Sample records for antifreeze protein prepared

  1. Protein-water dynamics in antifreeze protein III activity

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


    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

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


    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.

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


    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

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


    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

    Ravi Gupta; Renu Deswal


    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

    Ma Xinyu; Shen Xin; Lu Cunfu


    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

    Kutschan, B; Thoms, S


    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

    夏露; 张超; 王立; 张晖


    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

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


    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

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


    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.

    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

    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

    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: [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of)


    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

    Li, Qianzhong; Luo, Liaofu


    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

    万军; 朱兴友; 张洋


    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.

    Ramya, L; Ramakrishnan, Vigneshwar


    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

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


    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

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


    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

    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.


    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?

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


    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

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


    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

    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

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


    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.

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


    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

    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

    Srisulak Dheeranupattana


    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

    Runtao Yang


    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.

    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

    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


    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).

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


    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

    Ulf Sorhannus


    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.

    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


    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


    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.

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


    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.

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


    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.

    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

    K Sivakumar; S Balaji; Gangaradhakrishnan


    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

    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

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


    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

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


    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...


    Ricardo Vera Bravo


    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

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


    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.

    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

    汪少芸; 赵珺; 吴金鸿; 陈琳


    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.

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


    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

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


    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.

    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


    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.

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


    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

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


    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.

    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

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


    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.

    Du, Lihui; Betti, Mirko


    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.

    Buch, J L; Ramløv, H


    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 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

    洪晶; 汪少芸; 吴金鸿; 饶平凡


    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.

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


    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.)

    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


    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

    蔡文萍; 马纪


    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

    Kesić Jelena


    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.

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


    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.

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


    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

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


    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

    尹明安; 崔鸿文; 樊代明; 郭立


    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

    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)


    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

    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


    A. Suha Yalçın; Murat Türkoğlu


    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

    Daraboina, Nagu; Perfeldt, Christine Malmos; von Solms, Nicolas


    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...


    A. Suha Yalçın


    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



    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

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


    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.

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


    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

    钟伯雄; 翁宏飚; 等


    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

    LIU Jun; LIU Runqing


    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.


    S. V. Gushchin


    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

    Liu Zhitong; Li Xiaolian; Zhao Guangming


    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

    LIU Junzhe; LI Yushun; LV Lihua


    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.

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


    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

    Zepeda, Salvador; Yokoyama, Etsuro; Furukawa, Yoshinor


    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

    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.

    Yanye Feng

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

  20. Preparation of encapsulated proteins dissolved in low viscosity fluids

    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

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


    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

    Murna Muzaifa; Novi Safriani; Fahrizal Zakaria


    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

    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:,


    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.

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


    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.

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


    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

    Graham Laurie A


    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)

    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

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


    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

    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

    Murna Muzaifa


    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

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


    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.

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


    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



    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.

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


    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


    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

    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)


    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.

    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

    Pecht, I.; Mazurek, N.


    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

    MIČULKOVÁ, Klára


    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

    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

    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

    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

    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

    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.

    Javanainen, Matti; Martinez-Seara, Hector


    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.

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


    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

    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

    Elisabeth I. Vogt


    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.

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


    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


    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.

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


    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

    Ahlin Grabnar, Pegi; Kristl, Julijana


    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

    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

    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

    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

    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

    Balslev, Y; Hansen, Gert Helge


    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

    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.

    Bandyopadhyay, Kakali; Misra, Gautam; Ghosh, Santinath


    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.

    Bittner, B; Kissel, T


    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.

    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

    Ace Baehaki


    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].

    Qin, Yujie; Zhang, Tinghong; Ye, Xin


    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


    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.

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


    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


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


    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

    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.


    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

    Šťastná, Miroslava; Šlais, Karel


    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

    ZhangWeinong; LiuDachuan


    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.

    Hong, Haiping; Zheng, Yingsong; Roy, Walter


    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.

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


    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

    郭睿; 杨晓泉; 杨熙


    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

    Jiping Cai


    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.

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


    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.

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


    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.

    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


    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

    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

    Kato, Katsuya, E-mail: [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)


    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.

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


    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



    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


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


    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

    Constantin Moldovanu; Cornel Laslo


    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

    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

    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

    ZHANG Yu


    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.

    Zhang, Wei-Nong; Liu, Da-Chuan


    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.

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


    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

    Kaivarainen, A


    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 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


    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

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


    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


    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.

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


    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.

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


    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.

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


    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.

    Qu, Xue; Kobayashi, Nao; Komatsu, Teruyuki


    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

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


    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.

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


    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


    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

    Turgay Tay; Ender Köse; Rüstem Keçili; Rıdvan Say


    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?

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


    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

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


    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.

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


    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

    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.

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


    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.

    Apinunjarupong, Suthep; Lapnirun, Surawoot; Theerakulkait, Chockchai


    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

    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

    Turgay Tay


    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

    Liu Yong


    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

    Buhrman Jason S


    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.

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


    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

    Zheng Xiaojuan


    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

    Prymula, Katarzyna; Sałapa, Kinga; Roterman, Irena


    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.

    Lee, R T; Lee, Y C


    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

    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.

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


    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.

    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


    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

    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)


    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?

    Gabriel N. Medoua


    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

    Mackin, Robert B.


    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.

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


    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

    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

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


    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

    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

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


    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

    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: [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); Liang, Yu-He, E-mail: [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)


    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

    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

    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

    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.

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


    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

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


    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.

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


    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

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


    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

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


    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.

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


    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

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


    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.

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


    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.

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


    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

    Ace Baehaki1); Shanti Dwita Lestari; Achmad Rizky Romadhoni


    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

    Maznah Ismail


    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

    Zhang Weinong; Liu Dachuan


    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

    Doll, Hans; Andersen, Bente


    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.

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


    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

    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

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


    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

    Olivieri Oliviero


    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

    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

    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

    Dyrberg, T; Billestrup, Nils


    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.

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


    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.

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


    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

    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.

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


    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

    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.

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


    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

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


    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

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


    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

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


    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

    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



    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

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


    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.

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


    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

    Yan Liu; Yongwei Wei; Xiaofeng Wu; Lian Yu


    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

    Aleksandra Zambrowicz


    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

    Jinju Wang


    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].

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


    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


    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.

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


    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

    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

    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

    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


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


    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.

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


    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

    Solanki Jitesh B.


    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, 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

    Hassan AH


    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.

    Gu, X X; Tsai, C M


    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

    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

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


    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

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


    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

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


    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

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


    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

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


    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

    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


    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

    Kreider, Alexej


    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

    N. Akashi


    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

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


    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.

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


    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

    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

    Lylo V. V.


    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

    Zhiqiang Tian


    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.

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


    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