity. The difference between syntegration and other group work is that the participants are evenly and uniquely distributed and will collectively have the means, the knowledge, the experience, the perspectives, and the expertise, to deal with the topic. In this paper the possibilities with using Team Syntegrity in preparation for the development of a netbased Agora is discussed. We have identified that Team Syntegrity could be useful in the steps User Integration, Designing the netAgora environment, developing Test Scenarios, and assessment of netAgora environment.

  5. Preparation of Simulated Waste Solutions

    Energy Technology Data Exchange (ETDEWEB)

    Walker, D.D.

    1999-06-08

    Waste Processing Technology personnel routinely prepare 0.5 to 10 L batches of salt solutions simulating Savannah River Site (SRS) soluble waste. This report describes the compositions and preparation methods.

  6. Seismic Network Deployment Preparations

    OpenAIRE

    Allen Husker; Igor Stubailo; Monica Kohler; Paul Davis

    2003-01-01

    Technological and scientific preparations are occurring for the development of a multi-hop radio-linked seismic array (MHRLSA) of 50 broadband stations (GURALP 3ts) and its first few deployments. A ruggedized data relay device (DRD) is being fabricated using Intel’¡Çs new low power, small form factor stargate motherboard. A DRD will be placed at each node of the array and configured as a local area network (LAN) with station spacing up to 10 km. The objective is to use protocols that have bee...

  7. Preparation of tungsten oxide

    Science.gov (United States)

    Bulian, Christopher J.; Dye, Robert C.; Son, Steven F.; Jorgensen, Betty S.; Perry, W. Lee

    2009-09-22

    Tungsten trioxide hydrate (WO.sub.3.H.sub.2O) was prepared from a precursor solution of ammonium paratungstate in concentrated aqueous hydrochloric acid. The precursor solution was rapidly added to water, resulting in the crash precipitation of a yellow white powder identified as WO.sub.3.H.sub.2O nanosized platelets by x-ray diffraction and scanning electron microscopy. Annealing of the powder at 200.degree. C. provided cubic phase WO.sub.3 nanopowder, and at 400.degree. C. provided WO.sub.3 nanopowder as a mixture of monoclinic and orthorhombic phases.

  8. Preparation of tritiated lysine

    International Nuclear Information System (INIS)

    Tritiated L-lysine is used to study the function and metabolism of lysine in vivo. Therefore, it is an important tracer in biochemical research. The precursor, chloro-lysine was obtained by the reaction of L-lysine hydrochloride with chlorine gas in concentrated hydrochloric acid medium under uv light radiation. Then tritiated L-lysine was prepared by catalysed halogen-tritium exchange. The specific activity of L(β, γ-3H) lysine was 5.9 TBq/mmol (∼16 Ci/mmol). The radiochemical purity was over 95%

  9. KANJI: AN AYURVEDIC FERMENTATIVE PREPARATION

    OpenAIRE

    Santhosh B; Jadar P. G.; Nageswara Rao

    2012-01-01

    Kanji – A unique Ayurvedic fermentative preparation was prepared as per the textual reference Rasayanasara which is mainly indicated for the Shodhana (purification) of Metals and also for various mercurial processing. But Kanji by this reference is rarely prepared and used. Hence, the pharmaceutical and preliminary physico-chemical findings of this Kanji are reported in this paper. The fermentation process started on 7th day and completed on 31st day. The prepared Kanji was golden brown color...

  10. Physiologic effects of bowel preparation

    DEFF Research Database (Denmark)

    Holte, Kathrine; Nielsen, Kristine Grubbe; Madsen, Jan Lysgård; Kehlet, Henrik

    2004-01-01

    healthy volunteers (median age, 63 years) underwent bowel preparation with bisacodyl and sodium phosphate. Fluid and food intake were standardized according to weight, providing adequate calorie and oral fluid intake. Before and after bowel preparation, weight, exercise capacity, orthostatic tolerance...... preparation has significant adverse physiologic effects, which may be attributed to dehydration. The majority of these findings is small and may not be of clinical relevance in otherwise healthy patients undergoing bowel preparation and following recommendations for oral fluid intake....

  11. End-preparation assessments and tests for compounded sterile preparations.

    Science.gov (United States)

    McElhiney, Linda F

    2013-01-01

    Outsourcing has become a necessity to obtain sterile products that are currently on backorder. Because of the expense of outsourcing sterile compounding, pharmacy leadership in health systems are now considering the option of insourcing and batch preparing compounded sterile preparations, which can be a viable option for a health system. It can significantly decrease drug-spending costs, and the pharmacy has a complete record of the compounding process. The key to preparing high-quality, safe, sterile preparations and meeting United States Pharmacopeia standards is end-preparation assessments and tests. PMID:24261146

  12. Thermoplastic starch materials prepared from rice starch

    International Nuclear Information System (INIS)

    Rice starch is a source still little studied for the preparation of thermoplastic materials. However, its characteristics, such as the presence of proteins, fats and fibers may turn into thermoplastics with a better performance. The present study intends the evaluation of the viability of making starch thermoplastic from rice starch and glycerol as plasticizer. The results of X-ray diffraction and scanning electronic microscopy demonstrate the thermoplastic acquisition. The increase of plasticizer content brings on more hydrophilic thermoplastics with less resistance to tension and elongation at break. (author)

  13. Prepare Healthy Foods with Toddlers

    Science.gov (United States)

    Izumi-Taylor, Satomi; Rike, Cheryl

    2011-01-01

    Toddlers--from about 16 to 36 months--can learn a variety of skills as they prepare food and follow recipes in developmentally appropriate ways. Early childhood teachers are encouraged to support young children's healthy eating habits by offering simple food preparation experiences. When toddlers--and preschoolers--safely prepare healthy snacks,…

  14. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2011-01-01

    The Human Resources Department is organizing a Preparation for Retirement Seminar, which will take place on 18 and 21 October 2011 in the afternoon in the Main Auditorium and on 19 October and 15 and 16 November 2011 in the afternoon in the Council Chamber. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members under the age of 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to availability of places. Registration: In view of the number of people concerned, you are ...

  15. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2011-01-01

    The Human Resources Department is organizing a Preparation for Retirement Seminar, which will take place on 18 and 21 October 2011 in the afternoon in the Main Auditorium and on 19 October and 15 and 16 November 2011 in the afternoon in the Council Chamber. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members under the age of 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to availability of places. Registration: In view of the number of people concerned, you are r...

  16. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2007-01-01

    The Department of Human Resources is organising a preparation for retirement seminar which will take place on the four successive afternoons of 2 to 5 October 2007. Similar seminars in the past have always proved highly successful. Retirement marks the end of one’s working life and the start of a new period of life. This period of transition and change is experienced differently from one individual to another. In any case, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above as well as those who have retired during the year have been sent a personal invitation to attend. Spouses are welcome. Staff members below 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to availability of places. Registration: In view of the number of people concerned and the limited capacity of th...

  17. Preparation for retirement seminar

    CERN Multimedia

    HR Department

      The Human Resources Department is organizing a preparation for retirement seminar, which will take place on the afternoons of the 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members under the age of 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to the availability of places. Registration: In view of the number of people concerned and the limited capacity of the Main Auditorium, you are requested to register in advance via Indico. &a...

  18. Integrated coal preparation

    International Nuclear Information System (INIS)

    Perceptions of quality have changed over the years. The attributes of a certain coal (its rank, slagging propensity, ash content etc) are traditionally referred to as its quality. However, the subject of this paper is quality in a much wider sense: quality as fitness for purpose: and all that such a wide definition entails. British Standard BS 5750 (ISO 9000) Quality Systems defines a systems approach to quality, and includes both the supplier of raw materials and the final customer within this boundary. Coal preparation starts at the production face. The greater the proportion of dirt in run-of-mine product the greater the challenge in satisfying the customer's needs. Significant advances have been made in minimizing mined dirt. For example, the sue of vertical steering on longwall faces improves productivity and quality. Unfortunately modern mining methods produce large quantities of fines, despite efforts to reduce them at the point of production and during transportation to the surface. Coal preparation also produces further fines. It has been estimated that fine coal costs 2.5 times as much to clean as large coal, and the costs of handing wet fine coal product will inflate this estimate. Handling considerations rightly concern our customers and are part of the wider meaning of quality. In this paper the authors address some novel solutions to the challenge posed by fines

  19. PREPARATION FOR RETIREMENT PROGRAMME

    CERN Multimedia

    Human Resources Division

    2001-01-01

    27 March 2001 from 2.00 p.m. to 5.30 p.m. 28 March 2001 from 2.00 p.m. to 5.30 p.m. 29 March 2001 from 2.00 p.m. to 5.30 p.m. 30 March 2001 from 2.00 p.m. to 4.45 p.m. Auditorium (Main Building) After the success of the preparation seminars held in recent years, it has been decided that the programme should continue. The forthcoming seminar has been prepared in close collaboration with the CERN Pensioners' Association. The programme will be organised over several half-day sessions. Once again this year, a special session will be devoted to the 10th revision of the Swiss state pension scheme, the 'AVS' (Assurance-Vieillesse et Survivants), and the consequences for international civil servants. A talk will be given by Mrs Danièle Siebold, Director of the Caisse Cantonale Genevoise de Compensation, aimed mainly at those residing in or intending to move to Switzerland, or who worked in Switzerland before joining CERN. To enable Mrs Siebold to respond to your concerns as effectively as possible, please ...

  20. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware...