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Sample records for antifreeze protein prepared

  1. Protein-water dynamics in antifreeze protein III activity

    Science.gov (United States)

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

    2016-03-01

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

  2. Plant Antifreeze Proteins and Their Expression Regulatory Mechanism

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  3. Antivirulence Properties of an Antifreeze Protein

    Directory of Open Access Journals (Sweden)

    Martin Heisig

    2014-10-01

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

  4. Molecular basis for antifreeze activity difference of two insect antifreeze protein isoforms

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The insect spruce budworm(Choristoneura fumiferana) produces antifreeze protein(AFP) to assist in the protection of the over-wintering larval stage and contains multiple isoforms. Structures for two isoforms,known as CfAFP-501 and CfAFP-337,show that both possess similar left-handed β-helical structure,although thermal hysteresis activity of the longer isoform CfAFP-501 is three times that of CfAFP-337. The markedly enhanced activity of CfAFP-501 is not proportional to,and cannot be simply accounted for,by the increased ice-binding site resulting from the two extra coils in CfAFP-501. In or-der to investigate the molecular basis for the activity difference and gain better understanding of AFPs in general,we have employed several different computational methods to systematically study the structural properties and ice interactions of the AFPs and their deletion models. In the context of intact AFPs,a majority of the coils in CfAFP-501 has better ice interaction and causes stronger ice lattice disruption than CfAFP-337,strongly suggesting a cooperative or synergistic effect among β-helical coils. The synergistic effect would play a critical role and make significant contributions to the anti-freeze activity β-helical antifreeze proteins. This is the first time that synergistic effect and its implica-tion for antifreeze activity are reported for β-helical antifreeze proteins.

  5. Molecular basis for antifreeze activity difference of two insect antifreeze protein isoforms

    Institute of Scientific and Technical Information of China (English)

    ZHOU YanXia; TAN HongWei; YANG ZuoYin; JIA ZongChao; LIU RuoZhuang; CHEN GuangJu

    2007-01-01

    The insect spruce budworm (Choristoneura fumiferana) produces antifreeze protein (AFP) to assist in the protection of the over-wintering larval stage and contains multiple isoforms. Structures for two isoforms, known as CfAFP-501 and CfAFP-337, show that both possess similar left-handed β-helical structure, although thermal hysteresis activity of the longer isoform CfAFP-501 is three times that of CfAFP-337. The markedly enhanced activity of CfAFP-501 is not proportional to, and cannot be simply accounted for, by the increased ice-binding site resulting from the two extra coils in CfAFP-501. In order to investigate the molecular basis for the activity difference and gain better understanding of AFPs in general, we have employed several different computational methods to systematically study the structural properties and ice interactions of the AFPs and their deletion models. In the context of intact AFPs, a majority of the coils in CfAFP-501 has better ice interaction and causes stronger ice lattice disruption than CfAFP-337, strongly suggesting a cooperative or synergistic effect among β-helical coils. The synergistic effect would play a critical role and make significant contributions to the antifreeze activity β-helical antifreeze proteins. This is the first time that synergistic effect and its implication for antifreeze activity are reported for β-helical antifreeze proteins.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-05-01

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

  8. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    Ravi Gupta; Renu Deswal

    2014-12-01

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

  9. Utilizing avidity to improve antifreeze protein activity: a type III antifreeze protein trimer exhibits increased thermal hysteresis activity.

    Science.gov (United States)

    Can, Özge; Holland, Nolan B

    2013-12-03

    Antifreeze proteins (AFPs) are ice growth inhibitors that allow the survival of several species living at temperatures colder than the freezing point of their bodily fluids. AFP activity is commonly defined in terms of thermal hysteresis, which is the difference observed for the solution freezing and melting temperatures. Increasing the thermal hysteresis activity of these proteins, particularly at low concentrations, is of great interest because of their wide range of potential applications. In this study, we have designed and expressed one-, two-, and three-domain antifreeze proteins to improve thermal hysteresis activity through increased binding avidity. The three-domain type III AFP yielded significantly greater activity than the one- and two-domain proteins, reaching a thermal hysteresis of >1.6 °C at a concentration of hysteresis activity.

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

    Institute of Scientific and Technical Information of China (English)

    Ma Xinyu; Shen Xin; Lu Cunfu

    2003-01-01

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

  11. Dynamical mechanism of antifreeze proteins to prevent ice growth

    CERN Document Server

    Kutschan, B; Thoms, S

    2014-01-01

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

  12. Construction of plant expression vector of Pseudopleuronectes americanus antifreeze protein gene

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and fi sequence and Kozak sequence that can improve the expression in translation level, the high expression cassette of antifreeze protein was constructed. This cassette was connected to pBI121.1 and finally got the high expression vector pBRTSAFP introduced into the maize callus. The expression of gus gene that linked to the antifreeze protein gene was detected, and the results was that the gus gene can express strongly and instantaneously.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Raimond Gordienko

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  16. Continuous production of CO2 hydrate slurry added antifreeze proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tokunaga, Y.; Ota, M.; Murakami, K. [Tokyo Metropolitan Univ., Tokyo (Japan). Dept. of Mechanical Engineering; Ferdows, M. [Dhaka Univ., Dhaka (Bangladesh). Dept. of Mathematics; Endou, H. [Technova Co. Ltd., Tokyo (Japan). Dept. of Mechanical Engineering

    2008-07-01

    Ocean storage of carbon dioxide (CO{sub 2}) hydrate is possible in deep seas where low temperature and high pressure conditions exist. However, when hydrates are produced in large quantities, they can plug pipelines. The addition of antifreeze proteins (AFPs) can prevent hydrate crystals from forming. The hydrate may then behave like a slurry which can be transported from a production place to a place of storage with minimal pressure loss. This study developed a production method for a CO{sub 2} hydrate slurry and presented the prospect of the inhibition effect for CO{sub 2} hydrate formation by adding AFPs. It revealed the shift in induction time, the formation rate and the torque of the agitator under conditions of AFPs at 0.01 mg/ml. It was concluded that compared to pure water, the induction time for hydrate production increased 244 per cent, the formation rate decreased 76 per cent and the ratio of the torque decreased 48 per cent by adding AFPs. The AFPs rendered the hydrate particles small and well dispersed. It was concluded that type 3 AFPs can effectively inhibit the production of structure s1 type hydrates. 4 refs., 6 figs.

  17. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

    Science.gov (United States)

    Yue, Chang-Wu; Zhang, Yi-Zheng

    2009-03-01

    A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-02-15

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

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

    Science.gov (United States)

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

    1999-10-01

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

  20. Cloning and expression of a novel antifreeze protein AFP72 from the beetle Tenebrio molitor.

    Science.gov (United States)

    Yan, Qing-Hua; Yang, Li; Wang, Qing; Zhang, Hui-Rong; Shao, Qiang

    2012-01-01

    A novel antifreeze protein AFP72 cDNA (GenBbank accession No. AY929389) was obtained by RT-PCR from Tenebrio molitor. The 216 bp fragment encodes a protein of 72 amino acid residues. Sequence analysis revealed that the cDNA displays a high degree of homology with T. molitor antifreeze proteins, ranging up to 90.78%. Recombinant plasmids pMAL-p2X-afp72 and pMAL-c2X-afp72 were transferred into E. coil TBI to induce a MBP fusion protein by IPTG. The target fusion protein was released from the periplasm and cytoplasm by the cold osmotic shock procedure and sonication respectively. The content of the fusion protein came up to 38.9 and 41.5% of the total dissolved protein, respectively. The fusion protein was purified through an amylose affinity column, and incised by factor Xa. Molecular sieve chromatography was used to achieve a high state of purity of the target protein. The purified target protein displayed a single band in SDS-PAGE. The fusion protein was shown to increase resistance to low temperatures in bacteria. This finding could help in further investigations of the properties and function of antifreeze proteins.

  1. Cloning,sequencing and prokaryotic expression of cDNAs for antifreeze protein family from Beetle Tenebrio molitor

    Institute of Scientific and Technical Information of China (English)

    Zhongyuan LIU; Yun WANG; Guodong LU; Xianlei WANG; Fuchun ZHANG; Ji MA

    2008-01-01

    Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank.The recombinant pGEX-4T-l-tmafp-XJ430 was introduced into E.coli BL21 to induce a GST fusion protein by IPTG.SDS-PAGE analysis for the fusion protein shows a band of 38 kDa.pCDNA3- tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA.The titer of the antibody was 1:2000.Western blot-ting analysis shows that the antiserum was specifically against the antifreeze protein.Our results laid the founda-tion for further studies on the properties and functions of insect antifreeze proteins.

  2. Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth.

    OpenAIRE

    Carpenter, J F; Hansen, T N

    1992-01-01

    Antifreeze proteins (AFPs) are extremely efficient at inhibiting ice recrystallization in frozen solutions. Knight and Duman [Knight, C. A. & Duman, J. G. (1986) Cryobiology 23, 256-263] have proposed that this may be an important function of the proteins in freeze-tolerant organisms. We have tested this proposal in vitro by characterizing the influence of AFP on the recovery of cryopreserved cells, which often can survive cooling and yet subsequently be damaged by ice crystal growth during w...

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    Antifreeze proteins (AFPs) are characterized by their ability to protect organisms from subfreezing temperatures by preventing tiny ice crystals in solution from growing as the solution is cooled below its freezing temperature. This inhibition of ice growth is called antifreeze activity, and in p......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...

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

    DEFF Research Database (Denmark)

    Kristiansen, E; Ramløv, Hans; Højrup, Peter

    2011-01-01

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

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

    Science.gov (United States)

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

    1994-01-01

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

  7. Intermolecular interaction studies of winter flounder antifreeze protein reveal the existence of thermally accessible binding state.

    Science.gov (United States)

    Nguyen, Dat H; Colvin, Michael E; Yeh, Yin; Feeney, Robert E; Fink, William H

    2004-10-05

    The physical nature underlying intermolecular interactions between two rod-like winter flounder antifreeze protein (AFP) molecules and their implication for the mechanism of antifreeze function are examined in this work using molecular dynamics simulations, augmented with free energy calculations employing a continuum solvation model. The energetics for different modes of interactions of two AFP molecules is examined in both vacuum and aqueous phases along with the water distribution in the region encapsulated by two antiparallel AFP backbones. The results show that in a vacuum two AFP molecules intrinsically attract each other in the antiparallel fashion, where their complementary charge side chains face each other directly. In the aqueous environment, this attraction is counteracted by both screening and entropic effects. Therefore, two nearly energetically degenerate states, an aggregated state and a dissociated state, result as a new aspect of intermolecular interaction in the paradigm for the mechanism of action of AFP. The relevance of these findings to the mechanism of function of freezing inhibition in the context of our work on Antarctic cod antifreeze glycoprotein (Nguyen et al., Biophysical Journal, 2002, Vol. 82, pp. 2892-2905) is discussed.

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

    Science.gov (United States)

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

    2015-10-01

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

  9. CryoProtect: A Web Server for Classifying Antifreeze Proteins from Nonantifreeze Proteins

    Directory of Open Access Journals (Sweden)

    Reny Pratiwi

    2017-01-01

    Full Text Available Antifreeze protein (AFP is an ice-binding protein that protects organisms from freezing in extremely cold environments. AFPs are found across a diverse range of species and, therefore, significantly differ in their structures. As there are no consensus sequences available for determining the ice-binding domain of AFPs, thus the prediction and characterization of AFPs from their sequence is a challenging task. This study addresses this issue by predicting AFPs directly from sequence on a large set of 478 AFPs and 9,139 non-AFPs using machine learning (e.g., random forest as a function of interpretable features (e.g., amino acid composition, dipeptide composition, and physicochemical properties. Furthermore, AFPs were characterized using propensity scores and important physicochemical properties via statistical and principal component analysis. The predictive model afforded high performance with an accuracy of 88.28% and results revealed that AFPs are likely to be composed of hydrophobic amino acids as well as amino acids with hydroxyl and sulfhydryl side chains. The predictive model is provided as a free publicly available web server called CryoProtect for classifying query protein sequence as being either AFP or non-AFP. The data set and source code are for reproducing the results which are provided on GitHub.

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

    Science.gov (United States)

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

    2013-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Srisulak Dheeranupattana

    2005-07-01

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

  12. Induced ice melting by the snow flea antifreeze protein from molecular dynamics simulations.

    Science.gov (United States)

    Todde, Guido; Whitman, Christopher; Hovmöller, Sven; Laaksonen, Aatto

    2014-11-26

    Antifreeze proteins (AFP) allow different life forms, insects as well as fish and plants, to survive in subzero environments. AFPs prevent freezing of the physiological fluids. We have studied, through molecular dynamics simulations, the behavior of the small isoform of the AFP found in the snow flea (sfAFP), both in water and at the ice/water interface, of four different ice planes. In water at room temperature, the structure of the sfAFP is found to be slightly unstable. The loop between two polyproline II helices has large fluctuations as well as the C-terminus. Torsional angle analyses show a decrease of the polyproline II helix area in the Ramachandran plots. The protein structure instability, in any case, should not affect its antifreeze activity. At the ice/water interface the sfAFP triggers local melting of the ice surface. Bipyramidal, secondary prism, and prism ice planes melt in the presence of AFP at temperatures below the melting point of ice. Only the basal plane is found to be stable at the same temperatures, indicating an adsorption of the sfAFP on this ice plane as confirmed by experimental evidence.

  13. Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

    Science.gov (United States)

    Mao, Yougang; Ba, Yong

    2006-09-01

    This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

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

    Directory of Open Access Journals (Sweden)

    Runtao Yang

    2015-09-01

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

  15. Structural basis for antifreeze activity of ice-binding protein from arctic yeast.

    Science.gov (United States)

    Lee, Jun Hyuck; Park, Ae Kyung; Do, Hackwon; Park, Kyoung Sun; Moh, Sang Hyun; Chi, Young Min; Kim, Hak Jun

    2012-03-30

    Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96-115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region ((243)PFVPAPEVV(251)). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn(185) provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins.

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

    Institute of Scientific and Technical Information of China (English)

    李金耀; 马纪; 张富春

    2005-01-01

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

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

    Science.gov (United States)

    Zepeda, Salvador; Orme, Christine; Yeh, Yin

    2002-03-01

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

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

    Science.gov (United States)

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

    2014-06-01

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

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

    Science.gov (United States)

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

    2016-09-12

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

  20. Expression and characterization of an antifreeze protein from the perennial rye grass, Lolium perenne.

    Science.gov (United States)

    Lauersen, Kyle J; Brown, Alan; Middleton, Adam; Davies, Peter L; Walker, Virginia K

    2011-06-01

    Antifreeze proteins (AFP) are an evolutionarily diverse class of stress response products best known in certain metazoans that adopt a freeze-avoidance survival strategy. The perennial ryegrass, Lolium perenne (Lp), cannot avoid winter temperatures below the crystallization point and is thought to use its LpAFP in a freeze-tolerant strategy. In order to examine properties of LpAFP in relation to L. perenne's life history, cDNA cloning, recombinant protein characterization, ice-binding activities, gene copy number, and expression responses to low temperature were examined. Transcripts, encoded by only a few gene copies, appeared to increase in abundance after diploid plants were transferred to 4°C for 1-2 days, and in parallel with the ice recrystallization inhibition activities. Circular dichroism spectra of recombinant LpAFP showed three clear folding transition temperatures including one between 10 and 15°C, suggesting to us that folding modifications of the secreted AFP could allow the targeted degradation of the protein in planta when temperatures increase. Although LpAFP showed low thermal hysteresis activity and partitioning into ice, it was similar to AFPs from freeze-avoiding organisms in other respects. Therefore, the type of low temperature resistance strategy adopted by a particular species may not depend on the type of AFP. The independence of AFP sequence and life-history has practical implications for the development of genetically-modified crops with enhanced freeze tolerance.

  1. Isolation and characterization of type I antifreeze proteins from cunner, Tautogolabrus adspersus, order Perciformes.

    Science.gov (United States)

    Hobbs, Rod S; Shears, Margaret A; Graham, Laurie A; Davies, Peter L; Fletcher, Garth L

    2011-10-01

    Antifreeze proteins (AFPs) are produced by many species of teleost fish that inhabit potentially lethal ice-laden seawater and afford them protection from freezing. To date type I AFPs have been fully characterized in two teleost orders: Pleuronectiformes and Scorpaeniformes. In this study, we report the isolation and complete characterization of a type I AFP present in fish from a third order: cunner (Tautogolabrus adspersus), order Perciformes (family Labridae). This protein was purified from blood plasma and found to belong to what is now known as classical type I AFP with their small size (mass 4095.16 Da), alanine richness (> 57 mol%), high α-helicity (> 99%) with the ability to undergo reversible thermal denaturation, 11 amino acid (ThrX(10)) repeat regions within the primary structure, the capacity to impart a hexagonal bipyramidal shaping to ice crystals and the conservation of an ice-binding site found in many of the other type I AFPs. Partial de novo sequencing of the plasma AFP accounted for approximately half of the peptide mass. Sequencing of a combined liver and skin cDNA library indicated that the protein is produced without a signal sequence. In addition the translated product of the AFP cDNA suggests that it codes for the AFP isolated from plasma. These results further solidify the hypothesis that type I AFPs are multiphyletic in origin and suggest that they represent remarkable examples of convergent evolution within three orders of teleost fish.

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

    Science.gov (United States)

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

    2013-05-13

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

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

    Science.gov (United States)

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

    2015-09-16

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

  4. Hydration behavior at the ice-binding surface of the Tenebrio molitor antifreeze protein.

    Science.gov (United States)

    Midya, Uday Sankar; Bandyopadhyay, Sanjoy

    2014-05-08

    Molecular dynamics (MD) simulations have been carried out at two different temperatures (300 and 220 K) to study the conformational rigidity of the hyperactive Tenebrio molitor antifreeze protein (TmAFP) in aqueous medium and the structural arrangements of water molecules hydrating its surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its nonice-binding surface (NIBS). The presence of a set of regularly arranged internally bound water molecules is found to play an important role in maintaining the flat rigid nature of the IBS. Importantly, the calculations reveal that the strategically located hydroxyl oxygens of the threonine (Thr) residues in the IBS influence the arrangements of five sets of ordered waters around it on two parallel planes that closely resemble the basal plane of ice. As a result, these waters can register well with the ice basal plane, thereby allowing the IBS to preferentially bind at the ice interface and inhibit its growth. This provides a possible molecular reason behind the ice-binding activity of TmAFP at the basal plane of ice.

  5. New insights into ice growth and melting modifications by antifreeze proteins.

    Science.gov (United States)

    Bar-Dolev, Maya; Celik, Yeliz; Wettlaufer, J S; Davies, Peter L; Braslavsky, Ido

    2012-12-07

    Antifreeze proteins (AFPs) evolved in many organisms, allowing them to survive in cold climates by controlling ice crystal growth. The specific interactions of AFPs with ice determine their potential applications in agriculture, food preservation and medicine. AFPs control the shapes of ice crystals in a manner characteristic of the particular AFP type. Moderately active AFPs cause the formation of elongated bipyramidal crystals, often with seemingly defined facets, while hyperactive AFPs produce more varied crystal shapes. These different morphologies are generally considered to be growth shapes. In a series of bright light and fluorescent microscopy observations of ice crystals in solutions containing different AFPs, we show that crystal shaping also occurs during melting. In particular, the characteristic ice shapes observed in solutions of most hyperactive AFPs are formed during melting. We relate these findings to the affinities of the hyperactive AFPs for the basal plane of ice. Our results demonstrate the relation between basal plane affinity and hyperactivity and show a clear difference in the ice-shaping mechanisms of most moderate and hyperactive AFPs. This study provides key aspects associated with the identification of hyperactive AFPs.

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

    Science.gov (United States)

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

    2014-01-15

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

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

    Science.gov (United States)

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

    2013-05-01

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

  8. X-ray Structure of Snow Flea Antifreeze Protein Determined by Racemic Crystallization of Synthetic Protein Enantiomers

    Energy Technology Data Exchange (ETDEWEB)

    Pentelute, Brad L.; Gates, Zachary P.; Tereshko, Valentina; Dashnau, Jennifer L.; Vanderkooi, Jane M.; Kossiakoff, Anthony A.; Kent, Stephen B.H. (UPENN); (UC)

    2008-08-20

    Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 {angstrom}. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.

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

    Indian Academy of Sciences (India)

    K Sivakumar; S Balaji; Gangaradhakrishnan

    2007-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Laurie A Graham

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

  11. Antifreeze protein-induced superheating of ice inside Antarctic notothenioid fishes inhibits melting during summer warming.

    Science.gov (United States)

    Cziko, Paul A; DeVries, Arthur L; Evans, Clive W; Cheng, Chi-Hing Christina

    2014-10-07

    Antifreeze proteins (AFPs) of polar marine teleost fishes are widely recognized as an evolutionary innovation of vast adaptive value in that, by adsorbing to and inhibiting the growth of internalized environmental ice crystals, they prevent death by inoculative freezing. Paradoxically, systemic accumulation of AFP-stabilized ice could also be lethal. Whether or how fishes eliminate internal ice is unknown. To investigate if ice inside high-latitude Antarctic notothenioid fishes could melt seasonally, we measured its melting point and obtained a decadal temperature record from a shallow benthic fish habitat in McMurdo Sound, Antarctica. We found that AFP-stabilized ice resists melting at temperatures above the expected equilibrium freezing/melting point (eqFMP), both in vitro and in vivo. Superheated ice was directly observed in notothenioid serum samples and in solutions of purified AFPs, and ice was found to persist inside live fishes at temperatures more than 1 °C above their eqFMP for at least 24 h, and at a lower temperature for at least several days. Field experiments confirmed that superheated ice occurs naturally inside wild fishes. Over the long-term record (1999-2012), seawater temperature surpassed the fish eqFMP in most summers, but never exceeded the highest temperature at which ice persisted inside experimental fishes. Thus, because of the effects of AFP-induced melting inhibition, summer warming may not reliably eliminate internal ice. Our results expose a potentially antagonistic pleiotropic effect of AFPs: beneficial freezing avoidance is accompanied by melting inhibition that may contribute to lifelong accumulation of detrimental internal ice crystals.

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

    CERN Document Server

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

    2012-01-01

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

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

    Science.gov (United States)

    Grabowska, Joanna; Kuffel, Anna; Zielkiewicz, Jan

    2016-08-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ricardo Vera Bravo

    2011-12-01

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

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

    Science.gov (United States)

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

    2015-10-26

    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.

  18. Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance {sup 13}C NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daley, Margaret E.; Sykes, Brian D. [University of Alberta, Department of Biochemistry, CIHR Group in Protein Structure and Function and Protein Engineering Network of Centres of Excellence (Canada)

    2004-06-15

    The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance {sup 13}C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the {sup 1}H-{sup 13}C NOE were determined in this study. The C{alpha}H relaxation measurements were compared to the previously measured {sup 15}N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the {chi}{sub 1} dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than {+-}25 deg.

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

    Directory of Open Access Journals (Sweden)

    Jaewang Lee

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

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Science.gov (United States)

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

    2012-06-12

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  3. Gene expression of different antifreeze proteins of Tenebrio molitor in response to cold acclimation%黄粉虫不同抗冻蛋白基因家族成员的低温应激表达

    Institute of Scientific and Technical Information of China (English)

    冯云甲; 徐洪富; 王宪辉

    2012-01-01

    黄粉虫Tenebrio molitorL.抗冻蛋白基因家族有多个成员,其氨基酸数量和蛋白结构存在差异.尽管有报道发现冷驯化后这些抗冻蛋白的表达量会升高,但不同家族成员是否存在功能分化尚不清楚.本研究中,检测了冷驯化对低温死亡率的效应和对不同类型的抗冻蛋白家族成员基因表达量的影响.结果表明,冷驯化可以显著降低黄粉虫幼虫的低温死亡率和提高不同类型抗冻蛋白基因的表达量.其中,长的抗冻蛋白和低温死亡率的相关关系最为明显.说明不同的抗冻蛋白家族成员的功能有明显的分化,为进一步理解抗冻蛋白的活性和利用抗冻蛋白提供了新的认识.%The Tenebrio molitor L. antifreeze protein gene family has multiple members which differ in the number of amino acids and protein structure. The mRNA level of antifreeze proteins is enhanced by cold conditions but functional differentiation of different members of this family is not clear. In this study, we examined the effects of mortality from cold exposure on the gene expression of different types of antifreeze protein members in the family. The results show that cold acclimation can significantly reduce larval mortality and improve expression of the antifreeze protein gene. The longest antifreeze protein is most important to low-temperature-related mortality. This result indicates that the function of different antifreeze protein family members differs and provides new insights for the use of antifreeze proteins and antifreeze protein activity.

  4. Tissue specific expression of antifreeze protein and growth hormone transgenes driven by the ocean pout (Macrozoarces americanus) antifreeze protein OP5a gene promoter in Atlantic salmon (Salmo salar).

    Science.gov (United States)

    Hobbs, Rod S; Fletcher, Garth L

    2008-02-01

    Previous research aimed at producing genetically improved salmon broodstock for aquaculture led to the creation of two lines of transgenic Atlantic salmon using gene constructs that were derived in part from the ocean pout OP5a antifreeze protein (AFP) gene. One of the lines was produced using an OP5a AFP gene in which the 5' region of the promoter was removed (termed t-OP5a-AFP), and the other line contains a growth hormone (GH) transgene (EO-1alpha) that consists of a chinook salmon GH cDNA driven by a truncated OP5a AFP promoter that is almost identical to that of the t-OP5a-AFP construct. The similarity of the promoter regions of these transgenes provided an opportunity to evaluate their tissue specific expression patterns. Expression of mRNA was evaluated using Northern blot and RT-PCR techniques. The results demonstrate that the AFP and GH trangenes were expressed in almost all body tissues, suggesting that the promoter region of the OP5a AFP gene lacks tissue specific elements. Northern analysis revealed that expression of the t-OP5a-AFP gene was considerably greater than that of the EO-1alpha GH transgene. Only the spleen tissue of the GH transgenics showed a visible band of hybridization. In contrast clear bands of hybridization were evident in all tissues, except for blood cells, of the AFP transgenics with heart, liver and brain tissue showing the highest levels of mRNA expression. This higher level of expression could be attributable to the presence of introns in the t-OP5a-AFP transgene. Since the GH transgenic salmon grow considerably faster than non-transgenics the low levels of GH transgene expression in this line were clearly sufficient to produce the desired rapid growth phenotype. In contrast the levels of AFP expression were inadequate to impart any improvement in the freeze resistance of the AFP transgenic salmon.

  5. Saccharide antifreeze compositions

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-12-10

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

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

    Directory of Open Access Journals (Sweden)

    Chin-Sheng Yu

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform......, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18...... equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge...

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

    Institute of Scientific and Technical Information of China (English)

    周晓蕾; 陈滔滔; 王保怀; 李芝芬; 费云标; 魏令波; 高素琴

    2001-01-01

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

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

    DEFF Research Database (Denmark)

    Lørup Buch, Johannes; Ramløv, H

    2016-01-01

    The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method....... The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing...... AFP could reliably be told apart from controls after only two minutes of recrystallisation. The goal of providing a fast, cheap and easy method for detecting antifreeze proteins in solution was met, and further development of the system can be followed at https://github.com/pechano/cryostage....

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

    Science.gov (United States)

    Buch, J L; Ramløv, H

    2016-06-01

    The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method. The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing AFP could reliably be told apart from controls after only two minutes of recrystallisation. The goal of providing a fast, cheap and easy method for detecting antifreeze proteins in solution was met, and further development of the system can be followed at https://github.com/pechano/cryostage.

  11. Antifreeze glycopeptide diastereomers.

    Science.gov (United States)

    Nagel, Lilly; Budke, Carsten; Dreyer, Axel; Koop, Thomas; Sewald, Norbert

    2012-01-01

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

  12. Antifreeze glycopeptide diastereomers

    Directory of Open Access Journals (Sweden)

    Lilly Nagel

    2012-10-01

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

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

    Institute of Scientific and Technical Information of China (English)

    王颖; 杨光伟

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2016-01-01

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

  15. Hydration layer dynamics and association mechanisms of food and antifreeze proteins : A Molecular Dynamics and Transition Path Sampling study

    NARCIS (Netherlands)

    Brotzakis, Z.F.

    2017-01-01

    By the time the reader reads this line, billions of protein association events just occurred in our body, such as the ones regulating cell communication, signaling pathways, or in initiating a self-assembly processes, such as tissue fabrication, etc. The timescale of such transitions is slow, compar

  16. Antifreeze life cycle assessment (LCA

    Directory of Open Access Journals (Sweden)

    Kesić Jelena

    2005-01-01

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

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-04-07

    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.

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

    Institute of Scientific and Technical Information of China (English)

    曹文红; 赵子科; 田申; 陈良

    2015-01-01

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

  20. Cloning and bioinformatics analysis of antifreeze protein from Tenebrio molitor%黄粉甲抗冻蛋白基因克隆及生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    任谦; 熊鸿燕; 朱才众; 张世界

    2009-01-01

    目的:获得黄粉甲抗冻蛋白基因afpTx及相关生物信息学资料.方法:从黄粉甲幼虫中提取总RNA,通过RT-PCR合成黄粉甲抗冻蛋白基因afpTx的eDNA片段,克隆入载体pMDl9-T,进行测序分析.酶切后将其亚克隆入表达栽体pET32a(+),构建表达质粒pET32a-afpllx,并转化到大肠杆菌DL21后提取质粒,双酶切鉴定.采用MEGA 4.0,BioEdit 5.0.6软件对本研究克隆的抗冻蛋白基因afpTx进行氨基酸序列同源性变异及进化分析.结果:测序结果afpTx的cDNA长度为336 bp;编码112个氨基酸;酶切、电泳结果表明克隆和亚克隆获得成功.抗冻蛋白氨基酸序列相似性分析表明afpTx与GenBank上提交的23条黄粉甲抗冻蛋白的氨基酸序列平均一致性为88%;与11条赤翅甲抗冻蛋白氨基酸序列的平均一致性为67%,2种甲虫的平均一致性为63%.进化树分析结果显示黄粉甲与赤翅甲抗冻蛋白序列是同源序列.赤翅甲的序列趋异度显著大于黄粉甲抗冻蛋白基因序列.结论:成功克隆了本地黄粉甲的afpTx基因,该序列是GenBank上提交的黄粉甲与赤翅甲抗冻蛋白的同源序列.%AIM: To obtain sequence coding gene for the antifreeze proteins (AFP) from local Tenebrio molitor and to elucidate the related bioinformatics data. METHODS: After the total RNA was isolated, from the larva of Tenebrio molitor. cDNA encoding the afpTx was synthesized by RT-PCR, and the PCR products were inserted into the vector pMD19-T simple, which were subcloned into pET-32a( + ) and transformed into E. coli and identified with restriction enzyme analysis. Then the sequencing result was analyzed by MEGA 4. 0 and BioEdit 5. 0. 6 computer program for amino acid sequence homology and evolutionary variance. RESULTS: Sequencing result showed a correctly constructed vector that containing 336 bp antifreeze protein cDNA. Digestion and electrophoresis results confirmed that gene was successfully cloned and subcloned into pET32a

  1. Cloning and Sequencing of Antifreeze Protein Gene in Daucus carota var.sativus Hoffm Deutschl%胡萝卜Daucus carota var.sativus Hoffm Deutschl抗冻蛋白基因的克隆及测序

    Institute of Scientific and Technical Information of China (English)

    尹明安; 崔鸿文; 樊代明; 郭立

    2001-01-01

    以宁夏吴忠胡萝卜、陕西华县胡萝卜和陕西汉中胡萝卜3个地方品种为材料,用PCR(polymerase chain reaction)的方法克隆了中国胡萝卜的抗冻蛋白基因(afp),测定了其核苷酸序列,并和英国胡萝卜的afp序列进行了对比。在所测1004个核苷酸中,两变种碱基不同者有36个,占3.6%,其中无义突变21个,有义突变15个。按有义突变计,同源性为98.5%。%Three carrot cultivar Wuzhong Ningxia,Huaxian Shaanxi and Hanzhong Shaanxi were used as test material and antifreeze protein gene(a fp)of Chinese carrot(Daucus carota var.sativus Hoffm Deutschl)was clon ed and sequenced by PCR(polymerase chain reaction).Obtained sequence was compare d with that of British carrot(Daucus carota var.autumn King).There were 36 different bases in 1004 nucleotides(3.6%)between the two va-r ieties. Among the different bases there were 21 nonsense mutations and 15 sens e mutations.According to sense mutations,homology was 98.5%.

  2. Preparing and evaluating delivery systems for proteins

    DEFF Research Database (Denmark)

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

    2006-01-01

    From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping...

  3. Cloning of a carrot gene encoding antifreeze protein and construction of its plant expression vector%胡萝卜抗冻蛋白基因克隆及植物表达载体构建

    Institute of Scientific and Technical Information of China (English)

    尹明安; 崔鸿文; 樊代明; 郭立

    2001-01-01

    以胡萝卜品种Autumn King 的幼苗为材料,用CTAB法提取其基因组DNA,以PCR (Polymerase Chain Reaction)的方法在体外扩增出胡萝卜抗冻蛋白基因(afp),以pUCm-T Vector为载体构建成胡萝卜afp的克隆载体pTAF,用EcoRⅠ消化重组质粒pTAF使其线性化,再用DNA聚合酶Ⅰ Klenow大片段补平末端,然后用XbaⅠ消化,获得一末端粘,一末端平的目的片段(afp)。植物表达载体pBI121用XbaⅠ和SmaⅠ双酶切,获得一末端粘,一末端平的线性质粒。将目的片段与线性质粒在T4 DNA连接酶的作用下进行定向连接,构建成胡萝卜afp的植物表达载体pBAF。%Genomic DNA in the seedlings of carrot cultivar Autumn King was extracted with CTAB method.The carrot antifreeze protein gene (afp) was amplified by PCR(Polymerase Chain Reaction).Cloning vector pTAF of carrot afp was constru cted with pUCm-T Vector.PTAF was digested with EcoRⅠ and became linear.Its ends were filled with DNA Polymerase Ⅰ Klenow fragment.Then it was digested wi th XbaⅠ and a designed fragment (afp) with a cohesive end and a blunt end was released.Plant expression vector pBI121 was digested with XbaⅠ and SmaⅠand a linear plasmid with a cohesive end and a blunt end was obtained.The linear plasmid and the designed fragment (afp) were directively ligated with T4 DNA ligase,and the plant expression vector of carrot afp was constructed.

  4. Testing antifreeze protein from the longhorn beetle Rhagium mordax as a kinetic gas hydrate inhibitor using a high-pressure micro differential scanning calorimeter

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    F ÁLVAREZFLÓREZ

    2006-06-01

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

  6. 天然抗冻多肽的制备、分离及细菌低温保护活性研究%Preparation, isolation and hypothermia protection activity on bacteria of natural antifreeze peptides

    Institute of Scientific and Technical Information of China (English)

    赵珺; 汪少芸; 李晓坤

    2013-01-01

    The gelatin of shark skin was extracted by hot water. The most appropriate protease and hydrolysis time were selected with the index of hypothermia protection activity on bacteria. Then the hydro-lyzate was added on to Sephadex G - 50 gel filtration chromatography and SP - Sephadex C - 25 strong cation exchange chromatography to acquire high activity fractions. The results indicated that the most appropriate protease is acid protease, and the hydrolysis condition is as follows, hydrolysis temperature 50 °C. pH 3.0, proportion of acid protease to substratum 3 000 U · g-1 per substratum, substratum concentration 3%. hydrolysis time 1 h. The cationic fraction termed P2 shows higher antifreeze activity after Sephadex G -50 gel filtration chromatography and SP - Sephadex C -25 strong cation exchange chromatography. The hypothermia protection assay shows that the survival rate of E. coll was 80. 8% when the concentration of peptide complexes was 500 μg · mL-1. The antifreeze peptides obtained after isolation shows higher antifreeze activity, and can be applied into food and medicine industry.%利用热水抽提法提取鲨鱼皮明胶,控制酶法水解条件制备天然抗冻多肽.以细菌低温保护活性为指标,筛选蛋白水解酶种类及酶解时间,利用SephadexG-50凝胶过滤色谱、SP-SephadexC-25强阳离子交换色谱等分离手段进行分离,得到细菌低温保护高活性组分.结果表明,酶解鲨鱼皮明胶得到高活性抗冻多肽的最适蛋白水解酶为酸性蛋白酶,酶解温度50℃、pH 3.0、酶/底物3000U ·g-1、底物浓度3%、酶解时间1h;经过Sephadex G-50凝胶过滤色谱、SP-Sephadex C-25强阳离子交换色谱等分离手段得到阳离子的P2组分,经过细菌低温保护测试,当浓度为500 μg·mL-1时,大肠杆菌的存活率达到80.8%.分离得到的抗冻多肽具有较高的抗冻活性,可望应用于食品、医药等产业中.

  7. Antifreeze coatings for rotor blades of wind turbines - Final report; Antifreeze Beschichtungen fuer Rotorblaetter von Windenergieanlagen - Schlussbericht

    Energy Technology Data Exchange (ETDEWEB)

    Siegmann, K.; Meola, G.; Hirayama, M.

    2009-08-15

    Iced rotor blades drastically reduce the energy production of wind turbines. In addition, ice throw from the iced blades can be dangerous. There are yet no convincing solutions for the icing problem. An interesting approach is the use of a rotor blade coating. We have developed a coating which influences the freezing behaviour of water. In this report, we describe tests on the antifreeze-coatings developed by us. It is shown that water droplets on the antifreeze coating freeze later than droplets on the untreated glass. This effect could lead to a non-icing of coated rotor blades, because the droplets could be blown of the blade before they can freeze. Additionally, the ice adhesion to the antifreeze coatings is measured. Ice adheres to the antifreeze coating about as good as to bare aluminium and better than to adhesion reducing coatings. (authors)

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

    Institute of Scientific and Technical Information of China (English)

    钟伯雄; 翁宏飚; 等

    2002-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    钟伯雄; 翁宏飚; 方维焕

    2002-01-01

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

  10. Assessing the ability of a short fluorinated antifreeze glycopeptide and a fluorinated carbohydrate derivative to inhibit ice recrystallization.

    Science.gov (United States)

    Chaytor, Jennifer L; Ben, Robert N

    2010-09-01

    A short fluorinated antifreeze glycopeptide (2) was synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The activity of 2 was compared to native biological antifreeze AFGP 8 and a rationally designed C-linked AFGP analogue (OGG-Gal, 1). In addition, a simple fluorinated galactose derivative was prepared and its IRI activity was compared to non-fluorinated compounds. The results from this study suggest that the stereochemistry at the anomeric position in the carbohydrate plays a role in imparting ice recrystallization inhibition activity and that incorporation of hydrophobic groups such as fluorine atoms cause a decrease in IRI activity. These observations are consistent with the theory that fluorine atoms increase ordering of bulk water resulting in a decrease of IRI activity, supporting our previously proposed mechanism of ice recrystallization inhibition.

  11. Preparation of Functional Soybean Protein Isolate

    Institute of Scientific and Technical Information of China (English)

    Liuzhitong; LiXiaolian; 等

    2000-01-01

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

  12. Preparation of Functional Soybean Protein Isolate

    Institute of Scientific and Technical Information of China (English)

    Liu Zhitong; Li Xiaolian; Zhao Guangming

    2000-01-01

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

  13. Bioinspired Antifreeze Secreting Frost-Responsive Pagophobic Coatings

    Science.gov (United States)

    Sun, Xiaoda; Damle, Viraj; Rykaczewski, Konrad

    2014-11-01

    Prevention of ice and frost accumulation is of interest to transportation, power generation, and agriculture industries. Superhydrophobic and lubricant impregnated pagophobic coatings have been proposed, however, they both fail in frosting conditions. Inspired by functional liquid secretion in natural systems, such as toxin secretion by poison dart frost in response to predator presence, we developed frost-responsive antifreeze secreting pagophobic coatings. These are bi-layered coatings with an inner superhydrophilic ``dermis'' infused with antifreeze and an outer permeable superhydrophobic ``epidermis.'' The superhydrophobic epidermis separates the antifreeze from the environment and prevents ice accumulation by repelling impinging water droplets. In frosting conditions, the antifreeze is secreted from the dermis through pores in the epidermis either due to contact with condensed droplets or temporary switch of the epidermis wettability from hydrophobic to hydrophilic caused by surface icing. Here we demonstrate superior performance of this multifunctional coating in simulated frosting, freezing mist/fog, and freezing spray/rain conditions. KR acknowledges startup funding from ASU.

  14. RAPID TEST METHOD FOR EVALUATION OF ANTIFREEZE ADDITIVE EFFICIENCY

    Directory of Open Access Journals (Sweden)

    S. V. Gushchin

    2015-01-01

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

  15. The Antifreeze Critical Strength of Low-temperature Concrete Effected by Index

    Institute of Scientific and Technical Information of China (English)

    LIU Jun; LIU Yu; LIU Runqing

    2011-01-01

    The antifreeze critical strength and the pre-curing time of low-temperature concrete were studied by means of guaranteed rate of compressive strength and antifreeze performance for the structural safety requirement of concrete engineering, suffering once freeze damage under air environment. It is shown that the antifreeze critical strength is 3.7-4.4 MPa, pre-curing time is 18-32 h by guaranteed rate of compressive strength, and the antifreeze critical strength is 3.7-4.4 MPa, pre-curing time is 18-32 h by guaranteed rate of antifreeze performance. It can be found that the method of guaranteed rate of compressive strength is sensitive to the defect which generated by freeze damage in the concrete interior. The method is fit to evaluate the antifreeze critical strength of low-temperature concrete.

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

    Institute of Scientific and Technical Information of China (English)

    LIU Junzhe; LI Yushun; LV Lihua

    2005-01-01

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

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

    DEFF Research Database (Denmark)

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

    . The properties of the emulsifier used and the structure at the interface are therefore expected to be of great importance for oxidation in emulsions. This presentation will include results from mainly three different studies of lipid oxidation in omega-3 emulsions prepared with milk proteins and protein...... components. In these three studies different parameters that are expected to change the properties and structure of the proteins at the interface were investigated. The first study compares 70% emulsions with either sodium caseinate or whey protein isolate at two pH values with and without iron addition....... The second study evaluates the effect of two different high pressure homogenizers on oxidation in 10% emulsions with the same emulsifiers as in the first study. Finally, the third study considers the effect of changing pH on oxidation in emulsions prepared with different whey protein components. Results...

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

    Energy Technology Data Exchange (ETDEWEB)

    Engeler, L.; Salerno, B.

    2003-12-15

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

  19. Potential Antifreeze Compounds in Present-Day Martian Seepage Groundwater

    Directory of Open Access Journals (Sweden)

    Jiin-Shuh Jean

    2008-01-01

    Full Text Available Is the recently found seepage groundwater on Mars pure H2O, or mixed with salts and other antifreeze compounds? Given the surface conditions of Mars, it is unlikely that pure water could either exist in its liquid state or have shaped Mars¡¦ fluid erosional landforms (gullies, channels, and valley networks. More likely is that Mars¡¦ seepage groundwater contains antifreeze and salt compounds that resist freezing and suppress evaporation. This model better accounts for Mars¡¦ enigmatic surface erosion. This paper suggests 17 antifreeze compounds potentially present in Martian seepage groundwater. Given their liquid state and physical properties, triethylene glycol, diethylene glycol, ethylene glycol, and 1,3-propylene glycol are advanced as the most likely candidate compounds. This paper also explores how a mixing of glycol or glycerol with salts in the Martian seepage groundwater may have lowered water¡¦s freezing point and raised its boiling point, with consequences that created fluid gully and channel erosion. Ethylene glycol and related hydrocarbon compounds have been identified in Martian and other interstellar meteorites. We suggest that these compounds and their proportions to water be included for detection in future explorations.

  20. Bacterial Ice Crystal Controlling Proteins

    Directory of Open Access Journals (Sweden)

    Janet S. H. Lorv

    2014-01-01

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

  1. Preparation of Modified Films with Protein from Grouper Fish

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-01-19

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

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

    Directory of Open Access Journals (Sweden)

    Yanye Feng

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hyun-Cheol Kim

    2013-06-01

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

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

    Science.gov (United States)

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

    2009-11-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-07-01

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

  11. Nonequilibrium antifreeze peptides and the recrystallization of ice.

    Science.gov (United States)

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

    1995-02-01

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

  12. Electron Cryomicroscopy of Membrane Proteins: Specimen Preparation for Two-Dimensional Crystals and Single Particles

    OpenAIRE

    Schmidt-Krey, Ingeborg; Rubinstein, John L.

    2010-01-01

    Membrane protein structure and function can be studied by two powerful and highly complementary electron cryomicroscopy (cryo-EM) methods: electron crystallography of two-dimensional (2D) crystals and single particle analysis of detergent-solubilized protein complexes. To obtain the highest-possible resolution data from membrane proteins, whether prepared as 2D crystals or single particles, cryo-EM samples must be vitrified with great care. Grid preparation for cryo-EM of 2D crystals is possi...

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

    Science.gov (United States)

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

    2005-12-28

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

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

    Science.gov (United States)

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

    2016-07-01

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Graham Laurie A

    2012-09-01

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

  18. Qualification and quantification of fish protein in prepared surimi crabstick.

    Science.gov (United States)

    Reed, Z H; Park, J W

    2008-06-01

    Species identification and protein quantification in surimi crabstick were achieved using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). When the Lowry and Kjeldahl protein determination methods were compared, the former showed more consistent results. Densitometric scanning of the gels was used for quantification of total fish protein as well as total egg white protein. The lower molecular weight proteins, 30 kDa and lower, proved to be the most useful in fish species identification as well as egg white protein addition. Using a combination of the myosin heavy chain band and the species-specific myosin light chain (Alaska pollock: 22.5 kDa; Pacific whiting: 24.4 kDa) proved the most accurate in calculating fish protein content of the crabstick sample, while for those samples that contained egg white, quantification was accomplished from the densitometric analysis of the overlapping bands of actin (45 kDa) from fish and ovalbumin from egg white. Lysozyme (14.3 kDa) proved to be a unique protein band in determining the presence of egg white when the content of dried egg white was equal to or exceeded 0.5% of the total weight of the final crabstick.

  19. Oscillations and accelerations of ice crystal growth rates in microgravity in presence of antifreeze glycoprotein impurity in supercooled water

    Science.gov (United States)

    Furukawa, Yoshinori; Nagashima, Ken; Nakatsubo, Shun-ichi; Yoshizaki, Izumi; Tamaru, Haruka; Shimaoka, Taro; Sone, Takehiko; Yokoyama, Etsuro; Zepeda, Salvador; Terasawa, Takanori; Asakawa, Harutoshi; Murata, Ken-ichiro; Sazaki, Gen

    2017-01-01

    The free growth of ice crystals in supercooled bulk water containing an impurity of glycoprotein, a bio-macromolecule that functions as ‘antifreeze’ in living organisms in a subzero environment, was observed under microgravity conditions on the International Space Station. We observed the acceleration and oscillation of the normal growth rates as a result of the interfacial adsorption of these protein molecules, which is a newly discovered impurity effect for crystal growth. As the convection caused by gravity may mitigate or modify this effect, secure observations of this effect were first made possible by continuous measurements of normal growth rates under long-term microgravity condition realized only in the spacecraft. Our findings will lead to a better understanding of a novel kinetic process for growth oscillation in relation to growth promotion due to the adsorption of protein molecules and will shed light on the role that crystal growth kinetics has in the onset of the mysterious antifreeze effect in living organisms, namely, how this protein may prevent fish freezing. PMID:28262787

  20. Recent advances in the preparation progress of protein/peptide drug loaded PLA/PLGA microspheres.

    Science.gov (United States)

    Xu, Feng-Hua; Zhang, Qiang

    2007-01-01

    Sustained release drug delivery from microparticles is an excellent alternative for daily protein/peptide drug administration protocol. Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) are the most commonly used polymer carriers in the development of protein/peptide microspheres. Basically there are three preparation methods for PLA/PLGA microspheres: the solvent extraction/evaporation based multiple emulsion (W/O/W emulsion) method, the phase separation method and the spray drying method. The stability of the protein/pipetide loaded, encapsulation efficiency, and the burst effect of the microspheres are key problems usually met in the preparation of microspheres. In this review the preparation techniques and progress in the development of protein/pipetide microspheres which aimed to stabilize protein/peptide structural integrity, keep the bioactivity of drugs, increase the encapsulation efficiency and improve the release profile were summarized and evaluated.

  1. DSCG binding protein and process for preparing same

    Energy Technology Data Exchange (ETDEWEB)

    Pecht, I.; Mazurek, N.

    1987-07-28

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

  2. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

    Science.gov (United States)

    Weber, Daniela; Davies, Michael J; Grune, Tilman

    2015-08-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples.

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

  5. Preparative Procedures Markedly Influence the Appearance and Structural Integrity of Protein Storage Vacuoles in Soybean Seeds

    Science.gov (United States)

    In legumes, vacuoles serve as the final depository for storage proteins. The protein storage vacuoles (PSVs) of soybean contain electron-transparent globoid regions in which phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate) is sequestered. Here, I report the effect of preparative procedures o...

  6. Preparative Purification of Recombinant Proteins: Current Status and Future Trends

    Directory of Open Access Journals (Sweden)

    Mayank Saraswat

    2013-01-01

    Full Text Available Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications.

  7. A review on preparative and semi-preparative offgel electrophoresis for multidimensional protein/peptide assessment.

    Science.gov (United States)

    Moreda-Piñeiro, Antonio; García-Otero, Natalia; Bermejo-Barrera, Pilar

    2014-07-11

    Mass spectrometry (MS) techniques are commonly used for protein identification and further analysis of selected protein spots after high resolution 2-D electrophoresis. Complementary gel-free approaches have been developed during the last few years and have shown to be useful tools in modern proteomics. The development and application of various gel-free electrophoresis devices for performing protein fractionation according to the pI differences is therefore a topic of interest. This review describes the current state of isoelectric focusing (IEF) gel-free electrophoresis based on the Agilent offgel 3100 fractionator. The review includes, therefore, (i) an overview on IEF as well as other previous IEF gel-free electrophoresis developments; (ii) offgel fundamentals and future trends; (iii) advantages and disadvantages of current offgel procedures; (iv) requirements of isolated protein pellets for further offgel fractionation; (v) offgel fraction requirements to perform the second dimensional analysis by advance electrophoresis and chromatographic techniques; and (vi) effect of the offgel operating conditions on the stability of metal-protein complexes.

  8. Preparative displacement electrophoresis (isotachophoresis) of proteins on cellulose columns.

    Science.gov (United States)

    Johansson, G; Ofverstedt, L G; Hjertén, S

    1987-11-01

    This paper describes the separation of proteins by displacement electrophoresis on columns packed with cellulose powder as a stabilizing medium. Cellulose has virtually no molecular sieving properties and thus differs from dextran, polyacrylamide, and agarose in this respect. Therefore, without the risk of unstacking, columns packed with cellulose permit conventional elution of the protein zones and the use of a counter flow (to increase the effective length of the bed). For the same reason, electroosmotic flow is less disturbing. A continuous elution-migration technique adapted to suit the special requirements of displacement electrophoresis gave better separation than was obtainable by conventional elution. Normal human serum and a fresh hemolysate from human erythrocytes were used as samples. An expression for the volume velocity of the boundaries is derived. This parameter can be used to determine the maximum duration of a run and a suitable pump speed when continuous elution or a counter flow is employed. The special advantages of displacement electrophoresis in cellulose beds are discussed as well as general disadvantages of the displacement technique, including the risk that proteins precipitate during a run.

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  10. Flavour aspects of pea and its protein preparations in relation to novel protein foods

    NARCIS (Netherlands)

    Heng, L.

    2005-01-01

    This research is part of the multidisciplinary program, PROFETAS (PROtein Foods Environment Technology And Society), which aimed to feasibly shift from animal proteins to pea proteins for the development of Novel Protein Foods (NPFs) with desirable flavour. The aim of this research is to investigate

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Elisabeth I. Vogt

    2016-06-01

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

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

    Science.gov (United States)

    Lynn, R. O. L.

    1981-01-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2000-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Syed Hussinien H Shah

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

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

    Science.gov (United States)

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

    1985-01-01

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

  18. Physicochemical and structural properties of composite gels prepared with myofibrillar protein and lard diacylglycerols.

    Science.gov (United States)

    Diao, Xiaoqin; Guan, Haining; Zhao, Xinxin; Diao, Xinping; Kong, Baohua

    2016-11-01

    The objective of this study was to investigate the physicochemical and structural properties of composite gels prepared with porcine myofibrillar protein (MP) and lard, glycerolized lard (GL) or purified glycerolized lard (PGL). The gels prepared with MP and GL or PGL had significantly higher penetration force and water-holding capacity (WHC) than the gel with lard (Pgel, T21 and T22 of the gels that were prepared with GL or PGL moved in the direction of slower relaxation time, which suggests that the water mobility in the gel system was restricted. The presence of lard, GL and PGL did not affect the participating proteins in composite gels. The presence of GL and PGL altered the secondary and tertiary structures of MP in composite gels, which changed the gel properties. In general, the composite gels that were prepared with MP and GL or PGL showed improved gel quality.

  19. Preparation of the Mgm101 recombination protein by MBP-based tagging strategy.

    Science.gov (United States)

    Wang, Xiaowen; Mbantenkhu, MacMillan; Wierzbicki, Sara; Chen, Xin Jie

    2013-06-25

    The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

  20. Preparation and functional analysis of recombinant protein transduction domain-metallothionein fusion proteins.

    Science.gov (United States)

    Lim, Kwang Suk; Won, Young-Wook; Park, Yong Soo; Kim, Yong-Hee

    2010-08-01

    In order for proteins to be used as pharmaceuticals, delivery technologies need to be developed to overcome biochemical and anatomical barriers to protein drug transport, to protect proteins from systemic degradation, and to target the drug action to specific sites. Protein transduction domains (PTDs) are used for the non-specific transduction of bio-active cargo, such as proteins, genes, and particles, through cellular membranes to overcome biological barriers. Metallothionein (MT) is a low molecular weight intra-cellular protein that consists of 61 amino acids, including 20 cysteine residues, and is over-expressed under stressful conditions. Although MT has the potential to improve the viability of islet cells and cardiomyocytes by inhibiting diabetic-induced apoptosis and by removing reactive oxygen species (ROS), and thereby prevent or reduce diabetes and diabetic complications, all MT applications have been made for gene therapy or under induced over-expression of endogenous MT. To overcome the drawbacks of ineffective intra-cellular MT protein uptake, a human MT gene was cloned and fused with protein transduction domains (PTDs), such as HIV-1 Tat and undeca-arginine, in a bacterial expression vector to produce PTD-MT fusion proteins. The expression and purification of three types of proteins were optimized by adding Zn ions to maintain their stability and functionality mimicking intra-cellular stable conformation of MT as a Zn-MT cluster. The Zn-MT cluster showed better stability than MT in vitro. PTD-MT fusion proteins strongly protected Ins-1 beta cells against oxidative stress and apoptosis induced by glucolipotoxicity with or without hypoxia, and also protected H9c2 cardiomyocytes against hyperglycemia-induced apoptosis with or without hypoxia. PTD-MT recombinant fusion proteins may be useful protein therapeutics for the treatment or prevention of diabetes and diabetes-related complications.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Ouyang, Ruizhuo; Lei Jianping; Ju Huangxian, E-mail: jpl@nju.edu.cn, E-mail: hxju@nju.edu.cn [Key Laboratory of Analytical Chemistry for Life Science (Education Ministry of China), Department of Chemistry, Nanjing University, Nanjing 210093 (China)

    2010-05-07

    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 10{sup 18} g{sup -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.

  3. Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode.

    Science.gov (United States)

    To, Brian C S; Lenhoff, Abraham M

    2011-01-21

    The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.

  4. Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    Science.gov (United States)

    Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

    2010-01-01

    Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

  5. Separation and sequencing of familiar and novel murine proteins using preparative two-dimensional gel electrophoresis.

    Science.gov (United States)

    Merrick, B A; Patterson, R M; Witcher, L L; He, C; Selkirk, J K

    1994-05-01

    Strategies are needed for rapid protein isolation in order to identify disease-related proteins and facilitate the design of oligonucleotides for further molecular inquiry. In our laboratory, C3H10T1/2 murine fibroblasts have been found to express a variety of proteins in various subcellular fractions which are relevant to experimental transformation and carcinogenesis. Preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) procedures were developed to identify major cytoplasmic proteins by electroblotting and microsequencing. Isoelectric focusing tube gels were enlarged to 6 mm ID to accommodate larger protein loads at 0.5 to 2 mg protein. Separated proteins were electrotransferred from 6 mm thick slab gels onto 0.22 mu polyvinylidene difluoride membranes. Nearly 100 prominent blotted proteins were stained with Coomassie Brilliant Blue between pI 4.5-7.0 and 18-106 kDa and, of these, 27 prominent and well-resolved proteins were selected for sequencing. Sequences of 14 to 24 amino acid residues in length were obtained from 11 proteins which were identified from computerized databases. Some of these identified proteins had structural or enzymatic functions while others had only recently been discovered, including a newly reported Hsp 70 class member and a novel calcium-binding protein, reticulocalbin. The new heat shock protein has a molecular mass of 75 kDa and has been designated as Grp75, PBP74, CSA or p66mot-1 in mice and humans with purported roles in transformation and antigen processing. Reticulocalbin is an endoplasmic reticular protein which contains six domains of the EF-hand motif associated with high-affinity calcium-binding proteins. It may be involved in protein transport and luminal protein processing. In addition, sequences of 5 to 11 residues in length were also obtained from six other unidentified proteins. Thus, we have found that preparative 2-D PAGE serves as a powerful one-step purification method for protein isolation and

  6. Fibrous scaffolds loaded with protein prepared by blend or coaxial electrospinning.

    Science.gov (United States)

    Ji, Wei; Yang, Fang; van den Beucken, Jeroen J J P; Bian, Zhuan; Fan, Mingwen; Chen, Zhi; Jansen, John A

    2010-11-01

    The aim of the present study was to fabricate polycaprolactone-based nanofibrous scaffolds with incorporated protein via either the blend or coaxial electrospinning technique. Both techniques were compared with respect to processing set-up and scaffold characteristics as well as the release kinetics and biological activity of the loaded protein. Bovine serum albumin was used as a model protein to determine release profiles, while alkaline phosphatase was used to determine protein activity after the electrospinning process. Coaxial electrospinning resulted in a uniform fiber morphology with a core-shell structure, and a homogeneous protein distribution throughout the core of the fibers. In contrast, blend electrospinning formed bead-like fibers with a heterogeneous protein distribution in the fibers. The coaxial scaffold exhibited more sustained release profiles than the comparative blend scaffold, and the additive poly(ethylene glycol) (PEG) in the coaxial scaffold accelerated protein release. Both electrospinning processes decreased the biological activity of the incorporated protein, but coaxial electrospinning with PEG as an additive showed up to 75% preservation of the initial biological activity. Thus, coaxial electrospinning was demonstrated to be superior to blend electrospinning for the preparation of nanofibrous scaffolds with a uniform fibrous structure and protein distribution and sustained protein release kinetics as well as high preservation of the protein activity.

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

    DEFF Research Database (Denmark)

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

    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...... compared the effect of two different high pressure homogenizers on oxidation in caseinate and whey protein isolate emulsions. The second study evaluated the effect of homogenization pressure and temperature on emulsions prepared either with whey proteins or a combination of caseinate and β...

  8. Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags.

    Science.gov (United States)

    Mack, Laura; Brill, Boris; Delis, Natalia; Groner, Bernd

    2014-12-01

    The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction.

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

    Science.gov (United States)

    2003-02-01

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

  10. Microencapsulation of protein drugs for drug delivery: strategy, preparation, and applications.

    Science.gov (United States)

    Ma, Guanghui

    2014-11-10

    Bio-degradable poly(lactide) (PLA)/poly(lactide-glycolide) (PLGA) and chitosan microspheres (or microcapsules) have important applications in Drug Delivery Systems (DDS) of protein/peptide drugs. By encapsulating protein/peptide drugs in the microspheres, the serum drug concentration can be maintained at a higher constant value for a prolonged time, or injection formulation can be changed to orally or mucosally administered formulation. PLA/PLGA and chitosan are most often used in injection formulation and oral formulation. However, in the preparation and applications of PLA/PLGA and chitosan microspheres containing protein/peptide drugs, the problems of broad size distribution and poor reproducibility of microspheres, and deactivation of protein during the preparation, storage and release, are still big challenges. In this article, the techniques for control of the diameter of microspheres and microcapsules will be introduced at first, then the strategies about how to maintain the bioactivity of protein drugs during preparation and drug release will be reviewed and developed in our research group. The membrane emulsification techniques including direct membrane emulsification and rapid membrane emulsification processes were developed to prepare uniform-sized microspheres, the diameter of microspheres can be controlled from submicron to 100μm by these two processes, and the reproducibility of products can be guaranteed. Furthermore, compared with conventional stirring method, the big advantages of membrane emulsification process were that the uniform microspheres with much higher encapsulation efficiency can be obtained, and the release behavior can be adjusted by selecting microsphere size. Mild membrane emulsification condition also can prevent the deactivation of proteins, which frequently occurred under high shear force in mechanical stirring, sonification, and homogenization methods. The strategies for maintaining the bioactivity of protein drug were

  11. High Performance Protein-Coated Microcrystals of Rhizomucor miehei Lipase: Preparation and Application for Organic Synthesis.

    Science.gov (United States)

    Kazlauskas, Simas; Kiriliauskaitė, Vita; Kalėdienė, Lilija; Bendikienė, Vida

    2015-05-01

    The goal of obtaining enzyme forms with higher catalytic activity, greater stability, and improved reusability has been pursued for the last few decades. Various novel biocatalyst designs have been created, and protein-coated microcrystals (PCMCs) are one of them. PCMC is an enzyme immobilization method based on simultaneous precipitation of protein and carrier, forming micron-sized enzyme-coated crystals. Highly active Rhizomucor miehei lipase (RML) PCMCs were prepared by immobilizing the protein onto K2SO4 as a carrier salt in acetone as a precipitating solvent. The formation of RML PCMCs was confirmed by scanning electron microscopy. Preparation of RML PCMCs was optimized by response surface methodology (RSM). Obtained PCMCs were found to be more active and stable during p-nitrophenyl palmitate hydrolysis in n-hexane, compared to liquid RML. The enzymatic activity and temperature optimum increased from 0.011 U/mg(soluble) lipase to 8.70 U/mg(immobilized) lipase and from 30 to 37 °C, respectively. Additionally, the ability of RML PCMCs to catalyze flavor ester 2-phenethyl octanoate synthesis was investigated. Some reaction parameters were optimized, resulting in 80 % conversion within 1 h with an enhanced reusability, compared to commercial immobilized RML preparation. Thus, PCMCs offer a cheap and effective technology for obtaining highly active lipase preparations for biocatalysis in organic media.

  12. Preparation

    Directory of Open Access Journals (Sweden)

    M.M. Dardir

    2014-03-01

    Full Text Available Some hexanamide-mono and di-linoleniate esters were prepared by the reaction of linolenic acid and hexanamide (derived from the reaction of hexanoic acid and diethanolamine. The chemical structure for the newly prepared hexanamide-mono and di-linoleniate esters were elucidated using elemental analysis, (FTIR, H 1NMR and chemical ionization mass spectra (CI/Ms spectroscopic techniques. The results of the spectroscopic analysis indicated that they were prepared through the right method and they have high purity. The new prepared esters have high biodegradability and lower toxicity (environmentally friendly so they were evaluated as a synthetic-based mud (ester-based mud for oil-well drilling fluids. The evaluation included study of the rheological properties, filtration and thermal properties of the ester based-muds formulated with the newly prepared esters compared to the reference commercial synthetic-based mud.

  13. Expression and purification of human ARP1 protein and rapid preparation of polyclonal antibody.

    Science.gov (United States)

    Sun, Mingjuan; Zou, Rongjiang; Dong, Xiaoyi; Zong, Ying; Gao, Yun; Wang, Lianghua; Jiao, Binghua

    2013-01-01

    Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

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

    Science.gov (United States)

    Bittner, B; Kissel, T

    1999-01-01

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

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

    Science.gov (United States)

    Qu, Jian-Bo; Chen, Yan-Li; Huan, Guan-Sheng; Zhou, Wei-Qing; Liu, Jian-Guo; Zhu, Hu; Zhang, Xiao-Yun

    2015-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Xabier Osteikoetxea

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

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

    Science.gov (United States)

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

    2005-06-01

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

  18. Viable transmembrane region mutants of bacteriophage M13 coat protein prepared by site-directed mutagenesis.

    Science.gov (United States)

    Li, Z; Deber, C M

    1991-10-31

    Bacteriophage M13 coat protein - a 50-residue protein located at the E. coli host membrane during phage reproduction - is subjected to cytoplasmic, membrane-bound, and DNA-interactive environments during the phage life cycle. In research to examine the specific features of primary/secondary structure in the effective transmembrane (TM) region of the protein (residues 21-39: YIGYAWAMVVVIVGATIGI) which modulate its capacity to respond conformationally to the progressive influences of these varying environments, we have prepared over two dozen viable mutant phages with alterations in their coat protein TM regions. Mutants were obtained through use of site-directed mutagenesis techniques in combination with three "randomized" oligonucleotides which spanned the TM region. No subcloning was required. Among mutations observed were those in which each of the four TM Val residues was changed to Ala, and several with increased Ser or Thr content, including one double Ser mutant (G23S-A25S). Polar substitutions arising at Gly23 and Tyr24-including G23D, Y24H, Y24D and Y24N-suggested that this local segment resides external to the host membrane. Milligram quantities of mutant coat proteins are obtained by growing M13 mutant phages in liter preparations, with isotopic (e.g., 13C) labelling at desired sites, for subsequent characterization and conformational analysis in membrane-mimetic media.

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

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

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

  20. Altered regulation of brain-derived neurotrophic factor protein in hippocampus following slice preparation.

    Science.gov (United States)

    Danzer, S C; Pan, E; Nef, S; Parada, L F; McNamara, J O

    2004-01-01

    Brain-derived neurotrophic factor (BDNF) and its cognate receptor tyrosine kinase B (TrkB) play important roles in regulating survival, structure, and function of CNS neurons. One method of studying the functions of these molecules has utilized in vitro hippocampal slice preparations. An important caveat to using slices, however, is that slice preparation itself might alter the expression of BDNF, thereby confounding experimental results. To address this concern, BDNF immunoreactivity was examined in rodent slices using two different methods of slice preparation. Rapid and anatomically selective regulation of BDNF content followed slice preparation using both methodologies; however, different patterns of altered BDNF immunoreactivity were observed. First, in cultured slices, BDNF content decreased in the dentate molecular layer and increased in the CA3 pyramidal cell layer and the mossy fiber pathway of the hippocampus after 30 min. Furthermore, an initially "punctate" pattern of BDNF labeling observed in the mossy fiber pathway of control sections changed to homogenous labeling of the pathway in vitro. In contrast to these findings, slices prepared as for acute slice physiology exhibited no change in BDNF content in the molecular layer and mossy fiber pathway 30 min after slicing, but exhibited significant increases in the dentate granule and CA3 pyramidal cell layers. These findings demonstrate that BDNF protein content is altered following slice preparation, that different methods of slice preparation produce different patterns of BDNF regulation, and raise the possibility that BDNF release and TrkB activation may also be regulated. These consequences of hippocampal slice preparation may confound analyses of exogenous or endogenous BDNF on hippocampal neuronal structure or function.

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  2. Preparation of a hydrophobic polythiophene film to improve protein adsorption and proliferation of PC 12 cells.

    Science.gov (United States)

    Li, Da-Feng; Wang, Hua-Jie; Fu, Jian-Xi; Wang, Wei; Jia, Xue-Shun; Wang, Jin-Ye

    2008-12-25

    High quality films of polythiophene with different alkyl side chains were successfully synthesized by a novel method in the presence of sodium dodecylbenzenesulonate (SDBS) under N2 atmosphere on the PTFE (polytetrafluorethylene) substrate. The as-prepared films were characterized by scanning electron microscopy (SEM), conductivity measurement, and water contact angle measurement. The morphologies of the films were homogeneous with micro-/nanostructures, and their conductivities were high enough for biomedical applications. Hydrophobicity of the films could be adjusted easily by inducing alkyl side chains with different length, which could control protein adsorption in succession. Hydrophobic polythiophene film with a long alkyl side chain had a higher ability of protein adsorption and PC 12 cell proliferation. The biocompatibility study of the synthesized films in vitro proved that the synthesized films were not cytotoxic to two cell lines used and could support cell attachment and proliferation well. Polythiophenes films prepared by in-situ deposition will be good candidates for biomedical applications.

  3. “Click chemistry” preparation of WCX packings for protein separation

    Institute of Scientific and Technical Information of China (English)

    Kai Lou Zhao; Chao Song; Fei Wang; Quan Bai

    2012-01-01

    Click chemistry was applied to immobilize three kinds of alkyne-carboxylic acids onto azide-modified silica gel to prepare three novel stationary phases for weak cation exchange chromatography (WCX).The developed protocol combines the benefits of operational simplicity,exceptionally mild conditions and high surface loadings.Six kinds of standard proteins were separated completely on the novel packings.Compared with commercial WCX columns,the three kinds of novel WCX packings prepared by click chemistry approach have better resolution and selectivity.Lysozyme was purified successfully from egg white with the novel WCX column by one step.The purity was more than 97% and a high specific activity was achieved to be 81,435 U/mg.The results illustrate the potential of click chemistry for preparation of stationary phase for IEC.

  4. Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

    Energy Technology Data Exchange (ETDEWEB)

    Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O. [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States); Tumpey, Terrence M. [Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Atlanta, GA (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States)

    2016-01-15

    Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.

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

    Institute of Scientific and Technical Information of China (English)

    傅强

    2009-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    ZhangWeinong; LiuDachuan

    2002-01-01

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

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

    Science.gov (United States)

    Kato, Katsuya; Nakamura, Hitomi; Yamauchi, Yoshihiro; Nakanishi, Kazuma; Tomita, Masahiro

    2014-07-01

    Mesoporous silica (MPS) thin film biosensor platforms were established. MPS thin films were prepared from tetraethoxysilane (TEOS) via using sol-gel and spin-coating methods using a poly-(ethylene oxide)-block-poly-(propylene oxide)-block-poly-(ethylene oxide) triblock polymer, such as P123 ((EO)20(PO)70(EO)20) or F127 ((EO)106(PO)70(EO)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.

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

    Science.gov (United States)

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

    2015-12-01

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

  9. Semiautomated Sample Preparation for Protein Stability and Formulation Screening via Buffer Exchange.

    Science.gov (United States)

    Ying, William; Levons, Jaquan K; Carney, Andrea; Gandhi, Rajesh; Vydra, Vicky; Rubin, A Erik

    2016-06-01

    A novel semiautomated buffer exchange process workflow was developed to enable efficient early protein formulation screening. An antibody fragment protein, BMSdab, was used to demonstrate the workflow. The process afforded 60% to 80% cycle time and scientist time savings and significant material efficiencies. These efficiencies ultimately facilitated execution of this stability work earlier in the drug development process, allowing this tool to inform the developability of potential candidates for development from a formulation perspective. To overcome the key technical challenges, the protein solution was buffer-exchanged by centrifuge filtration into formulations for stability screening in a 96-well plate with an ultrafiltration membrane, leveraging automated liquid handling and acoustic volume measurements to allow several cycles of exchanges. The formulations were transferred into a vacuum manifold and sterile filtered into a rack holding 96 glass vials. The vials were sealed with a capmat of individual caps and placed in stability stations. Stability of the samples prepared by this process and by the standard process was demonstrated to be comparable. This process enabled screening a number of formulations of a protein at an early pharmaceutical development stage with a short sample preparation time.

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

    DEFF Research Database (Denmark)

    Balslev, Y; Hansen, Gert Helge

    1989-01-01

    Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG-gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling...... was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase-isomaltase. The labelling efficiency of RPGG was compared to that of protein A-gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling...... intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12 nm particles, 10.3 and 6.2 particles/micron of length of microvillar membrane, 3.5 and 1.0 particles/micron2 of Golgi profile and 5...

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

    Institute of Scientific and Technical Information of China (English)

    郭睿; 杨晓泉; 杨熙

    2012-01-01

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

  12. Preparation of weak cation exchange packings for chromatographic separation of proteins using "click chemistry''.

    Science.gov (United States)

    Zhao, Kailou; Bai, Quan; Song, Chao; Wang, Fei; Yang, Fan

    2012-04-01

    "Click chemistry" is defined as a class of robust and selective chemical reactions affording high yields and is tolerant to a variety of solvents (including water), functional groups, and air. In this study, click chemistry was used as an effective strategy for coupling three alkyne-carboxylic acids onto the azide-silica to obtain three novel stationary phases of weak cation exchange chromatography, which were characterized with FTIR and elemental analysis. Six kinds of standard proteins, such as myoglobin, RNase A, RNase B, cytochrome C, α-chymotrypsin A, and lysozyme, were separated completely with the three novel weak cation exchange chromatography stationary phases. Compared with commercial weak cation exchange chromatography columns, the three kinds of novel weak cation exchange chromatography packings prepared by click chemistry approach have better resolution and selectivity. The mass recovery of more than 97% was obtained for all the tested proteins, and the bioactivity recovery of lysozyme on the prepared column was determined to be 96%. In addition, lysozyme was purified successfully from egg white with the novel weak cation exchange chromatography column by one step. The purity was more than 97% and a high specific activity was achieved to be 81 435 U/mg. The results illustrate the potential of click chemistry for preparing stationary phase for ion-exchange chromatography.

  13. High-throughput preparation methods of crude extract for robust cell-free protein synthesis.

    Science.gov (United States)

    Kwon, Yong-Chan; Jewett, Michael C

    2015-03-02

    Crude extract based cell-free protein synthesis (CFPS) has emerged as a powerful technology platform for high-throughput protein production and genetic part characterization. Unfortunately, robust preparation of highly active extracts generally requires specialized and costly equipment and can be labor and time intensive. Moreover, cell lysis procedures can be hard to standardize, leading to different extract performance across laboratories. These challenges limit new entrants to the field and new applications, such as comprehensive genome engineering programs to improve extract performance. To address these challenges, we developed a generalizable and easily accessible high-throughput crude extract preparation method for CFPS based on sonication. To validate our approach, we investigated two Escherichia coli strains: BL21 Star™ (DE3) and a K12 MG1655 variant, achieving similar productivity (defined as CFPS yield in g/L) by varying only a few parameters. In addition, we observed identical productivity of cell extracts generated from culture volumes spanning three orders of magnitude (10 mL culture tubes to 10 L fermentation). We anticipate that our rapid and robust extract preparation method will speed-up screening of genomically engineered strains for CFPS applications, make possible highly active extracts from non-model organisms, and promote a more general use of CFPS in synthetic biology and biotechnology.

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

    Science.gov (United States)

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

    2012-01-01

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

  15. [Expression and purification of GST-CML28 fusion protein and preparation of its polyclonal antibody].

    Science.gov (United States)

    Mao, Xia; Zhang, Bing; Bai, Xue-Ling; Liu, Long-Long; Zhang, Dong-Hua

    2012-12-01

    This study was aimed to investigate the expression of GST-CML28 in Escherichia Coli and to prepare its antibody. The constructed recombinant expression vectors CML28-pGEX-3X were transformed into Escherichia Coli BL21 under IPTG induction. The protein was abstracted from the transformers, and purified by a GSTrap FF column. The rabbits were immunized by the purified fusion protein to produce serum with anti-CML28 antibody. The serum was purified by chromatographic column stuffed with glutathione Sephamse 4B to get the antibody. The specific antibody against CML28 was further identified by ELISA, Western blot, immunohistochemistry and quantum dot luminescence. The results indicated that GST-CML28 fusion protein was expressed in Escherichia coli and its specific polyclonal antibody was obtained. It is concluded that the anti-CML28 polyclonal antibodies with high titer and specificity are successfully prepared. These antibodies provide an useful experimental tool to profoundly research the physiological significance and biological function of the CML28 gene.

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

    Science.gov (United States)

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

    2015-03-01

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

  17. Use of a purified outer membrane protein F (porin) preparation of Pseudomonas aeruginosa as a protective vaccine in mice.

    OpenAIRE

    Gilleland, H. E.; Parker, M G; Matthews, J.M.; Berg, R D

    1984-01-01

    The outer membrane protein F (porin) from the PAO1 strain of Pseudomonas aeruginosa was purified by two different methods. One procedure involved separation by column chromatography of proteins extracted from isolated outer membranes, whereas the other involved extraction from gels after slab polyacrylamide gel electrophoresis of proteins extracted from cell envelopes. Both procedures yielded protein F preparations which successfully immunized mice from subsequent challenge with the PAO1 stra...

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

    Science.gov (United States)

    Chen, Weixin; Inoue, Yuuki; Ishihara, Kazuhiko

    2015-11-01

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

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

    Science.gov (United States)

    Hodgman, C Eric; Jewett, Michael C

    2013-10-01

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

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

    Science.gov (United States)

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

    2013-09-20

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

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

    Science.gov (United States)

    Bachelard, H S

    1966-07-01

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

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

    Science.gov (United States)

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

    2011-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-01

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

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

    Institute of Scientific and Technical Information of China (English)

    乔洁

    2006-01-01

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

  5. The comparison of protein-entrapped liposomes and lipoparticles: preparation, characterization, and efficacy of cellular uptake

    Directory of Open Access Journals (Sweden)

    Chang WK

    2011-10-01

    Full Text Available Wei-Kuo Chang1, Yu-Ju Tai2, Chiao-Hsi Chiang3, Chieh-Shen Hu2, Po-Da Hong2, Ming-Kung Yeh3,4 1Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taiwan, ROC; 2Graduate Institute of Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan, ROC; 3School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, ROC; 4Institute of Preventive Medicine, National Defense Medical Center, Sanhsia, Taipei, Taiwan, ROC Abstract: Fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA-loaded polyethylene glycol (PEG-modified liposomes and lipoparticles with high protein entrapment were developed. The lipid formula of the liposomes contained PEGylated lipids and unsaturated fatty acids for enhancing membrane fluidity and effective delivery into cells. The preparation techniques, lipid content, and PEG-modified lipoparticle ratios were evaluated. The PEG-modified lipoparticles prepared by ethanol injection extrusion (100 nm pore size achieve a population of blank liposomes with a mean size of 125 ± 2.3 nm and a zeta potential of —12.4 ± 1.5 mV. The average particle size of the PEG-modified lipoparticles was 133.7 ± 8.6 nm with a zeta potential of +13.3 mV. Lipoparticle conformation was determined using transmission electron microscopy and field-emission scanning electron microscopy. The FITC-BSA encapsulation efficiency was dramatically increased from 19.0% for liposomes to 59.7% for lipoparticles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE results confirmed the preparation process, and an 8-hour leaching test did not harm the protein structure. Once prepared, the physical and chemical stability of the PEG-modified lipoparticle formulations was satisfactory over 90 days. In vitro retention tests indicated that the 50% retention time for the protein-containing lipoparticles was 7.9 hours, substantially longer than

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

    DEFF Research Database (Denmark)

    Doll, Hans; Andersen, Bente

    1981-01-01

    The extraction, reduction, and alkylation of barley hordein for routine electrophoresis in sodium dodecyl sulfate-polyacrylamide gels were studied to set up a simple preparation procedure giving well-resolved bands in the electrophoresis gel. Hordein was extracted from single crushed seeds or flour...... by aqueous 50% propan-2-ol containing a Tris-borate buffer, pH 8.6. The presence of the buffer facilitates the consecutive complete reduction of the extracted protein in the alcohol. Reduction and alkylation in the buffer containing propan-2-ol give sharper bands in the electrophoresis than reduction...

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated......, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates...... of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings...

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

    Institute of Scientific and Technical Information of China (English)

    江兵兵; 高长有; 胡玲; 沈家骢

    2004-01-01

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

  10. Preparation and characterization of whey protein hydrolysates: applications in industrial whey bioconversion processes.

    Science.gov (United States)

    Perea, A; Ugalde, U; Rodriguez, I; Serra, J L

    1993-05-01

    A whey protein hydrolysate was prepared by incubation of reconstituted whey or a whey protein concentrate with Alcalase 0.6L. The proteolytic degradation of alpha-lactalbumin and beta-lactoglobulin initially resulted in 6-kDa and, later, 2.5-kDa degradation products, quickly followed by the appearance of multiple peptides of 1 kDa or smaller. The hydrolysate showed a steady increase in solubility and a biphasic change in foaming characteristics with decreasing peptide size. At the highest degree of hydrolysis achieved (22%), the majority of the peptides were smaller than 1 kDa and could be efficiently assimilated by the yeast Kluyveromyces marxianus growing in a defined medium.

  11. PREPARATION AND PROPERTIES OF WHEAT GLUTEN/RICE PROTEIN COMPOSITES PLASTICIZED WITH GLYCEROL

    Institute of Scientific and Technical Information of China (English)

    Yan-yan Yang; Kai-zhou Zhang; Yi-hu Song; Qiang Zheng

    2011-01-01

    Environment friendly thermosetting composites were prepared by blending wheat gluten (WG) and rice protein (RP) at different weight ratios with glycerol as plasticizer followed by compression molding the mixture at 120C to crosslink the proteins. Reducing agent of sodium bisulfate and sodium sulfite and crosslinking agent formaldehyde were used to adjust the properties of the composites. Morphology, moisture absorption and tensile properties were evaluated. The results showed that formaldehyde could increase tensile strength of the composites without significant influence on Young's modulus and elongation at break. On the other hand, reducing agents could improve tensile strength and extensibility simultaneously, which was much marked at WG/RP ratios from 7/3 to 3/7.

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

    Institute of Scientific and Technical Information of China (English)

    江兵兵; 高长有; 胡玲; 沈家骢

    2004-01-01

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

  13. Determinants of protein elution rates from preparative ion-exchange adsorbents.

    Science.gov (United States)

    Angelo, James M; Lenhoff, Abraham M

    2016-04-01

    The rate processes involved in elution in preparative chromatography can affect both peak resolution and hence selectivity as well as practical factors such as facility fit. These processes depend on the physical structure of the adsorbent particles, the amount of bound solute, the solution conditions for operation or some combination of these factors. Ion-exchange adsorbents modified with covalently attached or grafted polymer layers have become widely used in preparative chromatography. Their often easily accessible microstructures offer substantial binding capacities for biomolecules, but elution has sometimes been observed to be undesirably slow. In order to determine which physicochemical phenomena control elution behavior, commercially available cellulosic, dextran-grafted and unmodified agarose materials were characterized here by their elution profiles at various conditions, including different degrees of loading. Elution data were analyzed under the assumption of purely diffusion-limited control, including the role of pore structure properties such as porosity and tortuosity. In general, effective elution rates decreased with the reduction of accessible pore volume, but differences among different proteins indicated the roles of additional factors. Additional measurements and analysis, including the use of confocal laser scanning microscopy to observe elution within single chromatographic particles, indicated the importance of protein association within the particle during elution. The use of protein stabilizing agents was explored in systems presenting atypical elution behavior, and l-arginine and disaccharide excipients were shown to alleviate the effects for one protein, lysozyme, in the presence of sodium chloride. Incorporation of these excipients into eluent buffer gave rise to faster elution and significantly lower pool volumes in elution from polymer-modified adsorbents.

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

    Science.gov (United States)

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

    2004-11-01

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

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

    Directory of Open Access Journals (Sweden)

    ZHANG Yu

    2013-08-01

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

  16. Cellular uptake of beta-carotene from protein stabilized solid lipid nano-particles prepared by homogenization-evaporation method

    Science.gov (United States)

    Using a homogenization-evaporation method, beta-carotene (BC) loaded nano-particles were prepared with different ratios of food-grade sodium caseinate (SC), whey protein isolate (WPI), or soy protein isolate (SPI) to BC and evaluated for their physiochemical stability, in vitro cytotoxicity, and cel...

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

    Science.gov (United States)

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

    2009-06-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  20. An Efficient Synthetic Strategy for the Preparation of Nucleic Acid-Encoded Peptide and Protein Libraries for In Vitro Evolution Protocols

    Directory of Open Access Journals (Sweden)

    Peter A. Lohse

    2000-12-01

    Full Text Available We describe an improved synthetic strategy for the preparation of nucleic acid encoded peptide and protein libraries. A solid-phase format was used to prepare and purify a novel type of mRNA-template for in vitro mRNA-protein fusion synthesis. The present protocol simplifies and accelerates the preparation of fusion libraries and should prove most useful for in vitro protein evolution procedures which involve repetitive cycles of fusion library preparation and selection.

  1. Manganese containing protein complex isolated from Photosystem II preparations of spinach

    Energy Technology Data Exchange (ETDEWEB)

    Frasch, W.D.; Bowlby, N.R.

    1986-05-01

    By using a ligand-receptor crosslinking method the authors have stabilized Mn associated with Photosystem II (PSII) in a protein complex with an apparent molecular weight of 75,000 and with 3-4 Mn per complex. To crosslink the proteins, purified 33 kDa protein (33) was modified to contain about 8 adducts of the heterobifunctional photoaffinity reagent N-succinimidyl-(4-azidophenyl-dithio)-propionate (SADP). The SADP-33 was reconstituted into PSII membranes which had been depleted of 33 by a 1M CaCl/sub 2/ wash and crosslinking was initiated by ultraviolet illumination. The crosslinked membranes were solubilized in lauryl sulfate (SDS) and the constituent proteins were identified by SDS polyacrylamide gel electrophoresis. Evidence which supports the hypothesis that the Mn associated with the crosslinked proteins has been retained at the site of the photosynthetic oxygen evolving system includes: 1, Scatchard analysis of (/sup 125/I)-33 binding to CaCl/sub 2/-washed PSII preparations revealed one tight binding site and several lower affinity sites; 2, reconstitution of O/sub 2/ evolving activity and binding to the tight site had the same concentration dependence on 33; 3, the SADP-33 was able to reconstitute O/sub 2/ evolution in CaCl/sub 2/ washed PSII membranes; 4, the percent of Mn retained by the crosslinked membranes after treatment with edetic acid (EDTA) was approximately equal to the percent reconstitution of O/sub 2/ evolving activity by SADP-33 before photoactivation of SADP.

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

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

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

  5. Expression of Catalytic Domain of Protein Tyrosine Phosphatase 1B and Preparation of Its Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study focuses on the expression of human protein tyrosine phosphatase 1B(PTP1B) catalytic domain (△PTP1B) and preparation of polyclonal antibody against △PTP1B. △PTP1B gene was PCR amplified with the cDNA of human PTP1B as the template, and cloned into the pT7 expression vector. The recombinant pT7-△PTP1B was expressed in E. coli Rosetta( DE3 ) host cells and purified. The antiserum was prepared by immunizing rabbit with purified recombinant △PTP1B. The polyclonal antibody against △PTP1B was purified by PVDF immobilized antigen affinity chromatography. △PTP1B was correctly cloned, expressed, and purified as confirmed by PCR, DNA sequence ratio) and 0. 1 ng, respectively. This study provides an important basis for further studying the biological function of PTP1B and its relationship with human diseases.

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

    Science.gov (United States)

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

    2013-03-01

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

  7. Preparative procedures markedly influence the appearance and structural integrity of protein storage vacuoles in soybean seeds.

    Science.gov (United States)

    Krishnan, Hari B

    2008-05-14

    In legumes, vacuoles serve as the final depository for storage proteins. The protein storage vacuoles (PSVs) of soybean contain electron-transparent globoid regions in which phytic acid ( myo-inositol-1,2,3,4,5,6-hexakisphosphate) is sequestered. This paper reports the effect of preparative procedures on the appearance and ultrastructural integrity of PSVs in soybeans. Electron microscopy examination of both developing and mature soybean seeds that were postfixed with osmium tetroxide revealed PSVs that had a homogeneous appearance with very few globoid crystals dispersed in them. Numerous electron-dense lipid bodies were readily seen in these cells. Omission of osmium tetroxide strikingly altered the appearance of PSVs and aided the visualization of the location of the globoids in the PSVs. In contrast to the osmicated tissue, lipid bodies appeared as electron-transparent spheres. The choice of dehydration reagent or staining procedure had little influence on the appearance of the PSVs. The results of this study demonstrate the profound effect of osmium tetroxide on the appearance and structural integrity of PSVs in soybean.

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

    Science.gov (United States)

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

    2015-12-01

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

  9. Preparation of protein imprinted materials by hierarchical imprinting techniques and application in selective depletion of albumin from human serum

    Science.gov (United States)

    Liu, Jinxiang; Deng, Qiliang; Tao, Dingyin; Yang, Kaiguang; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2014-06-01

    Hierarchical imprinting was developed to prepare the protein imprinted materials, as the artificial antibody, for the selective depletion of HSA from the human serum proteome. Porcine serum albumin (PSA) was employed as the dummy template for the fabrication of the recognition sites. To demonstrate the advantages of the hierarchical imprinting, molecularly imprinted polymers prepared by hierarchical imprinting technique (h-MIPs) were compared with those obtained by bulk imprinting (b-MIPs), in terms of the binding capacity, adsorption kinetics, selectivity and synthesis reproducibility. The binding capacity of h-MIPs could reach 12 mg g-1. And saturation binding could be reached in less than 20 min for the h-MIPs. In the protein mixture, h-MIPs exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. For the proteomic application, the identified protein group number in serum treated by h-MIPs was increased to 422, which is 21% higher than that obtained from the original serum, meanwhile the identified protein group number for the Albumin Removal kit was only 376. The results demonstrate that protein imprinted polymers prepared by hierarchical imprinting technique, might become the artificial antibodies for the selective depletion of high abundance proteins in proteome study.

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

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

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

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

    Science.gov (United States)

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

    2012-02-28

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

  12. The use of sodium carboxymethylcellulose in the preparation of spray-dried proteins for pulmonary drug delivery.

    Science.gov (United States)

    Li, Hao-Ying; Song, Xiaosong; Seville, Peter C

    2010-04-16

    The use of sodium carboxymethylcellulose (NaCMC) as a spray-drying excipient in the preparation of inhalable formulations of proteins was investigated, using alkaline phosphatase as a model functional protein. Two spray-dried powders were investigated: a control powder comprising 100% (w/w) alkaline phosphatase and a test powder comprising 67% (w/w) NaCMC and 33% (w/w) alkaline phosphatase. Following physicochemical characterisation, the powders were prepared as both dry powder inhaler (DPI) and pressurised metered dose inhaler (pMDI) formulations. The aerosolisation performance of the formulations was assessed using a Multi-Stage Liquid Impinger, both immediately after preparation and over a 16-week storage period. Formulating the control powder as a DPI resulted in a poor fine particle fraction (FPF: 10%), whereas the FPF of the NaCMC-modified DPI formulation was significantly greater (47%). When the powders were formulated as pMDI systems, the control and NaCMC-modified powders demonstrated FPFs of 52% and 55%, respectively. Following storage, reduced FPF was observed for all formulations except the NaCMC-modified pMDI system; the performance of this formulation following storage was statistically equivalent to that immediately following preparation. Co-spray-drying proteins and peptides with NaCMC may therefore offer an alternative method for the preparation of stable and respirable pMDI formulations for pulmonary delivery.

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

    Science.gov (United States)

    Ishchenko, Andrii; Cherezov, Vadim; Liu, Wei

    2016-09-20

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

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

    DEFF Research Database (Denmark)

    1997-01-01

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

  15. An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation

    NARCIS (Netherlands)

    Wagner, K.; Miliotis, T.; Marko-Varga, G; Bischoff, Rainer; Unger, K.K.

    2002-01-01

    A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary techniqu

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

    Directory of Open Access Journals (Sweden)

    Turgay Tay

    2016-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Liu Yong

    2014-01-01

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

  18. Preparation and evaluation of oleoyl-carboxymethy-chitosan (OCMCS) nanoparticles as oral protein carriers.

    Science.gov (United States)

    Liu, Ya; Cheng, Xiao Jie; Dang, Qi Feng; Ma, Fang Kui; Chen, Xi Guang; Park, Hyun Jin; Kim, Bum Keun

    2012-02-01

    Oleoyl-carboxymethy chitosan (OCMCS) nanoparticles based on chitosan with different molecular weights (50, 170 and 820 kDa) were prepared by self-assembled method. The nanoparticles had spherical shape, positive surface charges and the mean diameters were 157.4, 274.1 and 396.7 nm, respectively. FITC-labeled OCMCS nanoparticles were internalized via the intestinal mucosa and observed in liver, spleen, intestine and heart following oral deliverance to carps (Cyprinus carpio). Extracellular products (ECPs) of Aeromonas hydrophila as microbial antigen was efficiently loaded to form OCMCS-ECPs nanoparticles and shown to be sustained release in PBS. Significantly higher (P < 0.05) antigen-specific antibodies were detected in serum after orally immunized with OCMCS-ECPs nanoparticles than that immunized with ECPs alone and non-immunized in control group in carps. These results implied that amphiphilic modified chitosan nanoparticles had great potential to be applied as carriers for the oral administration of protein drugs.

  19. [Establishment of optimal conditions at laboratory and pilot plant levels for the preparation of a protein isolated from Lupinus mutabilis].

    Science.gov (United States)

    Rodríguez Pacheco, T; Aliaga, T; Schoeneberger, H; Gross, R

    1981-12-01

    Laboratory conditions were first investigated to determine are optimum processing parameters for the preparation of a protein isolate from the ground, defatted, commercial flakes of Lupinus mutabilis. The extraction variables were: pH (2-10); solvent to lupine ratio (5:1 to 40:1); temperature (28 degrees C - 60 degrees C) and time (10-50 min). The isoelectric point of the lupine protein was found to be pH 4.5 with a protein solubility higher than 80% above pH 8.0. Using 70-100 mesh, ground defatted flakes, and extracting at pH 8.7 for 60 min, a protein isolate was obtained on acidification to pH 4.5 which was 99.8 protein (dry basis), compared to 55.25% protein for the original material. This protein isolate represented 32% of the initial material and 57.6% of the initial nitrogen. When making pilot plant assays we found that the yield of protein isolate decreased to 20.4% of the original material and 36.4% of the initial nitrogen. The protein efficiency ratio for the protein isolate was 2.15 when supplemented with methionine, and had a digestibility of 89.33

  20. Preparation and application of hollow molecularly imprinted polymers with a super-high selectivity to the template protein.

    Science.gov (United States)

    Chen, Yang; He, Xi-Wen; Mao, Jie; Li, Wen-You; Zhang, Yu-Kui

    2013-10-01

    Protein-imprinted polymers with hollow cores that have a super-high imprinting factor were prepared by etching the core of the surface-imprinted polymers that used silica particles as the support. Lysozyme as template was modified onto the surface of silica particles by a covalent method, and after polymerization and the removal of template molecules, channels through the polymer layer were formed, which allowed a single-protein molecule to come into the hollow core and attach to the binding sites inside the polymer layer. The adsorption experiments demonstrated that the hollow imprinted polymers had an extremely high binding capacity and selectivity, and thus a super-high imprinting factor was obtained. The as-prepared imprinted polymers were used to separate the template lysozyme from egg white successfully, indicating its high selectivity and potential application in the field of separation of protein from real samples.

  1. Detecting thermal hysteresis activity of the total protein in insects with differential scanning calorimetry%应用差示扫描量热法检测昆虫总蛋白的热滞活性

    Institute of Scientific and Technical Information of China (English)

    崔宁宁; 宋希明; 邹元平; 郝树广; 许永玉; 王宪辉

    2013-01-01

    产生抗冻蛋白是寒带昆虫抵御低温的重要机制之一,但检测其活性仍存在一些困难,尤其对于个体较小的昆虫样品.为了探索差示扫描量热法是否适于检测昆虫总蛋白的热滞活性,本研究利用差示扫描量热法对黄粉虫Tenebrio molitor幼虫的总蛋白和血淋巴分别进行了热滞活性检测.结果表明:黄粉虫总蛋白的热滞活性(0.49~0.98℃)要低于血淋巴(2.54 ~4.34C).通过这种方法,进一步检测了3种在内蒙古大兴安岭林区采集到的越冬昆虫:稠李巢蛾Yponomeuta evonymallus幼虫、舞毒蛾Lymantria dispar卵和落叶松八齿小蠹Ips subelongatus成虫.结果发现,它们都存在热滞活性,其中稠李巢蛾的热滞活性为0.34 ~0.43℃,舞毒蛾的热滞活性为0.35~0.42℃,落叶松八齿小蠹的热滞活性为0.37 ~0.40℃,说明这3种昆虫能以产生抗冻蛋白的方式作为越冬策略之一.本研究表明通过差示扫描量热法检测昆虫总蛋白是否存在热滞活性来判断抗冻蛋白的存在是可行的.%Producing antifreeze proteins is one of the most important mechanisms underlying insect cold tolerance. However, detecting the activity of antifreeze proteins still has some difficulties, especially when only a few of insect samples are available from fields. In order to explore if differential scanning calorimetry (DSC) can be used to detect thermal hysteresis activity ( THA) of the total protein in insects, the THA of the total protein and hemolymph from Tenebrio molitor larvae was detected by DSC. The results showed that the THA of the total protein (0. 49 -0. 98t ) is lower than that of hemolymph (2. 54 -4. 341) in T. molitor. In addition, we collected three overwintering insect species (Lymantria dispar larvae, Yponomeuta evonymallus eggs and Ips subelongatus adults) in the Daxing' anling Forest Region in Inner Mongolia, and then prepared their total protein. Using DSC, the THA of the total protein from the three

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

    Science.gov (United States)

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

    2016-05-01

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

  3. Comparison of the amino acid and peptide composition and postprandial response of beef, hydrolyzed chicken, and whey protein nutritional preparations

    Directory of Open Access Journals (Sweden)

    Christopher J. Detzel

    2016-10-01

    Full Text Available Background: Increasing dietary protein intake synergistically improves the effect of exercise to stimulate muscle protein synthesis. The purpose of this study was to evaluate the plasma amino acid response of two novel protein nutritional preparations, beef protein isolate (BeefISO™ and hydrolyzed chicken protein isolate (MyoCHX™. Methods: The postprandial plasma amino acid response over 3 hours was monitored in young adults (n=6 following consumption of 23 grams of WPC, BeefISO™, or MyoCHX™. Amino acid compositional analysis and molecular weight distributions of each protein were performed by HPLC. Statistical analyses were performed using one-way or two-way ANOVA where appropriate and corrected for multiple comparisons to account for the cross-over design. Results: Compositional evaluations revealed similar levels of essential and branched-chain amino acids for WPC and MyoCHX™. While the results of this study predictably demonstrated plasma amino acids levels increased following consumption of the different proteins, the kinetics of the postprandial response was unique to each protein source. WPC and MyoCHX™ were rapidly absorbed with maximum plasma amino acid concentrations observed at 30 and 15 min, respectively. The slightly faster absorption of MyoCHX™ was associated with the increased peptide content of MyoCHX™ (greater than 76% of protein is <2kDa. BeefISO™ exhibited sustained release characteristics as evidenced by increased post prandial amino acid concentrations after 3 hours. Conclusions: The protein preparations studied each had different amino acid profiles and absorption kinetics. WPC and MyoCHX™ contained a higher essential amino acid content and were rapidly absorbed with plasma amino acid concentrations peaking within 30 minutes following consumption. BeefISO™ contained a higher proportion of conditionally essential amino acids that steadily increased in plasma over 3 hours, indicating a sustained release

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Buhrman Jason S

    2012-09-01

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

  6. Synergistic Activity Between S-Layer Protein and Spore-Crystal Preparations from Lysinibacillus sphaericus Against Culex quinquefasciatus Larvae.

    Science.gov (United States)

    Lozano, Lucía C; Dussán, Jenny

    2017-03-01

    Lysinibacillus sphaericus is used for the biological control of mosquitoes. The main toxicity mechanism of pathogenic strains is a binary toxin produced during sporulation. S-layer is a proteinaceous structure on the surface of bacteria; its functions have been involved in the interaction between bacterial cells and the environment, for example, as protective coats, surface recognition, and biological control. In L. sphaericus, S-layer protein (SlpC) is expressed in vegetative cells, and is also found in spore-crystal preparations; it has larvicidal activity in Culex spp. In this study, partial and completed sporulated culture toxicities were compared; also, S-layer protein and spore-crystal proteins were tested against Culex quinquefasciatus larvae for possible interactions. Larvicidal activity obtained with a combination of SlpC and spore-crystal proteins from strain III(3)7 showed no significant interaction, whereas, combinations of both preparations from strain 2362 showed synergistic effect. The highest synergistic activity observed was between spore protein complex from strain 2362 and SlpC from III(3)7. S-layer protein could be considered a good alternative in formulation improvement, for biological control of mosquitoes.

  7. Recombinant Staphylococcal protein A with cysteine residue for preparation of affinity chromatography stationary phase and immunosensor applications

    Directory of Open Access Journals (Sweden)

    Gorbatiuk O. B.

    2015-04-01

    Full Text Available Aim. Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys for preparation of affinity chromatography stationary phase and formation of bioselective element of immunosensor. Methods. DNA sequences encoding IgG-binding region of SPA, His-tag and cysteine were genetically fused and expressed in E. coli. SPA-Cys was immobilized on maleimide-functionalized silica beads for affinity chromatography stationary phase preparation and on a gold sensor surface as a bioselective element of immunosensor. Results. SPA-Cys was expressed at a high-level in a soluble form. The target protein was purified and showed a high IgG-binding activity. The capacity of the obtained SPA-Cys-based affinity chromatography stationary phase was 10–12 mg of IgG /ml. The purity of eluted IgG was more than 95 % in one-step purification procedure. The developed SPA-Cys-based bioselective element of immunosensor selectively interacted with human IgG and did not interact with the control proteins. Conclusions. The recombinant Staphylococcal protein A with cysteine residue was successfully used for the preparation of affinity chromatography stationary phase and formation of the bioselective element of immunosensor.

  8. POE-PEG-POE triblock copolymeric microspheres containing protein. I. Preparation and characterization.

    Science.gov (United States)

    Yang, Y Y; Wan, J P; Chung, T S; Pallathadka, P K; Ng, S; Heller, J

    2001-07-10

    Poly(ortho ester) (POE)-poly (ethylene glycol) (PEG) triblock copolymers (POE-PEG-POE) with different PEG contents were synthesised as carriers for controlled protein delivery. POE-PEG-POE microspheres containing bovine serum albumin (BSA) were prepared using a double-emulsion (water-in-oil-in-water) process. In this first paper of a two-part series, we report the fundamentals of the fabrication and characterization of POE-PEG-POE microspheres. Because the triblock copolymer is more hydrophilic than neat poly(ortho ester), the triblock copolymer yields a more stable first emulsion (water-in-oil) and a greater BSA encapsulation efficiency (90% vs. 30%). No BSA is found on POE-PEG-POE microsphere surfaces measured by X-ray photoelectron spectroscopy, while uniform BSA distributions are observed within the microspheres by confocal microscopy. SEM pictures show that an increase in PEG content results in microspheres with a denser cross-section because of a more stable first emulsion and better affinity between the copolymer and water. POE-PEG(20%)-POE suffers significant swelling during the fabrication process and yields the biggest microspheres. However, the POE-PEG(30%)-POE microspheres are much smaller since the dissolution loss of POE-PEG(30%)-POE in the external water phase may be much higher than that of POE-PEG(20%)-POE. The salt concentration in the external water phase significantly affects the morphology of the resultant microspheres. Microspheres with a dense wall are produced when using pure water as the external water phase. Polymer concentration has less impact on BSA encapsulation efficiency but has a considerable effect on microsphere size and morphology. Increasing the concentration of the polyvinyl alcohol emulsifier does not cause an obvious decrease in microsphere size. However, increased BSA loading results in bigger microspheres.

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

    Science.gov (United States)

    Balabushevich, Nadezda G; Larionova, Natalia I

    2009-11-01

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

  10. Protein standardization IV: Value transfer procedure for the assignment of serum protein values from a reference preparation to a target material.

    Science.gov (United States)

    Blirup-Jensen, S; Johnson, A M; Larsen, M

    2001-11-01

    A new approach for the assignment of values to serum proteins in a target material using a reference preparation has been developed. The procedure describes the general as well as the practical principles involved in the value assignment (with examples). Two models have been developed: 1) The direct value transfer between serum matrices and 2) the indirect value transfer from a pure protein preparation to a serum protein material. The necessary mathematical equations are developed and explained. The data reduction and statistical evaluation are discussed. The practical procedure (the transfer protocol) is based on six dilutions of the reference preparation assayed together with six dilutions of the target material. In this way imprecision is reduced and the proportionality of the two materials (i.e. the presence or absence of matrix effects) can be assessed directly by evaluating a single regression plot. If no matrix effects are found, the regression line will pass through zero with a slope equal to the ratio of the concentrations of the two materials. The transfer protocol is based on a multiple point value assignment obtained by several measurements a day repeated on several days, an important prerequisite being that all reconstitutions and dilutions are controlled by weighing.

  11. Preparation of protein-loaded microspheres with size ≤10 μm by electrostatic droplet generation technology

    Institute of Scientific and Technical Information of China (English)

    XUE Weiming; LIU Xiudong; YU Weiting; MA Xiaojun

    2006-01-01

    The development of non-injection route for protein drugs, especially oral administration, has been the main focus of controlled release of drugs. To overcome obstacles unsolved such as enzyme degradation and penetration barrier of intestinal epithelium, technologies using microspheres as carrier of protein drugs have been proven potential to realize oral administration. It has been demonstrated that microspheres can not only protect proteins, but also facilitate the penetration and absorption through Peyer's patches when the size is smaller than 10 μm. Therefore, the objective of this paper is to prepare protein-loaded microspheres with size ≤10 μm. Electrostatic droplet generation technology was used with insulin and hemoglobin as drug models and sodium alginate as microsphere material. By decreasing the surface tension of feed solution by adding surfactant, and improving electric field distribution by changing the shape of container and electrode for gelation solution, protein-loaded microspheres with mean size less than 10 μm were successfully produced through needle with diameter of 400 μm. The microspheres showed good sphericity and narrow size distribution. The mean standard variance of size distribution was 1.61. The encapsulation efficiency of proteins was over 70%. Moreover, the significance analysis of factors influencing the size of protein loaded microspheres was carried out through orthogonal experiments, which showed that output voltage (U), needle diameter (D) and the distance between needle tips to the surface of gelation solution (δ ) influenced significantly the size of microspheres. Finally, the statistic analysis showed that when confidence level wasα=0.05, and α=0.1, confidence interval of microsphere size can be (6.2545, 10.1735) and (6.6022, 9.8258) correspondingly, suggesting that there is good repeatability and reliability for improving electrostatic droplet generation technology to prepare protein-loaded microspheres with size

  12. Sample Preparation and Staining Methods for Two-Dimensional Polyacrylamide Gel Electrophoresis of Proteins from Animal Tissues

    Directory of Open Access Journals (Sweden)

    Levente Czegledi

    2010-05-01

    Full Text Available Proteomics in animal science as well as in other biological sciences is a significant tool in the post-genomic era. In proteomic studies the presence and relative abundance of expressed proteins of a cell, tissue or biological fluid is studied. Recently, the whole genome of more and more domestic animal species is known, but genes and the transcribed mRNA have no direct effect on biological systems as they are regulated by proteins, which explain the importance of proteomics. The most common tool in proteomic approach is the two-dimensional polyacrylamide gel electrophoresis (2D PAGE, when proteins are separated by their isoelectric point followed by their mass separation as a second dimension. In this study authors used different sample preparation and protein staining methods on meat,  liver and blood plasma and carried out 2D PAGE experiments. The most appropriate sample preparation methods are described in this paper. We concluded that depletion of major proteins in plasma is required but not necessary for meat and liver samples.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-03-15

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

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

    Directory of Open Access Journals (Sweden)

    Gabriel N. Medoua

    2013-10-01

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

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

    NARCIS (Netherlands)

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

    1999-01-01

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

  16. Method and validation of synaptosomal preparation for isolation of synaptic membrane proteins from rat brain

    Directory of Open Access Journals (Sweden)

    Pradip Kumar Kamat

    2014-01-01

    Further, the resulting pellet was discarded and suspended in RIPA buffer (mixed with protease inhibitor and PMSF only. The sample was immediately used for protein estimation and protein electrophoresis.

  17. Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition

    Science.gov (United States)

    Winzen, S.; Schoettler, S.; Baier, G.; Rosenauer, C.; Mailaender, V.; Landfester, K.; Mohr, K.

    2015-02-01

    Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A-I (ApoA-I)) adsorb and interact with hydroxyethyl starch (HES) nanocapsules possessing different functionalities. To analyse the hard protein corona we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a protein quantitation assay. No significant differences were observed with regards to the hard protein corona. For analysis of the soft protein corona we characterized the nanocapsule-protein interaction with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). DLS and ITC measurements revealed that a high amount of plasma proteins were adsorbed onto the capsules' surface. Although HSA was not detected in the hard protein corona, ITC measurements indicated the adsorption of an HSA amount similar to plasma with a low binding affinity and reaction heat. In contrast, only small amounts of ApoA-I protein adsorb to the capsules with high binding affinities. Through a comparison of these methods we have identified ApoA-I to be a component of the hard protein corona and HSA as a component of the soft corona. We demonstrate a pronounced difference in the protein corona observed depending on the type of characterization technique applied. As the biological identity of a particle is given by the protein corona it is crucial to use complementary characterization techniques to analyse different aspects of the protein corona.Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A

  18. Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition.

    Science.gov (United States)

    Winzen, S; Schoettler, S; Baier, G; Rosenauer, C; Mailaender, V; Landfester, K; Mohr, K

    2015-02-21

    Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A-I (ApoA-I)) adsorb and interact with hydroxyethyl starch (HES) nanocapsules possessing different functionalities. To analyse the hard protein corona we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a protein quantitation assay. No significant differences were observed with regards to the hard protein corona. For analysis of the soft protein corona we characterized the nanocapsule-protein interaction with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). DLS and ITC measurements revealed that a high amount of plasma proteins were adsorbed onto the capsules' surface. Although HSA was not detected in the hard protein corona, ITC measurements indicated the adsorption of an HSA amount similar to plasma with a low binding affinity and reaction heat. In contrast, only small amounts of ApoA-I protein adsorb to the capsules with high binding affinities. Through a comparison of these methods we have identified ApoA-I to be a component of the hard protein corona and HSA as a component of the soft corona. We demonstrate a pronounced difference in the protein corona observed depending on the type of characterization technique applied. As the biological identity of a particle is given by the protein corona it is crucial to use complementary characterization techniques to analyse different aspects of the protein corona.

  19. Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine.

    Science.gov (United States)

    Xie, Q M; Ji, J; Du, L Q; Cao, Y C; Wei, L; Xue, C Y; Qin, J P; Ma, J Y; Bi, Y Z

    2009-08-01

    Avian influenza is a severe disease among farmed poultry and free-living birds and a constant threat to the commercial chicken industry around the world. Hemagglutinin (HA) is the major immunogen on the envelope of influenza A virus and is the predominant inducer of neutralizing antibody. To obtain the bioactive antigen proteins in large quantities, a new protein expression vector pBCX was constructed, which is based on the pET32a vector. The HA gene of the H5N1 subtype of avian influenza virus (AIV) was inserted into the pBCX vector and expressed efficiently in Escherichia coli BL21 (DE3). Fused expression of the exogenous gene and msyB produced a 97-kDa msyB-HA fusion protein. Sodium dodecyl sulfate-PAGE combined with scanning analysis demonstrated that the msyB-HA fusion protein accounted for 29.5% of the total bacterial protein, 90.5% being soluble. The msyB-HA fusion protein was purified with nondenaturing 50% Ni-NTA column chromatography, and the result showed that 24 mg of purified msyB-HA fusion protein could be obtained from 1 L of induced expression bacterial culture medium. The comparative results in the present study showed that pBCX was superior to pET32a as a protein expression vector. Western blotting showed the recombinant msyB-HA (rHA) to have better antigenic activity, which may be the result from the better posttranslation protein modification and folding in the pBCX expression system. With the rHA fusion protein as antigen, we successfully prepared and screened specific monoclonal antibodys against the H5N1 subtype AIV, which indicated that the rHA had antigen epitopes and biofunctions. The immune test confirmed that the rHA protein vaccine could also induce high neutralizing antibodies, and the AIV challenge test proved that the rHA protein-based vaccine could prevent the corresponding infection. This study demonstrates that the recombinant HA protein produced by the pBCX expression system could be used as a recombinant protein-based vaccine

  20. Kinetic Effects on Self-Assembly and Function of Protein-Polymer Bioconjugates in Thin Films Prepared by Flow Coating

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Dongsook [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave Cambridge MA 02142 USA; Huang, Aaron [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave Cambridge MA 02142 USA; Olsen, Bradley D. [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave Cambridge MA 02142 USA

    2016-11-04

    The self-assembly of nanostructured globular protein arrays in thin films is demonstrated using protein–polymer block copolymers based on a model protein mCherry and the polymer poly(oligoethylene glycol acrylate) (POEGA). Conjugates are flow coated into thin films on a poly(ethylene oxide) grafted Si surface, forming self-assembled cylindrical nanostructures with POEGA domains selectively segregating to the air–film interface. Long-range order and preferential arrangement of parallel cylinders templated by selective surfaces are demonstrated by controlling relative humidity. Long-range order increases with coating speed when the film thicknesses are kept constant, due to reduced nucleation per unit area of drying film. Fluorescence emission spectra of mCherry in films prepared at <25% relative humidity shows a small shift suggesting that proteins are more perturbed at low humidity than high humidity or the solution state.

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

    Science.gov (United States)

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

    2016-02-01

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

  2. Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD.

    Science.gov (United States)

    Chen, Yujian; Liu, Yong; Guo, Jiayu; Tang, Tao; Gao, Jian; Huang, Tao; Wang, Bin; Liu, Shaojun

    2016-06-17

    Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and contains an additional neuronal insert (NINS) fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120). The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120.

  3. Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

    OpenAIRE

    Wang, Xiaowen; Mbantenkhu, MacMillan; Wierzbicki, Sara; Chen, Xin Jie

    2013-01-01

    The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has be...

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

    DEFF Research Database (Denmark)

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

    1998-01-01

    Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish...... the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were...

  5. [Preparation and characterization of the recombinant protein containing immunomimetic peptide of benzo[a]pyrene].

    Science.gov (United States)

    Apal'ko, S V; Lunin, V G; Filipenko, M L; Matveeva, V A; Liashchuk, A M; Lavrova, N V; Sherina, E A; Aver'ianov, A V; Kostianko, M V; Glushkov, A N

    2011-01-01

    Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encoding sequences. The second plasmid contained the hybrid gene composed of the signal peptide of the protein pIII of bacteriophage M13, of immunomimetic peptide of benzo[a]pyrene, of the protein pill of bacteriophage M13 and of cellulose binding domain sequences. The obtained recombinant plasmids were used in expression of chimeric protein containing immunomimetic peptide ofbenzo[a]pyrene based on strain E. coli M15. The lack of the recombinant protein expression using first plasmid was demonstrated. In the same time, it was shown that accumulation of recombinant protein contained immunomimetic peptide with signal peptide of the protein pIIIl of bacteriophage was present. This chimeric protein was produced in "mature" (without signal peptide) and "unprocessing" (with signal peptide) forms. Using the Western-blot analysis, it was shown that the "mature" form only specifically bound to the B2 monoclonal antibody against benzo[a]pyrene. Thus, we expressed, purified, and characterized the recombinant protein containing immunomimetic peptide of benzo[a]pyrene.

  6. Sample preparation techniques for peptides and proteins analyzed by MALDI-MS

    DEFF Research Database (Denmark)

    Kussmann, M; Roepstorff, Peter

    2000-01-01

    Proteome analysis offers a unique means of identifying important proteins, characterizing their modifications and beginning to describe their function. This is achieved through the combination of two technologies: protein separation and selection by two-dimensional gel electrophoresis, and protein...... by the characterization of genes, revealed by sequencing, but which have no--or only weak homology--to any other known genes. Other applications, for example the identification of protein markers for particular human diseases, are only referred to. The aim of the article is thus to provide the basis for a sound...

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

    Institute of Scientific and Technical Information of China (English)

    Xiao-quan Li; Shu-lin Zhang; Li-hua Zhong; Jun Cheng; Yuan Hong; Meng-dong Lan; Xiao-bin Chen; Cheng-fu Sun

    2009-01-01

    Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coil BL21, the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG), and it was analyzed with sodium dodecyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyelonai antibody, with which we studied the function of NSSATP5 by determining the different liver tissues with the streptavidin-perosidase (SP) immunohistochemistry method. Results Recombinant NS5ATP5 (molecular weight: 65 kD) and polyclonal antibody were successfully prepared. NS5ATP5 expression in the liver of patients with chronic HCV infection was much higher than that of a normal person, and it was detected mainly in the cytoplasm. Conclusion The findings of the expression difference between HCV patients and normal people led to a novel diagnostic marker to detect HCV infection.

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

    Science.gov (United States)

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

    2007-10-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  10. Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

    Science.gov (United States)

    Tanphaichitr, Nongnuj; Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark

    2015-01-01

    The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm-ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm-ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm-ZP binding step.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-10-01

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

  12. [Preparation, characterization and application of rice Qb-SNARE protein OsNPSN11 polyclonal antibody].

    Science.gov (United States)

    Bao, Yong-Mei; Liu, Yong-Hui; Xu, Dong-Qing; Huang, Ji; Wang, Zhou-Fei; Wang, Jian-Fei; Zhang, Hong-Sheng

    2010-09-01

    Membrane fusion in vesicle trafficking in the cells of eukaryotic organisms is mediated by soluble-N-ethyl- maleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins. OsNPSN11 is a member of Qb-SNARE gene family isolate from rice. The cDNA of OsNPSN11 was subcloned into pET-30a and fusion to the 6 × His tag. Induced by 0.5 mmol/L IPTG for four hours, the recombinant protein was highly expressed in Escherichia coli, which was purified by Ni2+ -NTA His-bind resin affinity chromatography column to be used as an antigen to raise the antibody in New Zealand rabbits. Western blotting analysis showed that the antibody can specifically recognize the expressed antigen and the OsNPSN11 in plasma membrane protein from various rice tissues. This indicated that the antibody can be used for expres-sion analysis in transgenic rice.

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

    DEFF Research Database (Denmark)

    Dyrberg, T; Billestrup, Nils

    1984-01-01

    Chloramine-T iodination of protein A from Staphylococcus aureus and gel electrophoretic purification of the iodination mixture results in a stable tracer of high specific and functional activity. Following repeated gel electrophoresis of the tracer only a single component was observed. The specific...... activity of the 125I-protein A was between 30 and 55 muCi/micrograms. 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...

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

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Huihui; Qian, Bin; Zhang, Wei [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); Lan, Minbo, E-mail: minbolan@ecust.edu.cn [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)

    2016-02-15

    Highlights: • Antifouling PVP brushes were successfully grafted on PU films by SI-ATRP. • The effect of polymerization time on surface property and topography was studied. • Hydrophilicity and protein fouling resistance of PVP–PU films were greatly promoted. • Competitive adsorption of three proteins on PVP–PU films was evaluated. - Abstract: 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/cm{sup 2}, 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.

  15. Characterisation of novel fungal and bacterial protease preparations and evaluation of their ability to hydrolyse meat myofibrillar and connective tissue proteins.

    Science.gov (United States)

    Ryder, Kate; Ha, Minh; Bekhit, Alaa El-Din; Carne, Alan

    2015-04-01

    The catalytic capability of four commercially available food-grade fungal and bacterial protease preparations (AFP, FPII, F60K and HT) was evaluated over a range of pH, temperature and substrate conditions using esterase and caseinolytic activity assays and time course hydrolysis over 120 and 60 min of myofibrillar and connective tissue proteins, respectively. The protease preparations displayed similar casein hydrolysis kinetics and were active in hydrolysing BODIPY-FL casein to varying extents at postmortem aging meat pH (5.0-6.0). All of the four proteases exhibited selective hydrolytic activity towards meat myofibrillar proteins including myosin and actin. Significant hydrolysis of two meat tenderisation protein markers troponin T and desmin by the four proteases was detected by western blot. The results obtained indicate that the new fungal protease preparations AFP and FPII, bacterial protease preparation HT and the new source of fungal protease preparation F60K have potential for use in meat tenderising applications.

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

  18. Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

    Science.gov (United States)

    Martin, Nicholas J.; Bunch, Josephine; Cooper, Helen J.

    2013-08-01

    Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

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

    Institute of Scientific and Technical Information of China (English)

    Zhang Weinong; Liu Dachuan

    2002-01-01

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

  20. Dependence of protein recognition of temperature-sensitive imprinted hydrogels on preparation temperature.

    Science.gov (United States)

    Turan, Eylem; Ozçetin, Gökçen; Caykara, Tuncer

    2009-05-13

    Temperature-sensitive imprinted and non-imprinted hydrogels composed of N-isopropylacrylamide (NIPA) and 2-acrylamido-2-methyl-propanosulfonic acid (AMPS) have been prepared by free-radical crosslinking copolymerization in aqueous solution at three different temperatures: 10 degrees C (below the lower critical solution temperature, LCST), 33 degrees C (at the LCST), and 40 degrees C (above the LCST). Myoglobin (Mb, MW 17 kDa) is used as the template biomolecule. The effects of the initial concentration and adsorption time over the Mb adsorption capacity of the hydrogels have been analyzed and found to be strongly dependent on the preparation temperature (T(prep)). The maximum Mb adsorption for the imprinted hydrogel prepared at 10 degrees C is 97.40 +/- 2.35 mg Mb x g(-1) dry gel in 0.32 mg x mL(-1) Mb solution at 22 degrees C. Moreover, batch adsorption equilibrium and selectivity studies have been performed using a reference molecule, hemoglobin (Hb, MW 65 kDa). The imprinted hydrogels have a 2.8-3.3 times higher adsorption capacity for Mb than the non-imprinted hydrogels prepared at the same T(prep)s, and also have a 1.8-2.7 times higher selectivity for the imprinted molecule.

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

    Science.gov (United States)

    Mackin, Robert B

    2014-01-01

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

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

    OpenAIRE

    2013-01-01

    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 s...

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

    Directory of Open Access Journals (Sweden)

    Maznah Ismail

    2013-03-01

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

  4. Extraction and characterization of Foeniculum vulgare pectins and their use for preparing biopolymer films in the presence of phaseolin protein.

    Science.gov (United States)

    Giosafatto, Concetta V L; Mariniello, Loredana; Ring, Steve

    2007-02-21

    Pectins from Foeniculum vulgare were extracted under acidic conditions. The obtained pectins were mainly composed of uronic acid but also contained traces of rhamnose, galactose, and arabinose. Extracted pectins were used as a carbohydrate source to prepare biopolymer films in the absence and in the presence of phaseolin protein. The swelling characteristics of the films were examined as a function of ionic strength, pH, and the applied osmotic stress. The swelling behavior was dominated by a Donnan-type effect, which decreases with increasing ionic strength and counterion valency. In all cases the swelling of films containing phaseolin was reduced, suggesting a network formation between protein and pectins. Mechanical property studies have also estimated the validity of the obtained novel biopolymer films in terms of mechanical resistance.

  5. A simple pressure cell and delivery system for the preparation of Xe derivatives for protein crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Stowell, M.H.; Soltis, S.M.; Kisker, C.; Peters, J.W.; Schindelin, H.; Rees, D.C.; Cascio, D.; Beamer, L.; Hart, J.; Whitby, F.G. [Carl F. and Winifred H. Braun Laboratories, Mail Stop 147-75CH, California Institute of Technology, Pasadena, CA 91125 (United States)]|[Stanford Synchrotron Radiation Laboratory, SLAC, P. O. Box 4349, Bin 69, Stanford University, CA 94309 (United States)]|[University of California Los Angeles, Institute of Molecular Biology, Department of Chemistry and Biochemistry, Los Angeles, CA 90024 (United States)]|[University of Utah Medical Center, Department of Biochemistry, 50 North Medical Drive, Salt Lake City, UT 84132 (United States)

    1996-09-01

    We have developed a simple device for preparing Xe derivatives under moderate gaseous pressure (1{endash}100 atm). The device employs a Cajon ultra-torr fitting to ensure a gas-tight seal around a standard x-ray capillary. As such, the cell can accommodate standard x-ray capillaries up to 1.5 mm in diameter without any modification. The device is straightforward to utilize, and samples can be mounted and pressurized in a matter of seconds. In addition, a simple and safe purging and pressurization system has been designed and constructed for the use at beamline 7-1 at the Stanford Synchrotron Radiation Laboratory (SSRL). We describe the construction of both the pressure cell and the delivery system and present results on the cells use in the preparation of xenon derivatives. {copyright} {ital 1996 American Institute of Physics.}

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Controlled release behaviour of protein-loaded microparticles prepared via coaxial or emulsion electrospray.

    Science.gov (United States)

    Wang, Ying; Yang, Xiaoping; Liu, Wentao; Zhang, Feng; Cai, Qing; Deng, Xuliang

    2013-01-01

    Biodegradable poly (lactic-co-glycolic acid) (PLGA) microparticles are an effective way to achieve sustained drug release. In this study, we investigated a sustained release model of PLGA microparticles with incorporated protein via either emulsion or coaxial electrospray techniques. PLGA (75:25) was used as the carrier, and bovine serum albumin as a model protein. Coaxial electrospray resulted in a type of core-shell structure with mean diameters of 2.41 ± 0.60 µm and a centralised protein distribution within the core. Emulsion electrospray formed bigger microparticles with mean diameters of 22.75 ± 8.05 µm and a heterogeneous protein distribution throughout the microparticles. The coaxial electrospray microparticles presented a much slighter burst release than the emulsion electrospray microparticles. Loading efficiency was significantly higher (p coaxial group than emulsion group. This indicated that both emulsion and coaxial electrospray could produce protein-loaded microparticles with sustained release behaviour, but the former revealed a superior approach for drug delivery.

  8. Protein encapsulated core-shell structured particles prepared by coaxial electrospraying: investigation on material and processing variables.

    Science.gov (United States)

    Zamani, Maedeh; Prabhakaran, Molamma P; Thian, Eng San; Ramakrishna, Seeram

    2014-10-01

    Biodegradable polymeric particles have been extensively investigated for controlled drug delivery of various therapeutic agents. 'Coaxial' electrospraying was successfully employed in this study, to fabricate core-shell PLGA particles containing bovine serum albumin (BSA) as the model protein, and the results were also compared to particles prepared by 'emulsion' electrospraying. Two different molecular weights of PLGA were employed to encapsulate the protein. Solution properties and processing parameters were found to influence the morphology of the core-shell particles. Depending on the type of solvent used to dissolve the polymer as well as the polymer concentration and molecular weight, the mean diameter of the particles varied between 3.0 to 5.5 μm. Fluorescence microscopic analysis of the electrosprayed particles using FITC-conjugated BSA demonstrated the core-shell structure of the developed particles. The encapsulation efficiency and release behavior of BSA was influenced by shell:core feeding ratio, protein concentration, and the electrospraying method. The encapsulation efficiency of BSA within the core-shell particles of high and low molecular weight PLGA was found 15.7% and 25.1% higher than the emulsion electrosprayed particles, respectively. Moreover, the total amount of BSA released from low molecular weight PLGA particles was significantly higher than high molecular weight PLGA particles within 43 days of release studies, with negligible effect on encapsulation efficiency. The technique of coaxial electrospraying has high potential for encapsulation of susceptible protein-based therapeutic agents such as growth factors for multiple drug delivery applications.

  9. Influence of Bovine Whey Protein Concentrate and Hydrolysate Preparation Methods on Motility in the Isolated Rat Distal Colon

    Science.gov (United States)

    Dalziel, Julie E.; Anderson, Rachel C.; Bassett, Shalome A.; Lloyd-West, Catherine M.; Haggarty, Neill W.; Roy, Nicole C.

    2016-01-01

    Whey protein concentrate (WPC) and hydrolysate (WPH) are protein ingredients used in sports, medical and pediatric formulations. Concentration and hydrolysis methods vary for whey sourced from cheese and casein co-products. The purpose of this research was to investigate the influence of whey processing methods on in vitro gastrointestinal (GI) health indicators for colonic motility, epithelial barrier integrity and immune modulation. WPCs from casein or cheese processing and WPH (11% or 19% degree of hydrolysis, DH) were compared for their effects on motility in a 1 cm section of isolated rat distal colon in an oxygenated tissue bath. Results showed that WPC decreased motility irrespective of whether it was a by-product of lactic acid or mineral acid casein production, or from cheese production. This indicated that regardless of the preparation methodology, the whey protein contained components that modulate aspects of motility within the distal colon. WPH (11% DH) increased contractile frequency by 27% in a delayed manner and WPH (19% DH) had an immediate effect on contractile properties, increasing tension by 65% and frequency by 131%. Increased motility was associated with increased hydrolysis that may be attributed to the abundance of bioactive peptides. Increased frequency of contractions by WPH (19% DH) was inhibited (by 44%) by naloxone, implicating a potential involvement of opioid receptors in modulation of motility. Trans-epithelial electrical resistance and cytokine expression assays revealed that the WPC proteins studied did not alter intestinal barrier integrity or elicit any discernible immune response. PMID:27983629

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

    Directory of Open Access Journals (Sweden)

    Olivieri Oliviero

    2009-02-01

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

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

    Science.gov (United States)

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

    2013-05-29

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

  12. Purification of Pregnancy-associated Plasma Protein-A and Preparation of Its Antibodies

    Institute of Scientific and Technical Information of China (English)

    CHENG; Bin-yan; LI; Zi-ying; YUAN; Zhi-gang; ZHANG; Xue-feng; LIU; Yi-bing

    2013-01-01

    Pregnancy-associated plasma protein-A(PAPP-A)is isolated from the plasma of pregnant women.It is producted by the syntrophoblast tissue of the placenta and decidual cells.PAPP-A belongs to macromolecular glycoprotein.As a sensitive serum marker,the decreased PAPP-A levels during the first

  13. Effects of seed preparation and oil pressing on milkweed (Asclepias spp.) protein functional properties

    Science.gov (United States)

    The effects of seed cooking and oil processing conditions on functional properties of milkweed seed proteins were determined to identify potential value-added uses for the meal. Milkweed seeds were flaked and then cooked in the seed conditioner at 82°C for 30, 60 or 90 min. Oil was extracted by scre...

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

    DEFF Research Database (Denmark)

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

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

  15. Fibrous scaffolds loaded with protein prepared by blend or coaxial electrospinning.

    NARCIS (Netherlands)

    Ji, W.; Yang, F.; Beucken, J.J.J.P. van den; Bian, Z.; Fan, M.; Chen, Z.; Jansen, J.A.

    2010-01-01

    The aim of the present study was to fabricate polycaprolactone-based nanofibrous scaffolds with incorporated protein via either the blend or coaxial electrospinning technique. Both techniques were compared with respect to processing set-up and scaffold characteristics as well as the release kinetics

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

    Science.gov (United States)

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

    2013-10-01

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

  17. Optimization of Proteomic Sample Preparation Procedures for Comprehensive Protein Characterization of Pathogenic Systems

    Science.gov (United States)

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

    2008-01-01

    Mass spectrometry-based proteomics is a powerful analytical tool for investigating pathogens and their interactions within a host. The sensitivity of such analyses provides broad proteome characterization, but the sample-handling procedures must first be optimized to ensure compatibility with the technique and to maximize the dynamic range of detection. The decision-making process for determining optimal growth conditions, preparation methods, sample analysis methods, and data analysis techniques in our laboratory is discussed herein with consideration of the balance in sensitivity, specificity, and biomass losses during analysis of host-pathogen systems. PMID:19183792

  18. Discussion on applying an analytical method to optimize the anti-freeze design parameters for underground water pipelines in seasonally frozen areas

    Institute of Scientific and Technical Information of China (English)

    Ji Chen; JingYi Zhao; Kun Li; Yu Sheng

    2016-01-01

    Adopting the quasi-three-dimensional (Quasi-3D) numerical method to optimize the anti-freeze design parameters of an underground pipeline usually involves heavy numerical calculations. Here, the fitting formulae between the safe con-veyance distance (SCD) of a water pipeline and six influencing factors are established based on the lowest water temper-ature (LWT) along the pipeline axis direction. With reference to the current widely used anti-freeze design approaches for underground pipelines in seasonally frozen areas, this paper first analyzes the feasibility of applying the maximum frozen penetration (MFP) instead of the mean annual ground surface temperature (MAGST) and soil water content (SWC) to calculate the SCD. The results show that the SCD depends on the buried depth if the MFP is fixed and the variation of the MAGST and SWC combination does not significantly change the SCD. A comprehensive formula for the SCD is estab-lished based on the relationships between the SCD and several primary influencing factors and the interaction among them. This formula involves five easy-to-access parameters:the MFP, buried depth, pipeline diameter, flow velocity, and inlet water temperature. A comparison between the analytical method and the numerical results based on the Quasi-3D method indicates that the two methods are in good agreement overall. The analytic method can be used to optimize the anti-freeze design parameters of underground water pipelines in seasonally frozen areas under the condition of a 1.5 safety coefficient.

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jinju Wang

    2013-02-01

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

  3. Characterizing protein activities on the lysozyme and nanodiamond complex prepared for bio applications.

    Science.gov (United States)

    Perevedentseva, E; Cai, P-J; Chiu, Y-C; Cheng, C-L

    2011-02-01

    Recently, nanodiamond particles have attracted increasing attention as a promising nanomaterial for its biocompatibility, easy functionalization and conjugation with biomolecules, and its superb physical/chemical properties. Nanodiamonds are mainly used as markers for cell imaging, using its fluorescence or Raman signals for detection, and as carriers for drug delivery. For the success of these applications, the biomolecule associated with the nanodiamond has to retain its functionality. In this work, the protein activities of egg white lysozyme adsorbed on nanodiamond particles of different sizes is investigated. The lysozyme nanodiamond complex is used here as a protein model for analyzing its structural conformation changes and, correspondingly, its enzymatic activity after the adsorption. Fourier-transform infrared spectroscopy (FTIR) is used for the analysis of the sensitive protein secondary structure. To access the activities of the adsorbed lysozyme, a fluorescence-based assay is used. The process of adsorption is also analyzed using UV-visible spectroscopic measurements in combination with analysis of nanodiamond properties with FTIR, Raman spectroscopy, and ζ-potential measurements. It is found that the activity of lysozyme upon adsorption depends on the nanodiamond's size and surface properties, and that the nanodiamond particles can be selected and treated, which do not alter the lysozyme functional properties. Such nanodiamonds can be considered convenient nanoparticles for various bioapplications.

  4. Determination of adsorption isotherm parameters for minor whey proteins by gradient elution preparative liquid chromatography.

    Science.gov (United States)

    Faraji, Naeimeh; Zhang, Yan; Ray, Ajay K

    2015-09-18

    Ion-Exchange Chromatography (IEC) techniques have been extensively investigated in protein purification processes, due to the more selective and milder separation steps. To date, existing studies of minor whey proteins fractionation in IEC have primarily been conducted as batch uptake studies, which require more experimental search space, time and materials. In this work, the selected resin's (SP Sepharose FF) equilibrium and dynamic binding capacity were first investigated. Next, adsorption of the pure binary mixture of lactoperoxidase and lactoferrin was studied to calibrate steric mass action (SMA) model using a simplified approach with data from single column experiments. The calibrated model was then verified by performing factorial-design based experiments for various process operating conditions assessing process performance on a larger bed height column. The model predicted results demonstrated a realistic agreement with the experiments providing reproducible column elution profile and reduced experimental work. Finally, whey protein isolate was used to evaluate model parameters in real conditions. Results obtained herein are suitable for future large scale applications.

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

    Directory of Open Access Journals (Sweden)

    Aleksandra Zambrowicz

    2012-12-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Yan Liu; Yongwei Wei; Xiaofeng Wu; Lian Yu

    2005-01-01

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

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

    Science.gov (United States)

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

    2015-11-27

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

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

    Science.gov (United States)

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

    2014-06-09

    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.

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

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

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

  10. Preparation and characterization of monodisperse large-porous silica microspheres as the matrix for protein separation.

    Science.gov (United States)

    Xia, Hongjun; Wan, Guangping; Zhao, Junlong; Liu, Jiawei; Bai, Quan

    2016-11-04

    High performance liquid chromatography (HPLC) is a kind of efficient separation technology and has been used widely in many fields. Micro-sized porous silica microspheres as the most popular matrix have been used for fast separation and analysis in HPLC. In this paper, the monodisperse large-porous silica microspheres with controllable size and structure were successfully synthesized with polymer microspheres as the templates and characterized. First, the poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) microspheres (PGMA-EDMA) were functionalized with tetraethylenepentamine (TEPA) to generate amino groups which act as a catalyst in hydrolysis of tetraethyl orthosilicate (TEOS) to form Si-containing low molecular weight species. Then the low molecular weight species diffused into the functionalized PGMA-EDMA microspheres by induction force of the amino groups to form polymer/silica hybrid microspheres. Finally, the organic polymer templates were removed by calcination, and the large-porous silica microspheres were obtained. The compositions, morphology, size distribution, specific surface area and pore size distribution of the porous silica microspheres were characterized by infrared analyzer, scanning-electron microscopy, dynamic laser scattering, the mercury intrusion method and thermal gravimetric analysis, respectively. The results show that the agglomeration of the hybrid microspheres can be overcome when the templates were functionalized with TEPA as amination reagent, and the yield of 95.7% of the monodisperse large-porous silica microspheres can be achieved with high concentration of polymer templates. The resulting large-porous silica microspheres were modified with octadecyltrichlorosilane (ODS) and the chromatographic evaluation was performed by separating the proteins and the digest of BSA. The baseline separation of seven kinds of protein standards was achieved, and the column delivered a better performance when separating BSA digests

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

    Science.gov (United States)

    McDonagh, P F; Williams, S K

    1984-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    1998-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hassan AH

    2015-07-01

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

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

    Science.gov (United States)

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

    1986-01-01

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

  15. Protein Compatible Polymer Brushes on Polymeric Substrates Prepared by Surface-Initiated Transfer Radica Polymerization

    DEFF Research Database (Denmark)

    Fristrup, Charlotte Juel; Eskimergen, Rüya; Burkrinsky, J.T.

    2008-01-01

    as coating materials. ATR FTIR, water contact angle measurements, Thermal Gravimetric Analysis (TGA), and X-ray Photoelectron Spectroscopy (XPS) confirmed that hydrophilic polymers have been grafted from the surface. The surface topography which was evaluated by Atomic Force Microscopy (AFM) did not change......Materials for insulin containers and delivery systems should comply with requirements like compatibility with proteins, sterilisability, 'good barrier properties towards preservatives, and no toxic leachables. The number of commercially available polymer materials which can be u sed is rather...... limited. Therefore, a polymer coating containing some of the required properties may expand the use ofpolymers in medical devices. 'The approach was to graft polymer brushes from initiator-functionalized substrates using Surface-Initiated Atom TnlJlsfer Radical Polymerization (SI ATRP). Initial studies...

  16. Preparation of the selenome-thionine derivative of tab- toxin resistance protein

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to obtain phase information for the X-ray diffraction of tabtoxin resistance protein (TTR) crystal using the MAD phasing method, a selenomethionine (Se- Met) derivative of TTR was overexpressed in E. coli strain M15, with pQE-30 plasmid, through IPTG induction in M9 medium containing Se-Met. The product was purified to an estimated homogeneity of greater than 95% according to SDS-PAGE, by a Ni-NTA metal affinity followed by a Mono Q anion exchange column chromatography. The successful substitution of Se-Met for methionine (Met) was confirmed by MALDI-TOF and ESI-Quadrupole Mass Spectrometry analysis. The derivative crystal was obtained using similar conditions as those for the native.

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

    Science.gov (United States)

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

    2016-03-01

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

  18. [Preparation of reactants for the detection of proteins from different animal species by immunodiffusion].

    Science.gov (United States)

    Cordal de Bobbi, M E; Petracca, A; Moro, A A

    1985-01-01

    Qualitative identification of fresh ground meat samples of beef, sheep, pork, horse and rabbit has been made by the double agar gel diffusion test (method of Ouchterlony) and by immunoelectrophoresis. The reagents used were: 1) Antigens: saline meat extracts from different animal species, saline meat mixture extracts and whole sera from the different animal species. Meat extracts and sera were controlled by electrophoresis and the amounts of protein were estimated by the method of Lowry; 2) Antibodies: multivalent antisera anti-bovine, anti-ovine, anti-porcine and anti-equine, were obtained by immunization of rabbits with an emulsion of 1 vol of whole serum and 1 vol of Freund complete adjuvant; anti-rabbit serum was obtained by immunization of rats; antisera were controlled by the double agar gel diffusion test and by immunoelectrophoresis. To remove cross-reacting antibodies, when it was necessary, antisera were adsorbed.

  19. Preparation of gluten-free rice spaghetti with soy protein isolate using twin-screw extrusion.

    Science.gov (United States)

    Detchewa, Pakkawat; Thongngam, Masubon; Jane, Jay-Lin; Naivikul, Onanong

    2016-09-01

    The objective of this study was to investigate the effect of soy protein isolate on functional properties and consumer acceptance of gluten-free rice spaghetti (GFRS) made from rice flour. Dry-milled high-amylose (Chai Nat 1) rice flour was premixed with dry-milled waxy (RD 6) rice flour at a ratio of 90:10 (w/w) with the soy protein isolate (SPI) concentration varying between 0, 2.5, 5.0, 7.5, 10.0 %, db. The GFRS formulation was processed using a co-rotating twin-screw extruder up to 95 °C with a screw speed of 220 rpm, 32 % moisture content, and then dried at 40 °C. The GFRS samples were analyzed by differential scanning calorimetry (DSC), X-ray diffraction, scanning electron microscopy (SEM) and texture parameters. Increasing SPI decreased the starch retrogradation of GFRS, whereas the enthalpy change of the amylose-lipid complex increased and crystallinity decreased. SEM revealed that the surface of GFRS containing SPI was much more porous than that of GFRS without SPI. The cooked GFRS containing 5.0 % SPI showed the best eating quality with increased firmness and tensile strength, and decrease stickiness. The GFRS samples were evaluated on the bases of cooking qualities and sensory evaluation. The results showed that the GFRS containing 5.0 % SPI decrease the cooking time from 17.6 to 13.7 min and cooking loss from 25.4 to 17.0 %. Overall acceptability of cooked GFRS containing 5.0 % SPI was the highest among all GFRS samples.

  20. Preparation and Characterization of Soluble Eggshell Membrane Protein/PLGA Electrospun Nanofibers for Guided Tissue Regeneration Membrane

    Directory of Open Access Journals (Sweden)

    Jun Jia

    2012-01-01

    Full Text Available Guided tissue regeneration (GTR is a widely used method in periodontal therapy, which involves the placement of a barrier membrane to exclude migration of epithelium and ensure repopulation of periodontal ligament cells. The objective of this study is to prepare and evaluate a new type of soluble eggshell membrane protein (SEP/poly (lactic-co-glycolic acid (PLGA nanofibers using electrospinning method for GTR membrane application. SEP/PLGA nanofibers were successfully prepared with various blending ratios. The morphology, chemical composition, surface wettability, and mechanical properties of the nanofibers were characterized using scanning electron microscopy (SEM, contact angle measurement, Fourier transform-infrared spectroscopy (FTIR, and a universal testing machine. L-929 fibroblast cells were used to evaluate the biocompatibility of SEP/PLGA nanofibers and investigate the interaction between cells and nanofibers. Results showed that the SEP/PLGA electrospun membrane was composed of uniform, bead-free nanofibers, which formed an interconnected porous network structure. Mechanical property of SEP has been greatly improved by the addition of PLGA. The biological study results showed that SEP/PLGA nanofibers could enhance cell attachment, spreading, and proliferation. The study indicated the potential of SEP/PLGA nanofibers for GTR application and provided a basis for future optimization.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  4. Influence of calcium, magnesium, or potassium ions on the formation and stability of emulsions prepared using highly hydrolyzed whey proteins.

    Science.gov (United States)

    Ramkumar, C; Singh, H; Munro, P A; Singh, A M

    2000-05-01

    Oil-in-water emulsions (4 wt % soy oil) containing 4 wt % whey protein hydrolysate (WPH) (27% degree of hydrolysis) and different levels of calcium, magnesium, or potassium chloride were prepared in a two-stage homogenizer. Other emulsions containing 4 wt % WPH but including 0.35 wt % hydroxylated lecithin and different levels of the above minerals were similarly prepared. The formation and stability of these emulsions were determined by measuring oil droplet size distributions using laser light scattering and by confocal scanning laser microscopy and a gravity creaming test. Both lecithin-free and lecithin-containing emulsions showed no change in droplet size distributions with increasing concentration of potassium in the range 0-37.5 mM. In contrast, the diameter of emulsion droplets increased with increasing calcium or magnesium concentration >12.5 mM. Emulsions containing hydroxylated lecithin were more sensitive to the addition of calcium or magnesium than the lecithin-free emulsions. Storage of emulsions at 20 degrees C for 24 h further increased the diameter of droplets and resulted in extensive creaming in emulsions containing >25 mM calcium or magnesium. It appears that both flocculation and coalescence processes were involved in the destabilization of emulsions induced by the addition of divalent cations.

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

    Directory of Open Access Journals (Sweden)

    Liyue Wang

    2014-01-01

    Full Text Available 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 was inserted into Nsp2 gene of the FL12 plasmid by Spe I and Xho I sites to generate PRRSV infectious recombinant plasmid (FL12-GFP containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP was rescued in baby hamster kidney-21 (BHK-21 cells by transfecting PRRSV mRNA synthesized in vitro and amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs. This study provides essential conditions for further investigation on PRRSV.

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

    Directory of Open Access Journals (Sweden)

    N. Akashi

    2015-01-01

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

  7. The preparation of HL-60 cells vaccine expressing BCG heat shock protein 70 and detection of its immunogenicity in vitro.

    Science.gov (United States)

    Li, Xiao-Ling; Zhao, Yan-Xia; Sun, Li-Rong; Yang, Jing; Xu, Hui-Juan

    2012-10-01

    Gene-modified cell vaccines are the best way to achieve the immunotherapy for all types of acute leukemia. In this study, the recombinant eukaryotic expression vector (pDisplay-HSP70) of heat shock protein 70 (HSP70) of Bacille Calmette-Guérin (BCG) was constructed by amplifying the whole BCG HSP70 gene using polymerase chain reaction (PCR) and sub-cloning into the polyclone endonuclease sites in pDisplay. Then the HL-60 cell vaccine expressing the protein onto the cell surface was prepared by lipofectamine transfection and its anti-tumor effect and mechanism were further studied. Results showed that the fragment of BCG HSP70 was consistent with Mycobacterium tuberculosis HSP70 gene published in GeneBank. DNA sequencing showed that the recombinant vector was correctly constructed and named pDisplay-HSP70. After BCG HSP70 gene transfection, the yellow-green fluorescence on the HL-60 cells surface was observed under a fluorescence microscope. The immunogenicity of HSP70-transfected HL-60 cells exhibited upregulated proliferation of lymphocytes, increased cytokine secretion (IFN-γ) and enhanced killing activity. These results suggested that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of HL-60 cells. It may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.

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

    Directory of Open Access Journals (Sweden)

    Lylo V. V.

    2014-11-01

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

  9. High-resolution, preparative purification of PEGylated protein using a laterally-fed membrane chromatography device.

    Science.gov (United States)

    Madadkar, Pedram; Nino, Sergio Luna; Ghosh, Raja

    2016-11-01

    We discuss the use of a laterally-fed membrane chromatography (or LFMC) device for single-step purification of mono-PEGylated lysozyme. Recent studies have shown such LFMC devices to be suitable for high-resolution, multi-component separation of proteins in the bind-and-elute mode. The device used in this study contained a stack of rectangular cation-exchange membranes having 9.25mL bed volume. PEGylation of lysozyme was carried out in batch mode using 5kDa methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) in the presence of sodium cyanoborohydride as reducing agent. Membrane chromatographic separation was carried out at 1.62 membrane bed volumes per minute flow rate, in the bind-and-elute mode. When a salt gradient was applied, the higher PEGylated forms of lysozyme (i.e. the byproducts) eluted earlier than mono-PEGylated lysozyme (the target product), while lysozyme eluted last. Under elution conditions optimized for resolution and speed, the separation could be carried out in less than 15 membrane bed volumes. High purity and recovery of mono-PEGylated lysozyme was obtained. The resolution of separation of mono-PEGylated lysozyme obtained under the above condition was comparable to that reported in the literature for equivalent cation-exchange resin columns while the flow rate expressed in bed volumes/min was 21.7 times higher. Also, the number of theoretical plates per meter was significantly higher with the LFMC device. Therefore the LFMC based purification process discussed in this paper combined high-productivity with high-resolution.

  10. Zilascorb(2H), a new reversible protein synthesis inhibitor: clinical study of an oral preparation.

    Science.gov (United States)

    Semb, K A; Fodstad, O; Klem, B; Bibow, K; Osmundsen, K; Aamdal, S

    1997-03-01

    The new anti-cancer drug zilascorb(2H) has shown promising activity in preclinical models. Its putative mechanism of action is reversible protein synthesis inhibition and long-term treatment is required. As a clinical treatment modality, long-term daily zilascorb(2H) infusions, as used in previous studies, are not regarded feasible. Therefore, an oral formulation of the drug was developed, and pharmacokinetic profile, toxicity and antitumor activity of zilascorb(2H) tablets were studied. Thirteen patients with advanced solid cancer not amenable to established therapy, but with adequate performance status and organ functions, were included. The treatment was given as a daily i.v. zilascorb(2H) infusion for 5 days, followed by zilascorb(2H) tablets twice daily for 3 months. Blood and urine sampling was performed when estimated plasma steady-state level was reached for each formulation, respectively. Analyses of drug concentrations in plasma and urine were performed by high performance liquid chromatography. Zilascorb(2H) in tablet formulation had a bioavailability of 32%, was quickly absorbed and slowly eliminated. Concomitant use of the H2-blocker ranitidine possibly enhanced bioavailability. Zilascorb(2H) was well tolerated. Two patients experienced drug-related fever, disturbing the treatment schedule for one of them. Moderate nausea was reported. One objective response was obtained. The bioavailability of zilascorb(2H) tablets was satisfactory. The principle of oral administration of zilascorb(2H) is feasible for long-term treatment and the side effects are acceptable. The mechanisms of action and the very low toxicity of the drug makes it a candidate for combination with other anticancer agents.

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

    Directory of Open Access Journals (Sweden)

    Zhiqiang Tian

    2011-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Waldemar Gustaw

    2016-03-01

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

  13. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

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

    Science.gov (United States)

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

    2000-08-01

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

  15. The effects of antifreeze peptide III (AFP) and insulin transferrin selenium (ITS) on cryopreservation of chimpanzee (Pan troglodytes) spermatozoa.

    Science.gov (United States)

    Younis, A I; Rooks, B; Khan, S; Gould, K G

    1998-01-01

    We investigated the effects of antifreeze peptides (AFP) and insulin transferrin selenium (ITS) on the motility and membrane integrity of chimpanzee (Pan troglodytes) spermatozoa after chilling (0-5 degrees C) and thawing. The effects of three thawing procedures, in the presence or absence of AFP and ITS, on sperm motility and on the status of the plasma membrane and acrosome were also examined. During chilling, AFP and ITS seem mildly cytotoxic, as the progressive motility and velocity (curvilinear and straight line) declined significantly at AFP concentrations of 1, 10, and 100 microg/ml and at ITS concentrations of 1 and 10 microg/ml. However, at a concentration of 100 microg/ml, ITS was able to protect sperm during short-term hypothermic storage. Addition of AFP or ITS at 100 microg/ml to test egg yolk-glycerol extender during freezing significantly (P < 0.05) increased postthaw motility, plasma membrane integrity, and acrosome integrity. The mean (+/-SE) motility recovery rate increased from 28.9 +/- 3.9%, for the untreated control, to 59.2 +/- 5.8% and 67.8 +/- 7.4%, for ITS and AFP, respectively. The effects of the thawing procedure were influenced by the presence of AFP during the freezing cycle. An improved motility recovery rate of 67 +/- 4.2% was obtained when chimpanzee sperm frozen in test egg yolk-glycerol extender supplemented with AFP were thawed rapidly at 37 degrees C, compared to 47 +/- 5.2% and 44 +/- 8.2% for slow (23 degrees C) and ultrarapid (75 degrees C) thawing, respectively. The motility recovery after thawing of ITS-treated semen at 23 degrees C, 37 degrees C, or 75 degrees C was not significantly different. Semen frozen without AFP or ITS and thawed at 75 degrees C was seriously (P < 0.05) damaged. This study provides evidence that AFP- or ITS-supplemented semen extender improves postthaw sperm motility in the chimpanzee.

  16. Preparation and in vivo evaluation of a novel stabilized linker for {sup 211}At labeling of protein

    Energy Technology Data Exchange (ETDEWEB)

    Talanov, Vladimir S. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)]. E-mail: vstalanov@msrce.howard.edu; Garmestani, Kayhan [Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Regino, Celeste A.S. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Milenic, Diane E. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Plascjak, Paul S. [Department of Nuclear Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Waldmann, Thomas A. [Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Brechbiel, Martin W. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)]. E-mail: martinwb@mail.nih.gov

    2006-05-15

    Significant improvement of in vivo stability of {sup 211}At-labeled radioimmunoconjugates achieved upon employment of a recently reported new linker, succinimidyl N-2-(4-[{sup 211}At]astatophenethyl)succinamate (SAPS), prompted additional studies of its chemistry. The {sup 211}At radiolabeling of succinimidyl N-2-(4-tributylstannylphenethyl)succinamate (1) was noted to decline after storage at -15{sup o}C 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 {sup 211}At generates succinimidyl N-2-(4-[{sup 211}At]astatophenethyl)-N-methyl succinamate (Methyl-SAPS), with yields being consistent for greater than 1 year. Radiolabelings of 1 and SPEMS with {sup 125}I generated succinimidyl N-2-(4-[{sup 125}I]iodophenethyl)succinamate (SIPS) and succinimidyl N-2-(4-[{sup 125}I]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.

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

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

    DEFF Research Database (Denmark)

    Stensballe, A; Jensen, Ole Nørregaard

    2001-01-01

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

  19. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    Science.gov (United States)

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

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

    Science.gov (United States)

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

    2015-04-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  2. Preparation of poly(N-isopropylacrylamide)-grafted well-controlled 3D skeletal monolith based on E-51 epoxy resin for protein separation.

    Science.gov (United States)

    Xin, Peiyong; Shen, Ying; Qi, Li; Yang, Gengliang; Chen, Yi

    2011-08-15

    A novel type of poly(N-isopropylacrylamide) grafted E-51 epoxy-based monoliths in a 100 mm × 4.6mm I.D. stainless steel column with well-controlled three-dimensional skeletal structures has been prepared and proposed for the separation of proteins. The grafted PNIPAAm chain via surface-initiated atom transfer radical polymerization was successfully performed. The proposed method provided a new route to modify the E-51 epoxy-based monoliths for widening their applications. Meanwhile, the temperature and the salt concentration responses of the grafted monolithic columns were investigated. Under the salt gradient, six proteins were well separated in hydrophobic interaction mode. Moreover, for further confirming the application of the prepared monolith was meaningful for proteome analysis in actual system, the separation of human serum sample was performed.

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

    Directory of Open Access Journals (Sweden)

    Guedj AS

    2015-09-01

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

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

    Directory of Open Access Journals (Sweden)

    David Ramirez BREWER

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-12-14

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    A new microfluidic sample-preparation system is presented for the structural investigation of proteins using small-angle X-ray scattering (SAXS) at synchrotrons. The system includes hardware and software features for precise fluidic control, sample mixing by diffusion, automated X-ray exposure...... control, UV absorbance measurements and automated data analysis. As little as 15 l of sample is required to perform a complete analysis cycle, including sample mixing, SAXS measurement, continuous UV absorbance measurements, and cleaning of the channels and X-ray cell with buffer. The complete analysis...... cycle can be performed in less than 3 min. Bovine serum albumin was used as a model protein to characterize the mixing efficiency and sample consumption of the system. The N2 fragment of an adaptor protein (p120-RasGAP) was used to demonstrate how the device can be used to survey the structural space...

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

    Institute of Scientific and Technical Information of China (English)

    郝凤; 刘晓静; 周爱琴; 于铁峰

    2009-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    王长松; 陈磊

    2012-01-01

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

  9. Calorimetric Analysis of Antifreeze Activity and the Absorption-inhibition Mechanism of Antifreeze Protein%抗冻蛋白活性的差示扫描量热测定及其吸附-抑制机制

    Institute of Scientific and Technical Information of China (English)

    任禾盛; 许娜飞; 华泽钊

    2004-01-01

    用差示扫描量热技术(DSC)测定了从黄粉虫(Tenebrio molitor)幼虫体内提取的抗冻蛋白(AFP)的活性,结果表明AFP活性随其浓度的增加及初始冰晶量的减少而增大,这与AFP对冰晶的吸附-抑制机制相一致.

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

    Science.gov (United States)

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

    2011-04-19

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

  11. Beta-carotene-rich carotenoid-protein preparation and exopolysaccharide production by Rhodotorula rubra GED8 grown with a yogurt starter culture.

    Science.gov (United States)

    Frengova, Ginka I; Simova, Emilina D; Beshkova, Dora M

    2006-01-01

    The underlying method for obtaining a beta-carotene-rich carotenoid-protein preparation and exopolysaccharides is the associated cultivation of the carotenoid-synthesizing lactose-negative yeast strain Rhodotorula rubra GED8 with the yogurt starter culture (Lactobacillus bulgaricus 2-11 + Streptococcus thermophilus 15HA) in whey ultrafiltrate (45 g lactose/l) with a maximum carotenoid yield of 13.37 mg/l culture fluid on the 4.5th day. The chemical composition of the carotenoid-protein preparation has been identified. The respective carotenoid and protein content is 497.4 microg/g dry cells and 50.3% per dry weight, respectively. An important characteristic of the carotenoid composition is the high percentage (51.1%) of beta-carotene (a carotenoid pigment with the highest provitamin A activity) as compared to 12.9% and 33.7%, respectively, for the other two individual pigments--torulene and torularhodin. Exopolysaccharides (12.8 g/l) synthesized by the yeast and lactic acid cultures, identified as acid biopolymers containing 7.2% glucuronic acid, were isolated in the cell-free supernatant. Mannose, produced exclusively by the yeast, predominated in the neutral carbohydrate biopolymer component (76%). The mixed cultivation of R. rubra GED8 with the yogurt starter (L. bulgaricus 2-11 + S. thermophilus 15HA) in ultrafiltrate under conditions of intracellular production of maximum amount of carotenoids and exopolysaccharides synthesis enables combined utilization of the culture fluid from the fermentation process.

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

    Directory of Open Access Journals (Sweden)

    Shahryar Shakeri

    2015-07-01

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

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

    Science.gov (United States)

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

    1987-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-02-25

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

  15. Effect of perioperative application ofL-asrginine combined with intacted protein compound preparations on postoperative antitumor immunity and tumor load in patients with gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Xiu-Lan Jiang

    2016-01-01

    Objective:To analyze the effect of perioperative application ofL-arginine combined with intacted protein compound preparations on postoperative antitumor immunity and tumor load in patients with gastric cancer.Methods:A total of 68 patients with gastric cancer received radical operation, and according to different perioperative nutrition intervention, they were divided into control group (normal glucose saline enteral nutrition) and observation group (L-arginine combined with intacted protein compound preparations enteral nutrition) by half. Postoperative short-term antitumor immune cell levels and serum levels of illness-related indexes, nutrition and inflammation indexes of two groups were detected, patients were followed up for 3 years and the gastric stump MRI changes were observed.Results:Venous blood CD4+T lymphocyte level and CD4+/CD8+ ratio of observation group 3 months after treatment were higher than those of control group while CD8+T lymphocyte and Treg cell levels were lower than those of control group; serum Pentraxin-3, CYFRA21-1, TTF-1 and HE4 levels were lower than those of control group; ALB, PA and IL-2 levels were higher than those of control group while IL-6 and IL-10 levels were lower than those of control group (P<0.05). Gastric stump MRI images 3 years after operation were significantly different between two groups.Conclusions:Perioperative application ofL-arginine combined with intacted protein compound preparations can optimize postoperative immune and nutritional state in patients with gastric cancer, and it also has positive effect on reducing the incidence of long-term gastric stump carcinoma and other aspects.

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

    Institute of Scientific and Technical Information of China (English)

    朱淳; 刘强; 左静南; 朱汉威; 马济民

    2002-01-01

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

  17. [Preparation and the biological effect of fusion protein GLP-1-exendin-4/ IgG4(Fc) fusion protein as long acting GLP-1 receptor agonist].

    Science.gov (United States)

    Zheng, Yun-cheng

    2015-12-01

    GLP-1 has a variety of anti-diabetic effects. However, native GLP-1 is not suitable for treatment of diabetes due to its short half-life (t½, 2-5 min). Exendin-4 is a polypeptide isolated from lizard saliva, which can bind to GLP-1 receptor, produce physiological effects similar to GLP-1, t½ up to 2.5 h, therefore, we developed a long-lasting GLP-1 receptor agonists and GLP-1-exendin-4 fusion IgG4 Fc [GLP-1-exendin-4/ IgG4(Fc)]. We constructed the eukaryotic expression vector of human GLP-1-exendin-4/IgG4(Fc)-pOptiVEC- TOPO by gene recombination technique and expressed the fusion protein human GLP-1-IgG4 (Fc) in CHO/DG44 cells. The fusion protein stimulated the INS-1 cells secretion of insulin, GLP-1, exendin-4 and fusion protein in CD1 mice pharmacokinetic experiments, as well as GLP-1, exendin-4 and fusion protein did anti-diabetic effect on streptozotocin induced mice. Results demonstrated that the GLP-1-exendin-4/IgG4(Fc) positive CHO/DG44 clones were chosen and the media from these positive clones. Western blotting showed that one protein band was found to match well with the predicted relative molecular mass of human GLP-1-exendin-4/IgG4(Fc). Insulin RIA showed that GLP-1-exendin-4/IgG4(Fc) dose-dependently stimulated insulin secretion from INS-1 cells. Pharmacokinetic studies in CD1 mice showed that with intraperitoneal injection (ip), the fusion protein peaked at 30 min in circulation and maintained a plateau for 200 h. Natural biological half-life of exendin-4 was (1.39 ± 0.28) h, GLP-1 in vivo t½ 4 min, indicating that fusion protein has long-lasting effects on the modulation of glucose homeostasis. GLP-1-exendin-4/IgG4(Fc) was found to be effective in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced diabetes in mice, longer duration of the biological activity of the fusion protein. The biological activity was significantly higher than that of GLP-1 and exendin-4. GLP-1-exendin-4/IgG4(Fc) has good anti-diabetic activity

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2016-07-27

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

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

    Institute of Scientific and Technical Information of China (English)

    潘晶; 陈思思; 张晖; 郭晓娜

    2011-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Esther T. L. Lau

    2015-01-01

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

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

    Science.gov (United States)

    Ding, Xueqin; Wang, Yuzhi; Wang, Ying; Pan, Qi; Chen, Jing; Huang, Yanhua; Xu, Kaijia

    2015-02-25

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

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-11-01

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

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

    Science.gov (United States)

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

    2013-08-01

    Polymer implants are promising systems for sustained release applications but their utility for protein delivery has been hindered because of concerns over drug stability at elevated temperatures required for processing. Using bovine serum albumin (BSA) as a model, we have assessed whether proteins can be formulated for processing at elevated temperatures. Specifically, the effect of trehalose and histidine-HCl buffer on BSA stability in a spray-dried formulation has been investigated at temperatures ranging from 80°C to 110°C. When both the sugar and buffer are present, aggregation is suppressed even when exposed to 100°C, the extrusion temperature of poly(lactide-co-glycolide) (PLGA), a bioresorbable polymer. Estimation of aggregation rate constants (k) indicate that though both trehalose and histidine-HCl buffer contribute to BSA stability, the effect because of trehalose alone is more pronounced. BSA-loaded PLGA implants were prepared using hot-melt extrusion process and in vitro release was conducted in phosphate buffered saline at 37°C. Comparison of drug released from implants prepared using four different formulations confirmed that maximal release was achieved from the formulation in which BSA was least aggregated. These studies demonstrate that when trehalose and histidine-HCl buffer are included in spray-dried formulations, BSA stability is maintained both during processing at 100°C and long-term residence within implants.

  8. The Preparation of Protein Anti-staining Agent for Reactive Dyes%活性染料蛋白类防沾色剂的制备

    Institute of Scientific and Technical Information of China (English)

    徐华凤; 王雪燕

    2015-01-01

    以明胶和自制的阳离子交联改性剂WLS为原料,制备了一种阳离子明胶蛋白助剂,并与聚乙烯基吡咯烷酮( PVP)复配,制成一种新型防沾色助剂,应用于活性染料染色棉织物皂洗后处理。以白布沾色K/S值和色布耐皂洗色牢度为评价指标,运用正交试验确定了有利于改善活性染料染色棉织物皂洗防沾色的阳离子明胶蛋白助剂的合成条件,其为:m(明胶蛋白)∶m (WLS)=1∶10,NaOH用量为WLS用量的1.8%,反应温度70℃,反应时间3h;PVP与阳离子明胶蛋白复配质量比为1:50。%Taking gelatin and self-made cationic crosslinking modifier WLS as raw materials, a kind of cat-ionic gelatin protein additive was prepared and was compounded with PVP to prepare a new anti-staining agent for after-soaping treatment of dyed cotton fabric with reactive dyes. Taking K/S value of stained cloth and color fast-ness to soaping of dyed cloth as evaluation indicators, the preparation conditions of cationic gelatin protein addi-tives was determined by using orthogonal test, which could improve soaping anti-staining property of cotton fabric dyed with reactive dyes. The preparation conditions were: m ( gelatin): m ( WLS) was 1∶14, the dosage of NaOH was1. 8% of that of WLS, the reaction temperature was 70℃ and time was 3h, the quality ratio of PVP to cationic gelatin protein was 1∶50 .

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

    Science.gov (United States)

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

    2016-06-01

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

  10. Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment

    NARCIS (Netherlands)

    Snippert, H.J.G.; Schepers, A.G.; Delconte, G.; Siersema, P.D.; Clevers, H.

    2011-01-01

    In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  13. A novel method to prepare gluten-free dough using a meso-structured whey protein particle system

    NARCIS (Netherlands)

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

    2011-01-01

    This paper presents a novel concept for making an elastic dough using a structured protein suspension. The idea behind it is based on the hypothesis that a number of gluten properties originate from a particle structure present in the gluten network. Three different mesoscopically structured whey pr

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

    NARCIS (Netherlands)

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

    2009-01-01

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

  15. Preparation of a novel weak cation exchange/hydrophobic interaction chromatography dual-function polymer-based stationary phase for protein separation using "thiol-ene click chemistry".

    Science.gov (United States)

    Yang, Fan; Bai, Quan; Zhao, Kailou; Gao, Dong; Tian, Lei

    2015-02-01

    A novel dual-function mixed-mode stationary phase based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) microspheres was synthesized by thiol-ene click chemistry and characterized by infrared spectroscopy and elemental analysis. The new system displays both hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase, and weak cation exchange (WCX) chromatography character in a low salt concentration mobile phase. It can be used to separate proteins in both ion-exchange chromatography (IEC) mode and HIC mode. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 96% can be achieved in both HIC mode and IEC mode using this column. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. On the basis of this dual-function mixed-mode chromatography column, a new two-dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-γ from inclusion bodies. The mass recovery, purity, and specific bioactivity obtained for the purified recombinant human interferon-γ were 87.2%, 92.4%, and 2.8 × 10(7) IU/mg, respectively, in IEC mode, and 83.4%, 95.2%, and 4.3 × 10(7) IU/mg, respectively, in HIC mode. The results indicate that the

  16. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of Smu.1475c from caries pathogen Streptococcus mutans.

    Science.gov (United States)

    Zhou, Yan-Feng; Mi, Wei; Li, Lanfen; Zhang, Xiaoyan; Liang, Yu-He; Su, Xiao-Dong; Wei, Shicheng

    2006-02-01

    The gene smu.1475c encodes a putative protein of 211 residues in Streptococcus mutans, a primary pathogen for human dental caries. In this work, smu.1475c was cloned into pET28a and expressed in good amount from the E. coli strain BL21 (DE3). Smu.1475c protein was purified to homogeneity in a two-step procedure of Ni2+ chelating and size exclusion chromatography. Crystals were obtained by hanging-drop vapor-diffusion method and diffracted to 2.7 angstroms resolution. The crystal belongs to orthorhombic space group P2(1)2(1)2(1) with cell dimension of a = 68.3 angstroms, b = 105.9 angstroms, c = 136.2 angstroms. The asymmetric unit is expected to contain four molecules with solvent content of 49.4%.

  17. Preparation of Sulfobetaine-Grafted PVDF Hollow Fiber Membranes with a Stably Anti-Protein-Fouling Performance

    Directory of Open Access Journals (Sweden)

    Qian Li

    2014-04-01

    Full Text Available Based on a two-step polymerization method, two sulfobetaine-based zwitterionic monomers, including 3-(methacryloylamino propyl-dimethyl-(3-sulfopropyl ammonium hydroxide (MPDSAH and 2-(methacryloyloxyethyl ethyl-dimethyl-(3-sulfopropyl ammonium (MEDSA, were successfully grafted from poly(vinylidene fluoride (PVDF hollow fiber membrane surfaces in the presence of N,N′-methylene bisacrylamide (MBAA as a cross-linking agent. The mechanical properties of the PVDF membrane were improved by the zwitterionic surface layers. The surface hydrophilicity of PVDF membranes was significantly enhanced and the polyMPDSAH-g-PVDF membrane showed a higher hydrophilicity due to the higher grafting amount. Compared to the polyMEDSA-g-PVDF membrane, the polyMPDSAH-g-PVDF membrane showed excellent significantly better anti-protein-fouling performance with a flux recovery ratio (RFR higher than 90% during the cyclic filtration of a bovine serum albumin (BSA solution. The polyMPDSAH-g-PVDF membrane showed an obvious electrolyte-responsive behavior and its protein-fouling-resistance performance was improved further during the filtration of the protein solution with 100 mmol/L of NaCl. After cleaned with a membrane cleaning solution for 16 days, the grafted MPDSAH layer on the PVDF membrane could be maintain without any chang; however, the polyMEDSA-g-PVDF membrane lost the grafted MEDSA layer after this treatment. Therefore, the amide group of sulfobetaine, which contributed significantly to the higher hydrophilicity and stability, was shown to be imperative in modifying the PVDF membrane for a stable anti-protein-fouling performance via the two-step polymerization method.

  18. The effect of the application of protein and cellulose preparations as iodine carriers on stability of thiamine in processed meats

    OpenAIRE

    Krystyna Szymandera-Buszka; Katarzyna Waszkowiak; Marzanna Hęś; Anna Jędrusek-Golińska

    2011-01-01

      Fortification of processed meat with iodised table salt was shown to increase thiamine losses, both during thermal processing and storage. Taking into consideration the fact, as well as the recommendation for reduction of consumption of table salt, alternative iodine carriers need to be searched for. Thus the aim of the study was to determine the effect of soy protein isolate (SPI) and wheat fibre (WF) as iodine salts’ (potassium iodide and iodate) carriers on thiamine stabil...

  19. The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

    Directory of Open Access Journals (Sweden)

    Cheng Xu

    2014-08-01

    Full Text Available Follicle-stimulating hormone receptor (FSHR, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein (FSHR-57aa as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a(+-FSHR-57aa plasmid was constructed and expressed in Escherichia coli strain BL21 Star TM (DE3 and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 (16 weeks after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

  20. Extraction of Chinese Chestnut Protein and Preparation of the Ploypeptides%板栗蛋白质的提取及多肽的制备

    Institute of Scientific and Technical Information of China (English)

    张帅; 张培宜; 冯翠萍

    2011-01-01

    Protein was extracted from Chinese chestnut, and the Chinese chestnut polypeptide was obtained by hydrolysis, with a substance to provide a scientific basis for the development and use of Chinese chestnut protein polypeptide.Molecular weight of the protein was determined through SDS-PAGE. The albumen hydrolysis special-purpose enzyme was selected to determine the optimum hydrolysis conditions about Chinese chestnut polypeptide, using three factors three levels orthogonal experimental design. The isoelectronic point of the protein is 4. 5 , and molecular weight is about 25000 Da; The best hydrolysis condition of albumen hydrolysis special-purpose enzyme is : Substrate concentration 2. 5 g ·L-1 , enzyme capacity 0. 3 % , pH 8. 5 , the degree of hydrolysis 20. 13 %. The protein and polypeptides of Chinese chestnut were preparated.%以板栗为原料,提取蛋白质并水解制备多肽.通过SDS-PAGE测定板栗蛋白质的分子量,以植物蛋白水解专用酶采用三因素三水平的正交试验筛选制备板栗多肽的最佳水解条件,旨在为板栗的开发,利用提供理论依据.板栗蛋白质的等电点为4.5;分子量约为25 000 Da;植物蛋白水解专用酶最佳的水解条件:底物浓度2.5 g·L-1,酶添加量0.3%,pH 8.5,水解度是20.13%.得到板栗蛋白并制备出板栗多肽.

  1. Preparation of denatured protein bone sterilized with gamma radiation; Preparacion de hueso desproteinizado esterilizado con radiacion gamma

    Energy Technology Data Exchange (ETDEWEB)

    Luna Z, D. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)]. e-mail: dlz@nuclear.inin.mx

    2005-07-01

    The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

  2. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

    Science.gov (United States)

    Drori, Ran; Celik, Yeliz; Davies, Peter L; Braslavsky, Ido

    2014-09-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

  3. Preparation and testing of a Vi conjugate vaccine using pneumococcal surface protein A (PspA) from Streptococcus pneumoniae as the carrier protein.

    Science.gov (United States)

    Kothari, Neha; Genschmer, Kristopher R; Kothari, Sudeep; Kim, Jeong Ah; Briles, David E; Rhee, Dong Kwon; Carbis, Rodney

    2014-09-29

    In the current study pneumococcal surface protein A (PspA) was conjugated to Vi capsular polysaccharide from Salmonella Typhi to make available a vaccine against typhoid fever that has the potential to also provide broad protection from Streptococcus pneumoniae. High yielding production processes were developed for the purification of PspAs from families 1 and 2. The purified PspAs were conjugated to Vi with high recovery of both Vi and PspA. The processes developed especially for PspA family 2 could readily be adapted for large scale production under cGMP conditions. Previously we have shown that conjugation of diphtheria toxoid (DT) to Vi polysaccharide improves the immune response to Vi but can also enhance the response to DT. In this study it was shown that conjugation of PspA to Vi enhanced the anti-PspA response and that PspA was a suitable carrier protein as demonstrated by the characteristics of a T-cell dependent response to the Vi. We propose that a bivalent vaccine consisting of PspA from families 1 and 2 bound to Vi polysaccharide would protect against typhoid fever and has the potential to also protect against pneumococcal disease and should be considered for use in developing countries.

  4. Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    CHEN Yang; YANG Su-juan; FU Yao; WANG Jia-peng; ZHAO Zhi-zhuang; FU Xue-qi

    2008-01-01

    A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPa.

  5. Preparation of egg white protein emulsion sausage%蛋清蛋白乳化香肠的研制

    Institute of Scientific and Technical Information of China (English)

    王可; 周玲; 汪学荣

    2012-01-01

    The influences of fat/lean ration,egg white protein and carrageen on the quality of emulsion sausage were studied.The results showed that fat/lean ration,egg white protein and carrageen had effects on the quality of emulsion sausage on varying degrees.The composition was explored by means of L9(34)orthogonal design,and the best ratio were as follows:Fat/lean ration 2∶8,egg white protein 6%,carrageen 0.3%.Under the best condition,sensory evaluation value was 8.0,water retaining property was 79.41%,shear force was 1.588kg·f.%主要研究了原料肉肥瘦比、蛋清添加量和卡拉胶添加量对乳化香肠品质的影响。实验结果表明,原料肉肥瘦比、蛋清添加量和卡拉胶添加量对乳化香肠品质有不同程度的影响,通过L9(34)正交实验得到最佳配比为:原料肉肥瘦比2∶8,蛋清蛋白添加量6%,卡拉胶添加量0.3%。在此最佳配比下,乳化香肠的感官评分为8.0,系水力为79.41%,剪切力为1.588kg.f。

  6. Preparation of protein-like silver-cysteine hybrid nanowires and application in ultrasensitive immunoassay of cancer biomarker.

    Science.gov (United States)

    Chen, Wenjuan; Zheng, Liyan; Wang, Meilan; Chi, Yuwu; Chen, Guonan

    2013-10-15

    Novel protein-like silver-cysteine hybrid nanowires (p-SCNWs) have been synthesized by a green, simple, nontemplate, seedless, and one-step aqueous-phase approach. AgNO3 and l-cysteine were dissolved in distilled water, forming Ag-cysteine precipitates and HNO3. Under vigorous stirring, the pH of the solution was rapidly adjusted to 9.0 by addition of concentrated sodium hydroxide solution, leading to quick dissolution of the Ag-cysteine precipitates and sudden appearance of white precipitates of p-SCNWs. The p-SCNWs are monodispersed nanowires with diameter of 100 nm and length of tens of micrometers, and have abundant carboxyl (-COOH) and amine (-NH2) groups at their surfaces, large amounts of peptide-linkages and S-bonding silver ions (Ag(+)) inside, making them look and act like Ag-hybrid protein nanostructures. The abundant -COOH and -NH2 groups at the surfaces of p-SCNWs have been found to facilitate the reactions between the p-SCNWs and proteins including antibodies. Furthermore, the fact that the p-SCNWs contain large amounts of silver ions enables biofunctionalized p-SCNWs to be excellent signal amplifying chemiluminescence labels for ultrasensitive and highly selective detection of important antigens, such as cancer biomarkers. In this work, the immunoassay of carcinoembryonic antigen (CEA) in human serum was taken as an example to demonstrate the immunoassay applications of antibody-functionalized p-SCNWs. By the novel p-SCNW labels, CEA can be detected in the linear range from 5 to 400 fg/mL with a limit of detection (LOD) of 2.2 fg/mL (at signal-to-noise ratio of 3), which is much lower than that obtained by commercially available enzyme-linked immunosorbent assay (ELISA). Therefore, the synthesized p-SCNWs are envisioned to be an excellent carrier for proteins and related immunoassay strategy would have promising applications in ultrasensitive clinical screening of cancer biomarkers for early diagnostics of cancers.

  7. Progress in Protein Polypeptides Preparations%蛋白多肽类药物制剂的研究进展

    Institute of Scientific and Technical Information of China (English)

    李盈; 娄月芬

    2012-01-01

    Compared with other routes of administration, the route of administration of protein peptide drugs including oral, nasal, pulmonary is more feasible and commercial value. The pharmaceutics methods can improve their bioavailabiliry. This paper evaluated the protein peptide drug delivery systems and reviewed the drugs in vivo stability and bioavailability research progress at home and abroad in recent years.%相对于其他的给药途径,蛋白质多肽类药物的口服、经鼻、肺部给药途径更具可行性和商业价值.利用制剂学方法可提高蛋白质多肽类药物生物利用度.通过蛋白多肽类给药系统的评价,对近年来国内外此类药物在剂型、体内外稳定性及生物利用度等方面的研究进展予以综述.

  8. Preparation of Polyclonal Antibody against Crystalline Inclusion Protein of Entomopathogenic Bacterium%昆虫病原细菌Cip蛋白多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    游娟; 黄建林; 曹莉; 韩日畴

    2013-01-01

    Entomopathogenic Photorhabdus bacteria produce two types of intracellular crystalline inclusion proteins,CipA and CipB,as the nutrient resource for the symbiotic nematode.Based on constructed high-level prokaryotic expression system,recombinant Cip proteins were purified with a warm extract method and used as the antigens to immune New Zealand rabbits,respectively.The polyclonal antibodies with the titers of 1:6400 were raised and monitored by indirect enzyme-linked immunosorbent assay (ELISA).Western blot assay confirmed that the antibodies could specifically recognize the inclusion proteins in Photorhabdus bacteria.Preparation of Cip polyclonal antibodies will be helpful to develop the immunological assay method of these proteins.%昆虫病原发光杆菌属(Photorhabdus)细菌产生2种胞内晶体蛋白CipA和CipB,为其共生线虫的生长发育提供营养.基于Cip蛋白已在大肠杆茵原核表达系统中得到稳定且高水平表达,以纯化的重组Cip蛋白为抗原,免疫新西兰大耳兔制备多克隆抗体,ELISA测定2种蛋白抗血清效价,均可达到1∶6400.Western blot分析显示,制备的抗体均可特异识别发光杆菌的胞内晶体蛋白.Cip蛋白多克隆抗体的制备为建立该类蛋白的免疫学检测方法奠定了基础.

  9. Studies on the Renaturation with Simultaneous Purification of Recombinant Human Proinsulin with Unit of Simultaneous Renaturation and Purification of Protein in Semi-preparative Scale

    Institute of Scientific and Technical Information of China (English)

    Quan BAI; Yu KONG; Xin Du GENG

    2003-01-01

    The renaturation and purification of recombinant human proinsulin (rh-proinsulin) expressed in E. Coli with the unit of simultaneous renaturation and purification of protein (USRPP) in semi-preparative scale was studied. The result shows that rh-proinsulin extracted with 8.0 mol/L urea can be renatured and purified simultaneously in 45 minutes with the USRPP (10×50 mm ID). The purity of rh-proinsulin was found to be more than 90% and the mass recovery to be more than 80%. The renaturation effect of rh-proinsulin with the USRPP was tested by enzyme cleavage for obtaining insulin. In addition, the result was further confirmed with RPLC, SDS-PAGE electrophoresis, and MALDI-TOF, respectively.

  10. Evaluation of protein structural changes and water mobility in chicken liver paste batters prepared with plant oil substituting pork back-fat combined with pre-emulsification.

    Science.gov (United States)

    Xiong, Guoyuan; Han, Minyi; Kang, Zhuangli; Zhao, Yingying; Xu, Xinglian; Zhu, Yingying

    2016-04-01

    Protein structural changes and water mobility properties in chicken liver paste batters prepared with plant oil (sunflower and canola oil combinations) substituting 0-40% pork back-fat combined with pre-emulsification were studied by Raman spectroscopy and low-field nuclear magnetic resonance (NMR). Results showed that pre-emulsifying back-fat and plant oil, including substituting higher than 20% back-fat with plant oil increased the water- and fat-binding (pproperties, formed more even and fine microstructures, and gradually decreased the NMR relaxation times (T21a, T21b and T22), which was related to the lower fluid losses in chicken liver paste batters. Raman spectroscopy revealed that compared with a control, there was a decrease (poil combined with pre-emulsification. Pre-emulsification and plant oil substitution changed tryptophan and tyrosine doublet hydrophobic residues in chicken liver paste batters.

  11. Preparation of a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine) by surface-initiated atom transfer radical polymerization and analysis of protein adsorption resistance.

    Science.gov (United States)

    Inoue, Yuuki; Onodera, Yuya; Ishihara, Kazuhiko

    2016-05-01

    The purpose of this study was to prepare a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) and assess its resistance to protein adsorption from the dissolved state of poly(MPC) chains in an aqueous condition. The thick poly(MPC) brush layer was prepared through the surface-initiated atom transfer radical polymerization (SI-ATRP) of MPC with a free initiator from an initiator-immobilized substrate at given [Monomer]/[Free initiator] ratios. The ellipsometric thickness of the poly(MPC) brush layers could be controlled by the polymerization degree of the poly(MPC) chains. The thickness of the poly(MPC) brush layer in an aqueous medium was larger than that in air, and this tendency became clearer when the polymerization degree of the poly(MPC) increased. The maximum thickness of the poly(MPC) brush layer in an aqueous medium was around 110 nm. The static air contact angle of the poly(MPC) brush layer in water indicated a reasonably hydrophilic nature, which was independent of the thickness of the poly(MPC) brush layer at the surface. This result occurred because the hydrated state of the poly(MPC) chains is not influenced by the environment surrounding them. Finally, as measured with a quartz crystal microbalance, the amount of protein adsorbed from a fetal bovine serum solution (10% in phosphate-buffered saline) on the original substrate was 420 ng/cm(2). However, the poly(MPC) brush layer reduced this value dramatically to less than 50 ng/cm(2). This effect was independent of the thickness of the poly(MPC) brush layer for thicknesses between 20 nm and about 110 nm. These results indicated that the surface covered with a poly(MPC) brush layer is a promising platform to avoid biofouling and could also be applied to analyze the reactions of biological molecules with a high signal/noise ratio.

  12. The preparation and culture of washed human sperm:A comparison of a suite of protein-free media with media containing human serum albumin

    Institute of Scientific and Technical Information of China (English)

    Kelli L Peirce; Peter Roberts; Jaffar Ali; Phillip Matson

    2015-01-01

    Objective:To compare two suites of culture media (one with HSA and one protein-free (PF) supplemented with methylcellulose) for washing human sperm in IVF.Methods:Semen samples (n=41) underwent parallel density gradient preparation using PF or HSA-supplemented culture medium and subsequent yield, survival, morphology and motility were compared.Results:The PF medium resulted in a significantly higher sperm yield (P<0.0001), but similar sperm morphology (P=0.822) and 24-hour survival (P=0.11). There was, however, a lower percentage of progressively motile sperm (P<0.0001) and a higher proportion of sperm demonstrating non-progressive motility (P<0.0001) in the PF medium when observed on a Makler Chamber, apparently an artefact as a similar sperm motility index was measured using a Sperm Quality Analyser (P=0.83). Attachment of sperm in PF medium to the glass chamber reduced with time and any differences had disappeared after 6 minutes on the counting chamber.Conclusion:These results support the use of PF media supplemented with methylcellulose as an alternative to HSA, although a modification to the manufacturer’s protocol of 6-minutes pre-incubation before assessing sperm motility must be used. Further studies should investigate the function of such sperm prepared in PF medium.

  13. EFFECTS OF STATINS AND OTHER BIOLOGICAL PREPARATIONS UPON ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASES IN PATIENTS WITH RHEUMATOID ARTHRITIS

    Directory of Open Access Journals (Sweden)

    I. V. Shirinsky

    2009-01-01

    Full Text Available Abstract. In this study, we evaluated effects of statins and other biological preparations upon spontaneous and stimulated activation of МАРК p38 and ERK1/2 in monocytes from the patients with rheumatoid arthritis (RA. We used peripheral blood mononuclear cells (PBMC from RA patients and healthy donors. PBMC were cultured in presence of 0, 0.1, 1 or 10 мM mevastatin, 10 мg/ml IL-1 receptor antagonist (IL-1Ra, 5 мg/ml infliximab, and 5 мg/ml soluble pegylated p55 TNF-receptor (r-met-Hu-sTNF-RI. To study the mechanisms of mevastatin effects upon МАРК p38 and ERK1/2 activities, L-mevalonate was added to the cultures. The cells were stained with anti-phospho-MAPK p38, or anti-phospho-ERK1/2, and analyzed with flow cytometry. We have shown that IL-1Ra and r-met-Hu-sTNF-RI inhibited spontaneous MAPK р38 activation. Mevastatin reduced spontaneous MAPK p38 and ERK1/2 phosphorylation. Mevastatininduced suppression of MAPK p38 and ERK1/2 activation was not dose-dependent. L-mevalonate completely prevented mevastatin-induced reduction of MAPK р38 phosphorylation and partially reversed inhibition of МАРК ERK1/2. In conclusion, decrease in MAPK activation represents a common mechanism of anti-inflammatory effects exerted by statins and some other biologicals.

  14. The effect of the application of protein and cellulose preparations as iodine carriers on stability of thiamine in processed meats

    Directory of Open Access Journals (Sweden)

    Krystyna Szymandera-Buszka

    2011-03-01

    Full Text Available   Fortification of processed meat with iodised table salt was shown to increase thiamine losses, both during thermal processing and storage. Taking into consideration the fact, as well as the recommendation for reduction of consumption of table salt, alternative iodine carriers need to be searched for. Thus the aim of the study was to determine the effect of soy protein isolate (SPI and wheat fibre (WF as iodine salts’ (potassium iodide and iodate carriers on thiamine stability in selected processed meats (steamed meatballs and burgers. The results were compared to the effect of iodised table salt. The highest thiamine losses were found in the presence of iodised table salt, both in the form of iodide and iodate. The application of iodised WF and SPI significantly limited thiamine losses in the course of steaming. It also made possible to reduce thiamine losses during storage in relation to iodised table salt. It was found that the application of WF and SPI as iodine carriers facilitates increased stability of thiamine in relation to table salt during processing and storage of the meat dishes.  

  15. Optimization of a biolistic transformation system for transfer of antifreeze gene KN2 and the bar herbicide resistance gene in common wheat.

    Science.gov (United States)

    Cai, L; Sun, D F; Sun, G L

    2014-04-30

    We studied the effects of different media for callus induction and differentiation, and pre-culture period of immature wheat embryo culture on biolistic transformation efficiency for including antifreeze gene KN2 and bar conferring resistance to the herbicide bialaphos. The percentage of plantlets generated from induction and differentiation media without Cu2+ was lower than those cultured on differentiation media with Cu2+ (71.15%) or induction media with Cu2+ (68.45%) and both induction and differentiation media with Cu2+ (52.17%). The combinations of Nor medium for callus induction and Cu2+ medium for regeneration, and Cu2+ medium for induction and R medium for regeneration were superior for biolistic transformation. The calli induced on Cu2+ medium and pre-cultured for 4 d before biolistic transformation, and cultured on R medium after biolistic transformation produced the highest percentage (65%) of transgenic plantlets with the KN2 gene. Overall, about 50% plantlets regenerated from calli pre-cultured 4d before bombardment carried the KN2 gene; 44.7% of the plantlets carried the bar gene, which was higher than for any other treatment, followed by pre-culture 1d with 31.43% transformation rate for the KN2 gene and 20% transformation rate for the bar gene.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-10-01

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

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

    Science.gov (United States)

    Huang, Zhan-Yun; Chen, Min; Pan, Shi-Rong; Chen, Di-Hu

    2010-10-01

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

  18. Pressure-assisted dissociation and degradation of "proteinase K-resistant" fibrils prepared by seeding with scrapie-infected hamster prion protein.

    Science.gov (United States)

    Akasaka, Kazuyuki; Maeno, Akihiro; Murayama, Taichi; Tachibana, Hideki; Fujita, Yuzo; Yamanaka, Hitoki; Nishida, Noriyuki; Atarashi, Ryuichiro

    2014-01-01

    The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP(C), into a fibrous pathogenic form, PrP(Sc), which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP(Sc) into PrP(C) is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP(Sc) from PrP(C) eventually in vivo. Here we show that, by applying pressures at kbar range, the "proteinase K-resistant" fibrils (rHaPrP(res)) prepared from hamster prion protein (rHaPrP [23-231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP(res) fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP(Sc) from PrP(C) is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP(Sc) from various systems of pathogenic concern.

  19. 月见草籽粕分离蛋白的制备%Study on preparation of protein isolation from primrose meal

    Institute of Scientific and Technical Information of China (English)

    马涛; 吕品

    2009-01-01

    Based on primrose meal, the process conditions and functional properties of protein isolated was prepared on alkali-extraction and acid-isolation.Through orthogonal tests,the best conditions of extraction were: the pH 12,the temperature 45℃, the times of extraction 3, the proportion of the material to liquid 1:12, 1:10, 1:8 respectively and protein deposited at pH6.0, pH3.8.In the product gained through spray drying process the content were 75.5%.%以月见草冷榨浸出粕为材料,初步探索碱提酸沉法制备月见草籽粕分离蛋白的工艺条件及其功能特性通过正交实验得到提取的最佳工艺条件为:pH12,温度45℃,提取3次,料液比分别1:12、1:10、1:8;调pH6.0、3.8二次沉淀,喷雾干燥后产品的粗蛋白含量达75.5%.

  20. 碎米蛋白可食用膜制备工艺的优化%Optimization for preparation of broken rice protein edible film

    Institute of Scientific and Technical Information of China (English)

    艾丹; 胡秋林; 王双; 高俊

    2013-01-01

    With broken rice protein as raw material to prepare edible films, the effects of pH, protein concentration, glycerol addition and the reaction temperature had been studied individually. Based on the single factor experiment, the method of response surface analysis(RSA) was adopted to optimize the preparing technology. The results were as that:pHll. 5,protein concentration of 5 g/(100 ml), glycerol addition of 2. 09 g/(100 ml), reaction temperature of 87℃. Under these conditions,for the films,the tension strength was 2. 47 MPa, the elongation at break was 175.68%, the water vapor permeability was 4. 84 g ·mm/(kPa ·d ·m2), the transparency was 86. 1 % , the solubility was 27. 88% and the synthesizing evaluation score was 78. 44. The difference was 0. 24% with the predicted value(78. 63).%以碎米蛋白为原材料制备可食用膜,分别探讨了膜液pH值、碎米蛋白溶液浓度、甘油添加量和热反应温度对蛋白膜性能的影响.在单因素实验的基础上,利用响应面法对碎米蛋白可食用膜的制备工艺进行优化,得到最佳工艺条件为:pH11.5、碎米蛋白溶液浓度为5 g/(100ml)、甘油添加量2.09 g/(100ml)、温度87℃.该条件下,蛋白膜的抗拉强度为2.47MPa,断裂伸长率为175.68%,水蒸气透过率为4.84 g·mm/(kPa·d·m2),透光率为86.1%,溶解性为27.88%,综合评分为78.44,与模型预测值(78.63)相差0.24%.

  1. 豇豆籽蛋白的制备及理化性质%Preparation and physi-chemical properties of cowpea seed protein

    Institute of Scientific and Technical Information of China (English)

    李安林; 熊双丽; 黄妮

    2011-01-01

    以豇豆籽为原料,采用碱萃取和酸沉淀方法提取分离得到豇豆籽分离蛋白,并采用Osborne法分级制备各类蛋白质。结果发现,豇豆籽分离蛋白必需氨基酸为30.26%,疏水性氨基酸的摩尔比为38.10%,疏水性值为5.01kJ/mol,大于大豆蛋白。其蛋白中清蛋白含量最高(67.56%),其次是谷蛋白(15.32%)和醇溶蛋白(6.65%),球蛋白含量最低(5.43%)。清蛋白和谷蛋白具有较好的持水性质,清蛋白和球蛋白具有较好的持油性。SDS分析各类蛋白质的相对分子质量均在100000~10000Da之间。%The cowpea seed isolated protein was prepared by extraction with alkali solution(pH8.0)and precipitated with hydrochloric acid.It was also fractionated according to Osborne methods.The ratio of total essential amino acids and hydrophobic amino acid in isolated protein were 30.26% and 38.10% respectively.The hydrophobicity values(Q value)of the isolated protein was 5.01kJ/mol,which was higher than that of soybean.In the cowpea seed protein,albumin was the predominant protein fraction(67.56%)followed by glutelin(15.32%),prolamin(6.65%)and globulin(5.43%).Albumin and glutelin had better water-holding capacities.Albumin and globulin had better oil-holding capacities.The results of SDS-PAGE exhibited that the molecular weight of the fractions were between 100000Da and 10000Da.

  2. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes.

    Science.gov (United States)

    Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L; Toyoda, Hiroo

    2011-12-01

    Arsenic trioxide (arsenite, As(III)) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As(III) on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As(III) on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As(III)-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As(III) were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As(III) than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As(III) in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As(III)-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As(III) cytotoxicity between these cells.

  3. Cloning of fish enzymes and other fish protein genes.

    Science.gov (United States)

    Macouzet, M; Simpson, B K; Lee, B H

    1999-01-01

    Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.

  4. Antifreeze effect and mechanism of sodium poly-γ-glutamate on backers' yeast cells%γ-聚谷氨酸钠对面包酵母的抗冻作用及其机理

    Institute of Scientific and Technical Information of China (English)

    时晓剑; 缪冶炼; 卫昊; 徐虹; 王冀宁

    2012-01-01

    γ-聚谷氨酸钠(γ—PGA钠)是由Y-聚谷氨酸与钠离子结合而成的水溶性物质,可以食用,无色无味。γ—PGA钠作为面包酵母抗冻剂,具有较强的实用性。在0.03~0.40℃/min的冷冻速率、-7~-60℃的冻藏温度条件下,探讨了γ—PGA钠对面包酵母的抗冻作用及其机理。实验结果表明,酵母细胞存活率在冻藏初期逐渐下降,6d后趋于稳定。γ—PGA钠对酵母的抗冻作用明显高于葡萄糖和谷氨酸。γ—PGA钠抑制了冷冻和冷藏过程中大冰晶的产生、以及冰晶的重结晶。在冷冻速率大于0.27℃/min、冻藏温度低于-30℃、冻藏时间6f1的条件下,添加1%的γ—PGA钠使酵母细胞存活率从无γ—PGA钠时的36.5%上升到67.5%。γ—PGA钠、葡萄糖和谷氨酸的抗冻活性分别为2.48、2.03和1.42。细胞存活率(η,%)随抗冻活性(Aa,-)的增加呈线性上升。γ—PGA钠具有较大抗冻活性的机理是,γ—PGA钠的解离度较大,并且Na+能够固定较多的水分子。%Sodium poly-γ-glutamate (Na-γ-PGA), a water-soluble polyamide composed of poly-γ- glutamic acid and sodium ion, is edible, tasteless, colorless and odorless. It is considered as a potential cryoprotectant for backers' yeast. In the present study, the antifreeze effect of Na-γ-PGA on bakers' yeast cells was investigated in the freezing rate range of 0.03-0.40 ℃/min and the storage temperature range of -7--60 ℃. The mechanism of the antifreeze effect was also discussed. The experimental results showed that the survival ratio of yeast cells decreased at the beginning, and was constant after 6 dduring frozen storage. Na-γ-PGA had greater antifreeze effect on the yeast cells than glutamic acid and glucose. It inhibited both the generation of big-size ice crystals during freezing and the recrystallization of ice crystals during freezing storage. Under the condition of a freezing rate at 0

  5. Effects of an exogenous enzyme preparation on microbial protein synthesis, enzyme activity and attachment to feed in the Rumen Simulation Technique (Rusitec).

    Science.gov (United States)

    Wang, Y; McAllister, T A; Rode, L M; Beauchemin, K A; Morgavi, D P; Nsereko, V L; Iwaasa, A D; Yang, W

    2001-03-01

    The effects of an exogenous enzyme preparation, the application method and feed type on ruminal fermentation and microbial protein synthesis were investigated using the rumen simulation technique (Rusitec). Steam-rolled barley grain and chopped alfalfa hay were sprayed with water (control, C), an enzyme preparation with a predominant xylanase activity (EF), or autoclaved enzyme (AEF) 24 h prior to feeding, or the enzyme was supplied in the buffer infused into the Rusitec (EI). Microbial N incorporation was measured using (15NH4)2SO4 in the buffer. Spent feed bags were pummelled mechanically in buffer to segregate the feed particle-associated (FPA) and feed particle-bound (FPB) bacterial fractions. Enzymes applied to feed reduced neutral-detergent fibre content, and increased the concentration of reducing sugars in barley grain, but not alfalfa hay. Ruminal cellulolytic bacteria were more numerous with EF than with C. Disappearance of DM from barley grain was higher with EF than with C, but alfalfa was unaffected by EF. Treatment EF increased incorporation of 15N into FPA and FPB fractions at 24 and 48 h. In contrast, AEF reduced the 24 h values, relative to C; AEF and C were similar at 48 h. Infused enzyme (EI) did not affect 15N incorporation. Xylanase activity in effluent was increased by EF and EI, compared to C, but not by AEF. Xylanase activity in FPA was higher at 48 h than at 24 h with all treatments; it was higher with EF than C at 24 and 48 h, but was not altered by AEF or EI. Applying enzymes onto feeds before feeding was more effective than dosing directly into the artificial rumen for increasing ruminal fibrolytic activity.

  6. Preparation of silver carp protein powder by enzymatic hydrolysis and spray drying%酶法制备白鲢鱼蛋白粉的研究

    Institute of Scientific and Technical Information of China (English)

    王丽; 姜启兴; 许艳顺; 夏文水

    2013-01-01

    Silver carp protein powder was prepared by enzymatic hydrolysis and spray drying.The effect on different temperature,papain dosage,hydrolysis time,substrate concentration and pH on the degree of hydrolysis of silver carp protein were determined.And the effects of the conditions of spray drying on the physicochemical properties and water-soluble properties of the protein powder were studied.The results showed that the optimum conditions of hydrolysis by papain were temperature 50℃,enzyme dosages 4000U/g protein,hydrolysis pH6.5,hydrolysis time 4h.The degree of hydrolysis was as high as 29.82%.The hydrolysate was concentrated to 25%~30% and it was dried to protein powder by spray drying which operating conditions were inlet air temperature t70~180℃ and outlet air temperature 80~90℃.The content of which the relative molecular weight were below 1000 accounted for 90.90%.The solubility of the fish protein powder was 98.52%.The product with high nutritional value and watersoluble properties were thus potential protein functional food ingredients.%对白鲢鱼肉进行酶水解经喷雾干燥以制备水溶性高的鱼蛋白粉.研究了木瓜蛋白酶的添加量、温度、时间、底物浓度和pH水解条件对鱼蛋白水解度的影响以及喷雾干燥条件对蛋白粉物性和水溶性的影响.结果表明:木瓜蛋白酶对鲢鱼蛋白的最适水解条件是温度50℃、加酶量4000U/g蛋白质、底物浓度为5.5%、pH6.5、时间4h,水解度达到29.82%;该水解液经浓缩至浓度25% ~30%时,通过控制进风温度170~180℃、出风温度80~90℃用喷雾干燥得到鱼蛋白水解产物,相对分子量在1000以下组分占90.90%,溶解度达98.52%,是一种营养价值高和溶解好的蛋白质功能性食品配料.

  7. Preparation of Protein Hydrolysates with Waste Brewer's Yeast%啤酒废酵母蛋白水解物的制取工艺

    Institute of Scientific and Technical Information of China (English)

    张晓鸣; 袁信华; 徐柔; 章克昌

    2001-01-01

    Protein hydrolysates were prepared by enzymatic hydrolysis ofpretreated waste brewer's yeast. The hydrolysis parameters were specified by response surface analysis. The Parameters are as follows: initial yeast concentration 15%, temperature 55~60 ℃, the β-glucanase-substrate ratio 15~20 U/g, and pH 6.6~6.8. When hydrolysed for 7 hour, more than 82% of the Kjeldahl protein in yeast was extracted. Measured by high-performance liquid chromatography(HPLC), 65.26% of the hydrolysates had molecular weights of 210~635 Dalton, which corresponded to dipeptides and tripeptides.%利用啤酒废酵母资源,通过破壁预处理与复合酶降解相结合的工艺,制取特定相对分子质量范围的蛋白水解物.响应面分析结果表明,在温度55~60℃,pH6.6~6.8,β-葡聚糖酶用量15~20U/g(以酵母计)的条件下,有最高的肽提取率.酶解7h,蛋白质提取率大于82%,经HPLC测定,水解产物中65%组分的相对分子质量在210~635之间,处于二肽和三肽相对分子质量范围.

  8. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides.

    Science.gov (United States)

    Mitchell, Daniel E; Gibson, Matthew I

    2015-10-12

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs.

  9. Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-△SHP-1 Antibodies

    Institute of Scientific and Technical Information of China (English)

    LI Wan-nan; ZHUANG Yan; LI He; SUN Ying; FU Yao; WU Xiao-xia; ZHAO Zhi-zhuang; FU Xue-qi

    2008-01-01

    This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated △SHP-1) and the preparation of its polyelonal antibodies.A cDNA fragment encoding △SHP-1 was amplified by PCR and then cloned into the pT7 expression vector.The recombinant pT7-△SHP-1 plasmid was used to transform Rosetta(DE3) E.coll cells.△SHP-1 was distributed in the exclusion body of E.coll cell extracts and was purified through a two-column chromatographic procedure.The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns.It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases.To generate polyclonal anti-△SHP-1 antibodies,purified recombinant △SHP-1 was used to immunize a rabbit.The resultant anti-serum was subjected to purification on △SHP-1 antigen affinity chromatography.The purified polyclonal antibody displayed a high sensitivity and specificity toward △SHP-1.This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.

  10. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshino, Yuta [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Yuan, Bo, E-mail: yuanbo@toyaku.ac.jp [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Kaise, Toshikazu [Laboratory of Environmental Chemodynamics, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Takeichi, Makoto [Yoneyama Maternity Hospital, 2-12 Shin-machi, Hachioji, Tokyo 192-0065 (Japan); Tanaka, Sachiko; Hirano, Toshihiko [Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Kroetz, Deanna L. [Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Toyoda, Hiroo [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)

    2011-12-15

    Arsenic trioxide (arsenite, As{sup III}) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As{sup III} on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As{sup III} on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As{sup III}-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As{sup III} were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As{sup III} than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As{sup III} in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As{sup III}-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As{sup III} cytotoxicity between these cells. -- Highlights: Black-Right-Pointing-Pointer Examination of effect of As{sup III} on primary cultured chorion (C) and amnion

  11. Anti-freezing System's Status and Improvement of Sluice Gate in Huangbizhuang Reservoir%黄壁庄水库闸门防冻系统的状况及改进

    Institute of Scientific and Technical Information of China (English)

    郭文宇

    2013-01-01

    Itcombinedwiththeoperationpracticecomparedthreekindsofanti-freezingmeasures(diggingiceditch,anti-freezingsystemofsub-mersible pump, and anti-freezing system of air compressor) of the sluice gate in winter. It puts forward the improvement measures and practica-bility of anti-freezing system of submersible pump in Huangbizhuang reservoir, and puts forward some ideas for the further improvement.%结合黄壁庄水库冬季闸门运行实践,对闸门的三种防冰冻措施(开凿冰沟、潜水泵防冰冻系统、压缩空气泡防冰冻系统)进行了比较。结合潜水泵式防冰冻系统在黄壁庄水库的应用实践,提出了此系统对于北方水库的实用性改进措施,并根据实际应用中积累的经验提出了进一步的改进设想。

  12. 干混抗冻混凝土配合比设计及抗冻性能研究%Dry-mixed antifreeze concretes design of mixing proportion and performance of winter

    Institute of Scientific and Technical Information of China (English)

    罗憨; 李延和; 夏永清; 范贤玉

    2014-01-01

    随着建筑市场的飞速发展,混凝土冬季施工在所难免。且我国北方地区气候寒冷,普通商品混凝土不能满足建筑工程要求。介绍一种特殊的混凝土---干混抗冻混凝土,通过模拟冬季施工环境,调整混凝土配合比,在满足混凝土强度要求前提下,找出最适宜的干混抗冻混凝土配合比。%With the rapid development of the construction market,concrete construction in winter is inevitable.Since the weather is cold,ordinary concrete construction can not meet the requirements.This paper describes a special concrete-dry-mixed concrete antifreeze. By simulating winter construction environment,adjust the concrete mix,concrete strength to meet the requirements of the premise,we want to identify the most suitable dry-mixed antifreeze concrete mix.

  13. Influence of frozen dough processing conditions on the activity of antifreeze yeast in fermented dough%冷冻工艺条件对发酵面团中抗冻酵母活性的影响

    Institute of Scientific and Technical Information of China (English)

    周璐; 孙冰玉; 石彦国; 梁金钟

    2011-01-01

    研究了冷冻面团的冷冻终结温度、冻藏温度、冻藏时间对抗冻酵母活性的影响,得出最有利于抗冻酵母活性保持的冷冻工艺条件:冷冻终结温度-18℃,冻藏温度应与冷冻终结温度保持一致,贮存时间不超过30d为宜。%The effects of the end of freezing temperature,freeze preservation temperature and freeze preservation time of frozen dough on the activity of antifreeze yeast were researched.The most beneficial freezing process conditions to keep the activity of antifreeze yeast were found.The end of freezing temperature was-18℃,freeze preservation temperature should be consistent with the end of the freezing temperature.And it's better to keep the freeze preservation time less than 30 days.

  14. 防冻剂对混凝土引气剂气泡稳定性能影响研究∗%Impact study of anti-freezing agent on bubble stability performance of concrete air-entraining agent

    Institute of Scientific and Technical Information of China (English)

    张向东; 李庆文; 李广华; 李桂秀

    2015-01-01

    为探究防冻剂对混凝土引气剂气泡稳定性能的影响,考虑4种防冻剂及不同掺量等敏感性因素进行水泥稀浆摇泡实验,分析其对引气剂起泡与稳泡能力影响及作用机理。结果表明,建立摇泡实验的气泡体积衰减指数模型,其参数能表征不同引气剂起泡与稳泡能力;硝酸钙对皂苷类引气剂稳泡能力有正作用,防冻剂对其起泡能力均有负作用;乙二醇对苯磺酸盐类引气剂起泡能力有正作用,对其稳泡能力有正作用,表现为亚硝酸钙>硝酸钙>乙二醇;4种防冻剂对掺皂苷类引气剂的新拌混凝土含气量均有负影响,其显著性为乙二醇最强,亚硝酸钙最弱,钙盐类防冻剂对掺苯磺酸盐类引气剂均有负作用,乙二醇对其有正作用。%In order to explore the influence of anti-freezing agent on bubble stability performance of concrete air-entraining agent,the cement slurry bubble test was carried out considering sensitivity factors which was four kinds of anti-freezing agents and different dosages,and analyzed its effects on foaming ability,stabilizing foam ability and mechanism.The results revealed that bubble volume attenuation index model of shake bubble test was set up,which parameters characterized the foaming ability and stabilizing foam ability of different air-en-training agents.Calcium nitrate had a positive effect on stabilizing foam ability of the saponins air-entraining agent,and anti-freezing agents had a negative effect on it.Ethylene glycol had a positive effect on foaming abili-ty of the benzene sulfonate air-entraining agent,the significance of improving the stabilizing foam ability was calcium nitrite>calcium nitrate>ethylene glycol.Four kinds of anti-freezing agents all had a negative impact on the air content of fresh concrete mixed with the saponins air-entraining agent,the most significant of it was ethylene glycol,and the least significant was calcium nitrite

  15. Preparation of Antihypertensive Peptides from Soy Protein Isolate%大豆分离蛋白ACE活性抑制肽的研究

    Institute of Scientific and Technical Information of China (English)

    王喜波; 迟玉杰; 孙波

    2009-01-01

    The pretreatment conditions of soy protein isolate (SPI) hydrolysis were tested,and alcalase was selected from six enzymes for the SPI hydrolysis to prepare antihypertensive peptides with high inhibitory activity for angiotensin converting enzyme (ACE).Orthogonal design of four factors three levels was taken to optimize the hydrolysis conditions of SPI with alcalase protease.Finally,eight kinds of macroporous adsorbing resins were compared for purification of the produced Sntihypertensive peptides,and D3520 was selected.Results: After the purification,the recovery rate of antihypertensive peptides is more than 90.6%,and the ACE inhibitory activity of the product is 84.1%.The result of HPLC shows that the relative molecular weights of the antihypertensive peptides mainly distribute from 200 to 800 ku.%研究了预处理条件对大豆分离蛋白水解效果的影响,热处理与微波辅助处理相结合能显著提高蛋白酶的水解效率.比较了6种酶解大豆蛋白产物的ACE抑制活性,选择碱性蛋白酶为最佳水解用酶,并优化了酶解条件.研究了8种大孔树脂对水解产物精制效果,结果表明:经D3520型大孔吸附树脂精制后,水解产物脱盐率89.4%,肽回收率90.6%,ACE抑制活性达84.1%.经HPLC法测定,大豆分离蛋白ACE活性抑制肽混合物分子质量主要分布在200~800 ku之间.

  16. Effects of a type I antifreeze protein (AFP) on the melting of frozen AFP and AFP+solute aqueous solutions studied by NMR microimaging experiment.

    Science.gov (United States)

    Ba, Yong; Mao, Yougang; Galdino, Luiz; Günsen, Zorigoo

    2013-01-01

    The effects of a type I AFP on the bulk melting of frozen AFP solutions and frozen AFP+solute solutions were studied through an NMR microimaging experiment. The solutes studied include sodium chloride and glucose and the amino acids alanine, threonine, arginine, and aspartic acid. We found that the AFP is able to induce the bulk melting of the frozen AFP solutions at temperatures lower than 0 °C and can also keep the ice melted at higher temperatures in the AFP+solute solutions than those in the corresponding solute solutions. The latter shows that the ice phases were in super-heated states in the frozen AFP+solute solutions. We have tried to understand the first experimental phenomenon via the recent theoretical prediction that type I AFP can induce the local melting of ice upon adsorption to ice surfaces. The latter experimental phenomenon was explained with the hypothesis that the adsorption of AFP to ice surfaces introduces a less hydrophilic water-AFP-ice interfacial region, which repels the ionic/hydrophilic solutes. Thus, this interfacial region formed an intermediate chemical potential layer between the water phase and the ice phase, which prevented the transfer of water from the ice phase to the water phase. We have also attempted to understand the significance of the observed melting phenomena to the survival of organisms that express AFPs over cold winters.

  17. 抗冷冻蛋白基因(afp)转化大豆的研究%Transformation of Soybean with Antifreeze Protein Gene

    Institute of Scientific and Technical Information of China (English)

    陈颖珊; 陈晟; 赵印华; 郭丽琼; 林俊芳

    2012-01-01

    目的:通过农杆菌介导法遗传转化大豆.方法:通过热激法将质粒pCAAFP66导入根癌农杆菌菌株EHA105中获得含有抗冷冻蛋白基因(afp)及除草剂抗性筛选标记基因(bar)的农杆菌工程菌株;以大豆品种华春6号和马祖1号种子的下胚轴为外植体,经过农杆菌介导将抗冷冻蛋白基因导人大豆基因组中,在含有除草剂草丁膦(PPT)的培养基中筛选、并经过PCR鉴定获得大豆转化植株.结果:PPT的最佳筛选浓度为1.0mg/L,华春6号和马祖1号的阳性植株数分别为6株和2株,转化效率分别为3.70%和0.94%.结论:不同基因型大豆的转化率存在差异,抗冷冻蛋白基因成功遗传转化进大豆细胞中.

  18. Probing protein-sugar interactions.

    Science.gov (United States)

    Ebel, C; Eisenberg, H; Ghirlando, R

    2000-01-01

    We have investigated the partial specific volumes (2) (ml/g), hydration, and cosolvent interactions of rabbit muscle aldolase by equilibrium sedimentation in the analytical ultracentrifuge and by direct density increment (partial differential/partial differentialc(2))(mu) measurements over a range of sugar concentrations and temperature. In a series of sugars increasing in size, glucose, sucrose, raffinose, and alpha-cyclodextrin, (partial differential/ partial differentialc(2))(mu) decreases linearly with the solvent density rho(0). These sugar cosolvents do not interact with the protein; however, the interaction parameter B(1) (g water/g protein) mildly increases with increasing sugar size. The experimental B(1) values are smaller than values calculated by excluded volume (rolling ball) considerations. B(1) relates to hydration in this and in other instances studied. It decreases with increasing temperature, leading to an increase in (2) due to reduced water of hydration electrostriction. The density increments (partial differential/ partial differentialc(2))(mu), however, decrease in concave up form in the case of glycerol and in concave down form for trehalose, leading to more complex behavior in the case of carbohydrates playing a biological role as osmolytes and antifreeze agents. A critical discussion, based on the thermodynamics of multicomponent solutions, is presented.

  19. Preparation and application of sericin protein modifier on polyester fabrics%丝胶改性剂的制备及对涤纶的改性研究

    Institute of Scientific and Technical Information of China (English)

    戴杰; 郭晓玲; 周青青; 本德萍; 崔贞; 申国栋

    2014-01-01

    以环氧氯丙烷、丝胶蛋白为原料,在碱性条件下制备新型丝胶改性剂.丝胶改性剂与涤纶织物上的羧基发生酯化反应并固着于涤纶织物上,改善涤纶织物的抗静电性、吸湿性等性能.对整理后涤纶织物的抗静电性、透气性、毛细效应、接触角、强力等进行测试.结果表明:改性涤纶织物的抗静电性得到改善,表面电荷由原来的0.036μC变为0.001μC;多次水洗后,涤纶织物表面电荷基本稳定,说明丝胶改性剂与涤纶织物上的羧基发生酯化反应,并固着在涤纶织物上;透气性由原来的19.43 mm/s变为46.29 mm/s.%A novel sericin modifier was prepared using epichlorohydrin and sericin protein as raw materi-als under alkaline condition. The sericin modifier reacted with the carboxyl groups on polyester fabric through esterification reaction and fixed onto the fabrics, improving the antistatic and hygroscopic properties of polyes-ter fabrics. The antistatic, gas permeability, capil ary effect, contact angle and strength of finished polyester fab-ric were tested. The results showed that the antistatic property of modified polyester fabric was improved and the surface charge changed from 0.036 μC to 0.001 μC. After repeated launderings, the surface charge on the finished polyester remained steady showing the esterification reaction between the sericin modifier with the carboxyl groups on polyester fabrics. The gas permeability also increased from 19.43 mm/s to 46.29 mm/s.

  20. 素混凝土抗冻性能试验研究%Experimental investigation on anti-freeze behavior of plain concrete

    Institute of Scientific and Technical Information of China (English)

    方从启; 张俊萌; 段桂珍

    2014-01-01

    In order to further study the antifreeze performance difference of confined concrete and plain concrete,in confined concrete model for reference,making the same ratio of plain concrete cubic block and under the same conditions were also tested under freeze-thaw cycle of different numbers as 0,20,40,60,100 and 120 times,As the second part,mainly analyzed the compressive strength of plain concrete under freeze-thaw test,mass loss of samples and water absorption performance changing with the number of freezing and thawing,and establish the freeze-thaw damage prediction model of concrete compressive strength and mass loss ratio.Experiment showed that with the increase of freeze-thaw cycles,the compressive strength of plain concrete decreased parabola,drying quality and full water mass linearly decreased,while water absorption decreased by double line.From the aspects of material level,fly ash had a greater influence on the loss of concrete compressive strength,mass loss rate and water absorption,while water cement ratio had little influence on the above indicators.The loss of compressive strength of mixing 15%fly ash concrete was about 20%to 30%higher than mixing no fly ash concrete;Mass loss rate was about 5%to 6%larger than not mixing fly ash ,fly ash is not conducive to the antifreeze performance of concrete.As the same fly ash,the loss of compressive strength,mass and bibulous rate in low water-cement ratio concrete changed lager than high water cement ratio.When using fly ash concrete,it is easy to reduce the water cement ratio.%为了进一步研究约束混凝土和无约束混凝土的抗冻性能差异,以约束混凝土模型为参照,制作相同配合比的素混凝土立方体块并在相同条件下进行0、20、40、60、100、120次的冻融腐蚀试验。主要分析冻融腐蚀下素混凝土试块抗压强度,质量损失以及吸水率等性能随冻融次数的变化规律,并建立素混凝土抗压强度和质量损失冻融损伤预测

  1. An immunocytochemical study of pulpal responses to cavity preparation by laser ablation in rat molars by using antibodies to heat shock protein (Hsp) 25 and class II MHC antigen.

    Science.gov (United States)

    Suzuki, Takeshi; Nomura, Shuichi; Maeda, Takeyasu; Ohshima, Hayato

    2004-03-01

    Initial responses of odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars. In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused the destruction of the odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site. Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation. By 3-5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp. The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity. These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.

  2. 小麦蛋白肽的酶解制备方法及研究进展%Enzymatic Hydrolysis Preparation Methods and the Research Progress of Wheat Protein Peptid

    Institute of Scientific and Technical Information of China (English)

    王明皓; 刘建龙; 史建国; 李亮; 王谦

    2016-01-01

    Multiple bioactivities have been found in the wheat protein peptide. The healthcare function of wheat protein peptide is considered to be good. In addition, it has the advantage of price. Therefore, the market prospects of it in functional food and health care products are deemed to be great. The preparation methods of wheat protein peptide have been changed from acid hydrolysis which is not so good in its effects to enzymatic hydrolysis. The research progress and enzymatic hydrolysis preparation methods to make wheat protein peptide are summed up in the paper.%小麦蛋白肽具有多种生物活性,具备很好的保健功能,且具有成本低的优势,因而在功能性食品领域、保健品领域市场前景广阔。其制备方法已由原本制备效果较差的酸水解逐渐转向酶解。综述国内外几种酶解小麦蛋白制备多肽的方法及研究进展。

  3. 登革病毒4型E蛋白的表达与多克隆抗体的制备%Expression of the envelope protein of Dengue virus type 4 and preparation of the polyclonal antibody

    Institute of Scientific and Technical Information of China (English)

    李竹石; 俞永新; 李玉华; 林华; 杨健; 杨会强; 曾献武; 赵宇; 刘俐; 刘莉娜; 康庄

    2012-01-01

    目的 在原核细胞中表达登革病毒4型E蛋白及E蛋白Ⅲ区,分别以两种蛋白免疫家兔,获得可检测登革病毒的多克隆抗体.方法 将登革病毒4型E蛋白与E蛋白Ⅲ区编码序列克隆到pET-32a(+)质粒中,分别构建两种蛋白的表达载体,以IPTG诱导其在Rosetta细胞中的大量表达,并进行SDS-PAGE检测.分别以两种蛋白免疫家兔,制备出针对E蛋白与E蛋白Ⅲ区的多克隆抗体,并对其进行Western blot鉴定.结果 登革病毒4型E蛋白与E蛋白Ⅲ区在Rosetta细胞中以包涵体的形式大量表达;利用所制备的多克隆抗体对登革病毒进行检测,出现了预期的条带.结论 本研究制备获得的多克隆抗体可用于登革病毒的检测,为登革病毒相关研究奠定了基础.%Objective To express full length (DENV4E) and domain III (DENV4EIII) of the envelope protein of Dengue virus type 4 in procaryotic cells respectively, and to prepare the polyclonal antibody against dengue virus by immunizing rabbits with the expressed proteins. Methods The sequences encoding DENV4E and DENV4EIII proteins were cloned into pET-32a ( + ) plasmids respectively to construct expression vectors. Then DENV4E or DENV4EIII proteins were expressed in Rosetta cells by IPTG induction and detected by SDS-PAGE. Polyclonal antibody against DENV4E or DENV4EIII proteins was obtained by immunizing rabbits with the expressed proteins and was identified by Western blot. Results DENV4E and DENV4EIII proteins were both successfully expressed in Rosetta cells mainly in the form of inclusion body. The prepared polyclonal antibody formed bands as expected when reacting with the dengue virus. Conclusions The polyclonal antibody is prepared and may be used in the detection of dengue virus.

  4. Graphite supported preparation (GSP) of α-cyano-4-hydroxycinnamic acid (CHCA) for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for peptides and proteins.

    Science.gov (United States)

    Gorka, Jan; Bahr, Ute; Karas, Michael

    2012-11-01

    Graphite as MALDI matrix or in combination with other substances has been reported in recent years. Here, we demonstrate that graphite can be used as target coating supporting the crystallization of the α-cyano-4-hydroxycinnamic acid matrix. A conventional dried-droplet preparation of matrix and analyte solution on a graphite-coated metal target leads to a thin, uniform layer of cubic crystals with about 1 μm edge length. Commercially available graphite powder of 1-2 μm particle size is gently wiped over the target using a cotton Q-tip, leading to an ultra-thin, not-visible film. This surface modification considerably improves analysis of peptides and proteins for MALDI MS using conventional dried-droplet preparation. Compared with untreated targets, the signal intensities of standard peptides are up to eight times higher when using the graphite supported crystallization. The relative standard deviation in peak area of angiotensin II for sample amounts between 1 and 50 fmol is reduced to about 15 % compared with 45 % for untreated sample holders. For a quantification of 1 fmol of the peptide using an internal standard the coefficient of variation is reduced to 3.5 % from 8 %. The new graphite supported preparation (GSP) protocol is very simple and does not require any technical nor manual skills. All standard solvents for peptides and proteins can be used.

  5. Cloning, expression and purification of orthologous membrane proteins: a general protocol for preparation of the histidine sensor kinase ETR1 from different species.

    Science.gov (United States)

    Classen, Elisa; Groth, Georg

    2012-03-01

    Orthologous proteins do not necessarily share the same function in all species and those sharing the same function might employ a modified catalytic mechanism. Thus, comparative analysis of homologous or orthologous proteins from different organisms can provide detailed information on the function and the mechanism of an entire protein family. The sensor kinase ETR1 from Arabidopsis thaliana has been well characterized by genetic, physiological and biochemical studies. However, as further model plants are coming into focus for plant hormone research, a general protocol for isolation and purification of orthologous ETR1 proteins seems instrumental for a detailed molecular analysis of this protein family. In this study, we describe the native purification of recombinant ETR1 from Arabidopsis thaliana by mild solubilization with the zwitter-ionic detergent Fos-Choline-14 and single-step purification by immobilized metal ion affinity chromatography. The same protocol was successfully applied for the purification of the orthologous proteins from the moss Physcomitrella patens subsp. patens and the tomato Lycopersicon esculentum. The successful transfer of the purification protocol to proteins of the same family which share sequence identity of 63-80% only suggests that this protocol presents a general purification strategy which is likely to apply also to the purification of other members of the sensor histidine kinase family.

  6. Preparation and characterization of antisera against recombinant E2 protein of bovine viral diarrhea virus%牛病毒性腹泻病毒E2蛋白的多克隆抗体制备及鉴定

    Institute of Scientific and Technical Information of China (English)

    高欲燃; 朱远茂; 康健; 史鸿飞; 李娇; 任宪刚; 冯军科; 于作; 薛飞

    2011-01-01

    To prepare the polyclonal antibody against recombinant E2 protein of bovine viral diarrhea virus (BVDV) in rabbits, E. coli BL21 (DE3) was transformed with the recombinant plasmid pET30a-E2. The recombinant E2 protein was expressed in E. coli after cultivation and induction. The purified recombinant E2 protein could be recognized by specific BVDV antisera in western blot. Then the purified recombinant E2 protein was used as antigen for immunizing rabbit to prepare polyclonal antibody against the recombinant E2 protein. The result of virus nentralization test showed that the titer of the polyclonal antibody to neutralize BVDV was 1:2,048. The polyclonal antibody against the recombinant E2 protein of BVDV also had highly reactivity and specialty in immunofluorescence analysis and western blot. The polyclonal antibody against recombinant E2 protein of BVDV developed in rabbits could be used in detection of BVDV in China and provided a good basis for establishing an ELISA for detecting of E2 protein of BVDV.%为制备牛病毒性腹泻病毒(BVDV)重组E2蛋白的兔源多克隆抗体,本研究利用表达BVDV E2蛋白的重组质粒pET30a-E2转化E.coli BL21(DE3),经诱导表达获得重组E2蛋白.Western blot检测显示纯化蛋白能够与BVDV参考阳性血清反应.以纯化的重组E2蛋白免疫新西兰白兔制备多克隆抗体,病毒中和试验测定其中和效价为1:2 048,间接免疫荧光和western blot试验表明其具有良好的反应性和特异性.本研究制备的BVDV重组E2蛋白兔源多克隆抗体可应用于BVDV的检测,同时为进一步建立检测BVDV E2蛋白的ELISA方法奠定基础.

  7. Preparation of a weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography stationary phase for protein separation using click chemistry.

    Science.gov (United States)

    Zhao, Kailou; Yang, Fan; Xia, Hongjun; Wang, Fei; Song, Qingguo; Bai, Quan

    2015-03-01

    In this study, 3-diethylamino-1-propyne was covalently bonded to the azide-silica by a click reaction to obtain a novel dual-function mixed-mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high-salt-concentration mobile phase and weak anion exchange character in a low-salt-concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed-mode chromatography stationary phase, a new off-line two-dimensional liquid chromatography technology using only a single dual-function mixed-mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.

  8. Silica as a Matrix for Encapsulating Proteins: Surface Effects on Protein Structure Assessed by Circular Dichroism Spectroscopy

    Directory of Open Access Journals (Sweden)

    Genet H. Zemede

    2012-08-01

    Full Text Available The encapsulation of biomolecules in solid materials that retain the native properties of the molecule is a desired feature for the development of biosensors and biocatalysts. In the current study, protein entrapment in silica-based materials is explored using the sol-gel technique. This work surveys the effects of silica confinement on the structure of several model polypeptides, including apomyoglobin, copper-zinc superoxide dismutase, polyglutamine, polylysine, and type I antifreeze protein. Changes in the secondary structure of each protein following encapsulation are monitored by circular dichroism spectroscopy. In many cases, silica confinement reduces the fraction of properly-folded protein relative to solution, but addition of a secondary solute or modification of the silica surface leads to an increase in structure. Refinement of the glass surface by addition of a monosubstituted alkoxysilane during sol-gel processing is shown to be a valuable tool for testing the effects of surface chemistry on protein structure. Because silica entrapment prevents protein aggregation by isolating individual protein molecules in the pores of the glass material, one may monitor aggregation-prone polypeptides under solvent conditions that are prohibited in solution, as demonstrated with polyglutamine and a disease-related variant of superoxide dismutase.

  9. Preparation and characterization of Neisseria meningitidis mutants deficient in production of the human lactoferrin-binding proteins LbpA and LbpB.

    Science.gov (United States)

    Bonnah, R A; Schryvers, A B

    1998-06-01

    Pathogenic members of the family Neisseriaceae produce specific receptors facilitating iron acquisition from transferrin (Tf) and lactoferrin (Lf) of their mammalian host. Tf receptors are composed of two outer membrane proteins, Tf-binding proteins A and B (TbpA and TbpB; formerly designated Tbp1 and Tbp2, respectively). Although only a single Lf-binding protein, LbpA (formerly designated Lbp1), had previously been recognized, we recently identified additional bacterial Lf-binding proteins in the human pathogens Neisseria meningitidis and Moraxella catarrhalis and the bovine pathogen Moraxella bovis by a modified affinity isolation technique (R. A. Bonnah, R.-H. Yu, and A. B. Schryvers, Microb. Pathog. 19:285-297, 1995). In this report, we characterize an open reading frame (ORF) located immediately upstream of the N. meningitidis B16B6 lbpA gene. Amino acid sequence comparisons of various TbpBs with the product of the translated DNA sequence from the upstream ORF suggests that the region encodes the Lf-binding protein B homolog (LbpB). The LbpB from strain B16B6 has two large stretches of negatively charged amino acids that are not present in the various transferrin receptor homologs (TbpBs). Expression of the recombinant LbpB protein as a fusion with maltose binding protein demonstrated functional Lf-binding activity. Studies with N. meningitidis isogenic mutants in which the lbpA gene and the ORF immediately upstream of lbpA (putative lbpB gene) were insertionally inactivated demonstrated that LbpA, but not LbpB, is essential for iron acquisition from Lf in vitro.

  10. Fast preparation of antioxidant proteins from ginkgo and soybean by using affinity chromatography%亲和层析快速提取白果和大豆的抗氧化蛋白

    Institute of Scientific and Technical Information of China (English)

    王桂珍; 凌辉生; 谢丽思; 李任强

    2012-01-01

    After Sephadex G-25 polymerbeads were activated by using epichlorohydrin,hemin was bound with them to prepare an immobilized hemin affinity chromatography column,which was used to prepare antioxidant proteins from ginkgo and soybean.Equilibrated with deionized water and eluted with pH3.6,0.2mol/L NaAc-HAc buffer,those proteins obtained from this column were demonstrated to possess high antioxidative activity(AA) after the measurement with linoleic acid-potassium thiocyanate.AA of crude proteins of ginkgo was 7.92% and two polypeptides of them were obtained with AA 17.45% after purification.In soybean,several polypeptides with AA 30.72% were obtained from the crude proteins with AA 10.52%.This method was a novel,rapid and effective method for preparation of antioxidant proteins from ginkgo and soybean.%用环氧氯丙烷活化法把血红素结合于惰性分子筛填料SephadexG-25上作为配基做成亲和层析柱,用于从白果或大豆中提取具有抗氧化活性的蛋白。此层析柱以去离子水为平衡液、0.2mol/L的NaAc-HAc缓冲液(pH3.6)为洗脱液,层析得到的蛋白经亚油酸-硫氰酸钾抗氧化活性测定证明具有较高的抗氧化活性。白果全蛋白的抗氧化活性为7.92%,经此方法提纯后得到2种抗氧化多肽,活性达到17.45%;而大豆中的抗氧化蛋白经纯化则得到多种,抗氧化活性由全蛋白时的10.52%提高至30.72%。这是一个新的、只需一个步骤即可从白果或大豆蛋白提取液中提取到具有高抗氧化活性蛋白的方法,具有成本低,快速高效的特点。

  11. Preparation and Physicochemical Properties of Whey Protein Isolate-soluble Starch Conjugate%乳清分离蛋白-可溶性淀粉接枝物的制备及其理化性质

    Institute of Scientific and Technical Information of China (English)

    罗志刚; 卢静静; 孙炜炜

    2011-01-01

    To provide a reference on the study of protein-starch conjugate, the formation and secondary structure of whey protein isolate-soluble starch(SS) conjugate prepared by dry-heating method were investigated. The indexes of significant changes in A294 , browning intensity, free amino groups content and SDS-PAGE showed that WPI-SS conjugate was successfully prepared based on Maillard reaction under dry-heating. The results also showed that more incubation time significantly promoted glycosylation in the WPI-SS mixture. Meanwhile, the secondary structure of whey protein isolate had a considerable loss due to the covalent attachment of high molecular weight starch ; the surface hydrophobicity of whey protein was reduced.%研究了干热法处理条件下乳清分离蛋白-可溶性淀粉接枝物的制备及其二级结构分析,为蛋白质-淀粉接枝物的研究提供参考。A294、褐变、游离氨基含量变化、电泳图谱等证实乳清分离蛋白与可溶性淀粉在干热处理下确实发生了以美拉德反应为基础的接枝反应,且反应天数的延长能够显著促进乳清分离蛋白-可溶性淀粉接枝物的生成。由于大分子淀粉的共价接入,乳清分离蛋白的二级结构遭到破坏;蛋白质表面疏水性指数降低。

  12. Preparation and Characterization of Wheat Protein/PVA Blend Composite Nanofiber%小麦蛋白/聚乙烯醇复合纳米纤维的制备及其表征

    Institute of Scientific and Technical Information of China (English)

    潘丽军; 张黎黎; 姜绍通; 王华

    2012-01-01

    Wheat protein/PVA composite nanofiber was prepared via electrospining technique. The effects of total concentration of the solution, voltages, tip to collector distance and structure of the nanofibers were investigated by scanning electron microscopy (SEM), fourier transform infratred (FT-IR) and X-ray diffraction (XRD). The results indicated that the uniform and smooth wheat protein/PVA composite nanofiber with average diameter of 280 nm could be prepared with following optimal process parameters: total concentration of the solution 10 %, weight ratio of wheat protein to PVA 8:2, applied voltage 12 kV, and the tip-to-collector distance 10 cm. There were hydrogen bonds between wheat protein and PVA in the composite nanofibers.%以小麦蛋白、聚乙烯醇(PVA)为原料,采用静电纺丝法制备小麦蛋白/PVA共混复合纳米纤维,重点研究纺丝液质量分数、电压、接收距离对纤维形态的影响,利用扫描电镜、傅里叶变换红外光谱、X-射线衍射光谱对纤维的形态与结构进行表征.结果表明:在纺丝液质量分数10%、小麦蛋白与PVA质量比8∶2、电压12 kV、接收距离10 cm的条件下,可以制备平均直径为280 nm左右的均一、表面光滑的纳米纤维.小麦蛋白与PVA复合后,分子间以氢键结合.

  13. Prokaryotic expression and polyclonal antibody preparation of novel ZLG10 protein involved in infection of RSV on SPC-A1 cells.

    Science.gov (United States)

    Li, Lei; Zhao, Dongchi; Zhang, Chuyu; Zhang, Qiwei; You, Shangyou

    2005-05-01

    Differentially expressed genes between normal SPC-A1 cells and SPC-A1 cells infected by RSV were investigated using differential display. The novel zlg10 gene codes for a novel protein, ZLG10, which has previously been reported to be up-regulated in RSV-infected SPC-A1 cells. Its putative open reading frame was also identified. To better understand the structure, function, and possible role of ZLG10 as a potential candidate for diagnosis and vaccine studies, the intact region encoding ZLG10 was obtained by PCR and expressed in Escherichia coli as a GST-fusion protein. After purification, GST-ZLG10 fusion protein was used to immunize the adult rabbits following standard protocols. Consequently, we found that the produced antiserum of the novel fusion protein significantly suppressed the infection by RSV on SPC-A1 cells by using neutral red uptake assay and quantitative measurement. Together, our data demonstrate that ZLG10, a novel protein expressed and purified in this report, might be a potential effective therapeutic candidate for treating RSV infections.

  14. 高纯菜籽蛋白的制备及其相关性质研究%Study on preparation of high-purity rapeseed protein and its properties

    Institute of Scientific and Technical Information of China (English)

    周俊; 潘丽军; 韩智宏

    2012-01-01

    Protein was extracted from coldpressed rapeseed meal which was detoxicated firstly,then the extract was ultrafiltered to obtain high-purity rapeseed protein,chemical components,amino acid and function of proteins were also studied.Extraction rate of protein from rapeseed meal after detoxication was 67.97%,rapeseed protein of different purity from 70% to 90% could be prepared by ultrafiltration.Phytic acid,glucosinolate were not detected in proteins,and content of polyphenol was decreased obviously with rise of protein purity.Sulphur-containing amino acid(Methionine+Cysteine) was the first limiting amino acid.Chemical scores of the other essential amino acids were higher than FAO/WHO standard,but most were inferior to standard of whole egg protein.Capacities of water-holding,fat-absorbing,emulsion,emulsion stability,foaming and foam stability correlated positively with purity of rapeseed protein.%对脱毒后的冷榨菜籽粕进行蛋白提取,超滤纯化提取液获得高纯度的蛋白成品,并研究分析了蛋白的成分、氨基酸及其功能特性。脱毒后菜籽粕中蛋白提取率为67.97%,超滤后蛋白纯度可达70%~90%。成品蛋白中植酸与硫苷未检出,多酚含量随蛋白纯度增加而显著下降,甲硫氨酸+半胱氨酸是第一限制性氨基酸,其余必需氨基酸化学评分均高于FAO/WHO标准,却大多低于全蛋蛋白标准。菜籽蛋白的持水性、吸油性、乳化性及乳化稳定性、起泡性及起泡稳定性均与蛋白纯度呈正相关。

  15. A comparative in vitro study of the digestibility of heat- and high pressure-induced gels prepared from industrial milk whey proteins

    Science.gov (United States)

    He, Jin-Song; Mu, Tai-Hua; Wang, Juan

    2013-06-01

    We undertook this study to compare the digestibility of heat- and high pressure-induced gels produced from whey protein isolate (WPI). To simulate in vivo gastrointestinal digestion of WPI gels, a pepsin-trypsin digestion system was used. The in vitro protein digestibility of WPI gels induced by high pressure (400 MPa and 30 min; P-gel) and those induced by heat (80°C and 30 min; H-gel) was compared using a protein concentration of 0.14 g mL-1. The in vitro protein digestibility of P-gels was significantly greater than that of H-gels (p<0.05). The size-exclusion chromatography profiles of the hydrolysates showed that the P-gel generated more and smaller peptides than natural WPI and H-gels. Furthermore, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed some soluble disulfide-mediated aggregation in the P-gel, while there was more insoluble aggregation in the H-gel than the P-gel. The P-gel was more sensitive to proteinase than the H-gel, which was related to the content of S-S bonds, and this in turn could be attributed to the differences in the gelation mechanism between the H-gel and P-gel.

  16. Study on preparation and purification of protein from pefilla seeds%紫苏籽蛋白的提取及纯化研究

    Institute of Scientific and Technical Information of China (English)

    石玮婷; 李荣; 姜子涛

    2013-01-01

    本文以紫苏籽为原料,利用石油醚对其进行脱脂.采用微波辅助提取紫苏籽蛋白.在单因素试验的基础上,采用L9 (34)正交试验设计,研究pH、微波功率、微波时间和固液比对紫苏籽蛋白提取率的影响.结果表明,在各影响因素中,影响程度依次为:pH>微波功率>微波时间>固液比,微波辅助提取紫苏籽蛋白的最佳工艺条件为:pH10.0、固液比为1∶10、微波时间8min、微波功率200W,在此条件下测得脱脂紫苏粉粗蛋白提取率为25.85%,其蛋白含量为43.01%.经超滤法处理后得到的蛋白提取率为46.90%,其蛋白含量为91.74%.%The pefilla seed is used as a raw material in this study,and use the petroleum ether to remove the oil in pefilla seeds.The protein was extracted from pefilla seeds by microwave-assisted.Based on single factor test,the effect of pH,microwave power,microwave time,solid/liquid ratio (w/v) on the extraction rate of protein was studied according to L9 (34) orthogonal test.The results showed that factors influenced the extraction of protein from pefilla seeds by microwave-assisted.The methods were in the order as follows:pH > microwave power > microwave time >solid/liquid ratio (w/v),the optimum extracting conditions of protein from pefilla seeds by microwave-assisted were adding ten times amount water solutions of pH 10.0 and extracting for 8min at 200W power by microwave.The extraction rate of pefilla seed protein was 25.85% under the above optimal extracting conditions and the protein purity is 43.01%.The extraction rate of the basil seed isolated protein purified by ultrafiltration is 46.90% and the purity is 91.74% .

  17. Prokaryotic expression and polyclonal antibody preparation of enterovirus 71 3A protein%人肠道病毒71型3A蛋白的原核表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    黄琴琴; 唐瑞; 杨勇波

    2013-01-01

    Objectives To construct a prokaryotic expression vector of the EV71 3a gene and prepare its recombinant protein and polyclonal antibody for subsequent study.Methods The 3a gene was amplified with PCR and cloned into the vector PET28a to yield PET28a-3a for the prokaryotic expression of 3A protein.The recombinant 3A protein was expressed in E.coli BL21 and was subsequently used to immunize mice.The resulting anti-sera were evaluated using immunofluorescence staining and Western blotting.Results The recombinant protein 3A was efficiently produced in E.coli in the form of inclusion bodies in the expressed protein.Western blot analysis indicated that the resulting mouse anti-3A sera reacted with the eukaryotically expressed EGFP-3A fusion protein.Moreover,the antisera positively recognized cells infected with EV71 according to immunofluorescence staining.Conclusion An anti-3A antibody was successfully prepared and may provide a foundation for subsequent study of the 3a gene.%目的 构建人肠道病毒71型3A基因原核表达质粒,制备重组3A蛋白及其抗体. 方法 PCR方法扩增人肠道病毒71型3A基因,构建原核表达质粒PET28a-3A并转化大肠埃希菌,诱导表达3A重组蛋白,免疫小鼠制备3A蛋白抗体.通过免疫荧光和Western blot方法鉴定抗体特异性. 结果 成功构建了PET28a-3A原核表达质粒并表达了3A重组蛋白,3A蛋白以包涵体的形式存在.细胞免疫荧光和Western blot检测显示,制备的鼠源3A蛋白抗体可识别真核表达的EGFP-3A融合蛋白以及EV71感染细胞表达的3A蛋白. 结论 成功构建了人肠道71型3A基因原核表达质粒,利用该质粒表达的重组蛋白成功制备了特异性的3A蛋白的鼠源抗体.

  18. Process of peanut protein milk powder by dry preparation%花生蛋白奶粉的干法生产工艺研究

    Institute of Scientific and Technical Information of China (English)

    章宝; 单杨; 李高阳

    2011-01-01

    以去壳花生仁为原料,经低温烘烤、脱红衣、冷榨脱脂、超微粉碎等工艺生产出花生蛋白粉,将其与全脂奶粉混合生产花生蛋白奶粉,所得产品溶解度高、冲调性好、口感细腻、营养均衡,同时具有奶味和花生特有的香味。%Peanut protein milk powder was made by mixing whole milk powder with peanut protein powder,which was made by the technologies of low-temperature baking,taking the peanut red skin off,cold-pressed degreasing and ultra-fine pulverizatio by using shelled pea

  19. Precipitation and ultimate pH effect on chemical and gelation properties of protein prepared by isoelectric solubilization/precipitation process from pale, soft, exudative (PSE)-like chicken breast meat.

    Science.gov (United States)

    Zhao, X; Xing, T; Chen, X; Han, M-Y; Li, X; Xu, X-L; Zhou, G-H

    2016-11-11

    Pale, soft, exudative (PSE)-like chicken breast is considered deteriorated raw material in the poultry meat industry that has inferior processing ability. The chemical and gelation properties of PSE-like chicken breast meat paste were studied. These pastes were prepared by the pH adjustment method and protein isolation using the isoelectric solubilization/precipitation (ISP) process from PSE-like chicken meat. The ISP-isolated samples were solubilized at pH 11.0 and recovered at pH 5.5 and 6.2. The ultimate pH of the ISP-isolated protein and meat paste was adjusted to 6.2 and 7.0. The ultimate pH in this article referred to the final pH of the extracted protein and meat paste. Higher reactive sulfhydryl content and surface hydrophobicity were found in the precipitation at pH 6.2 than at pH 5.5. However, various ultimate pH values showed no significant influence on the surface hydrophobicity. The hardness of gel, as measured by textural profile analysis, was improved using 6.2 as the precipitation pH compared with pH 5.5. The viscoelastic modulus (G(')) of gel pastes prior to the thermal gelation was higher with ISP treatment. However, lower G(') was seen after thermal gelation compared with the control. Dynamic rheological measurement demonstrated a different gel-forming mechanism for protein precipitated at pH values of 5.5 and 6.2 compared with the meat paste. The cooking loss showed that the recovered protein failed to form a gel with good water-retention capacity unless the ultimate pH was adjusted to 7.0. Gels made from protein extracted by the ISP method had higher yellowness and lower redness values, probably due to protein denaturation. Precipitation at pH 6.2 formed a harder gel with lower water-retention ability than that at pH 5.5, and this result was possibly due to higher surface hydrophobicity and S-S bridge formation. Overall, network characteristics of ISP-treated protein gels were strongly dependent on precipitation pH and ultimate pH.

  20. Preparation of concentrated hazelnut protein by ethanol extraction%醇提法制备榛仁浓缩蛋白的工艺

    Institute of Scientific and Technical Information of China (English)

    矫春娜; 芦明春; 张星

    2012-01-01

    以榛仁粕为原料,用乙醇浸出法生产榛仁浓缩蛋白.研究了乙醇体积分数、浸提温度、浸提时间、固液比、浸提次数等因素对榛仁浓缩蛋白中蛋白质量分数的影响.通过正交试验,确定了醇法制备榛仁浓缩蛋白的最佳工艺条件为:乙醇体积分数65%、固液比1:9、浸提温度55℃、浸提4次(30 min/次).由该条件制备的榛仁浓缩蛋白中粗蛋白质量分数为81.73%,其色浅味淡,无溶剂残留.%Concentrated hazelnut protein was extracted from hazelnut meal by ethanol. The effects of ethanol concentration, temperature, extraction time, solid-to-liquid ratio and extraction times on the content of concentrated hazelnut protein were studied by orthogonal experimental design. The optimized processing conditions were found to be ethanol concentration of 65%, extraction temperature of 55 ℃ , extraction time of 30 min, liquid-to-solid ratio of 1 : 9 and extracted 4 times, then the concentrated hazelnut protein acquired 81. 73%.

  1. Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

    Science.gov (United States)

    Choi, Kang-Seuk; Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

    2013-01-01

    A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 μL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

  2. Preparation of non-aggregated fluorescent nanodiamonds (FNDs) by non-covalent coating with a block copolymer and proteins for enhancement of intracellular uptake.

    Science.gov (United States)

    Lee, Jong Woo; Lee, Seonju; Jang, Sangmok; Han, Kyu Young; Kim, Younggyu; Hyun, Jaekyung; Kim, Seong Keun; Lee, Yan

    2013-05-01

    Fluorescent nanodiamonds (FNDs) are very promising fluorophores for use in biosystems due to their high biocompatibility and photostability. To overcome their tendency to aggregate in physiological solutions, which severely limits the biological applications of FNDs, we developed a new non-covalent coating method using a block copolymer, PEG-b-P(DMAEMA-co-BMA), or proteins such as BSA and HSA. By simple mixing of the block copolymer with FNDs, the cationic DMAEMA and hydrophobic BMA moieties can strongly interact with the anionic and hydrophobic moieties on the FND surface, while the PEG block can form a shell to prevent the direct contact between FNDs. The polymer-coated FNDs, along with BSA- and HSA-coated FNDs, showed non-aggregation characteristics and maintained their size at the physiological salt concentration. The well-dispersed, polymer- or protein-coated FNDs in physiological solutions showed enhanced intracellular uptake, which was confirmed by CLSM. In addition, the biocompatibility of the coated FNDs was expressly supported by a cytotoxicity assay. Our simple non-covalent coating with the block copolymer, which can be easily modified by various chemical methods, projects a very promising outlook for future biomedical applications, especially in comparison with covalent coating or protein-based coating.

  3. Extraction of phytic acid and preparation of protein isolates from rapeseed meal%菜籽粕植酸提取和分离蛋白的制备

    Institute of Scientific and Technical Information of China (English)

    潘丽军; 周俊; 姜绍通; 孙汉巨; 罗水忠; 韩智宏

    2011-01-01

    植酸和蛋白是菜籽粕中2种极具经济价值的成份.为提高菜籽粕的综合利用效果,该文以双低冷榨菜籽粕为原料,采用醋酸溶液提取植酸,在膜分离精制植酸粗提液过程中同时回收蛋白;再对植酸提取后的残余物进行蛋白分离,超滤纯化后获得高纯度的蛋白成品.响应面优化的植酸最适提取条件为:醋酸质量分数0.7%,提取温度48℃,液料比10 mL/g,提取时间1.6 h,该条件下植酸得率为1.865%.植酸粗提液中回收出的蛋白和损失植酸分别占菜籽粕的3.63%和0.395%.超滤精制的分离蛋白可达到70%~90%不同纯度的要求,蛋白中多酚含量显著减少,且植酸与硫苷未检出.%Phytic acid and protein are two kinds of valuable components in rapeseed meal. To improve comprehensive utilization in this research, phytic acid was extracted with acetum from double-low coldpressed rapeseed meal, and protein was recovered from the crude extract of phytic acid by membrane separating technology. The meal residue was dried to extract protein, which was then purified by ultrafiltration to obtain high purity product. Extraction conditions of phytic acid were optimized by response surface methodology (RSM) as follows: mass fraction of acetic acid 0.7%,extraction temperature 48℃, liquid-to-solid ratio 10 mL/g, extraction time 1.6 h. Under this condition, extraction yield was 1.865%. Protein recovered and phytic acid loss accounting for rapeseed meal were 3.63% and 0.395% respectively in crude extract of phytic acid. Purity of the protein refined by ultrafiltration was between 70% and 90%, in which the content ofpolyphenols was significantly reduced and no phytic acid and glucosinolates could be detected.

  4. 基因重组蛛丝蛋白-聚乙烯醇复合支架材料的制备%Preparation of recombinant spider silk protein-polyvinyl alcohol composite scaffold material

    Institute of Scientific and Technical Information of China (English)

    魏梅红; 涂桂云; 陈登龙; 李敏

    2008-01-01

    目的探讨致孔剂NaCl粒径和比例、变性剂和聚乙烯醇(PVA)等因素对基因重组蛛丝蛋白-PVA复合支架材料形态及性能的影响.方法基因重组蛛丝蛋白溶解于98%甲酸,采用冷冻干燥粒子滤沥法制备重组蛛丝蛋白-PVA复合多孔支架;采用扫描电子显微镜观察支架的形态;采用单纤维强力试验机测试支架机械性能.结果乙醇作变性剂制得的多孔支架力学性能较好,支架的断裂应力、断裂比强度均提高5倍以上,断裂伸长率可达12.21%.以粒径<500μm的NaCl为致孔剂制得的多孔支架力学性能较好.高分子材料PVA能明显改善重组蛛丝蛋白多孔支架的件能.结论重组蛛丝蛋白-PVA复合支架材料有望在组织工程领域得以应用.%Objective To investigate the effects of many factors such as porogen, denaturant and polyvinyl alcohol (PVA) polymer on the structure and properties of recombinant spider silk protein-PVA composite scaffold material. Methods The silk fibrous protein solution was prepared by dissolving the recombinant spider silk protein in 98% formic acid. Recombinant spider silk protein-PVA composite porous scaffolds were prepared by freeze-drying and particle-leaching method. Scanning electron microscope was used to observe the conformation of the scaffolds. Single strand strength testing machine was used to test the mechanical property of the scaffolds. Results Mechanical property of the scaffolds was better with ethanol as denaturant. Breaking stress and break specific tenacity enhanced by over 5 times. Breaking elongation ratio could reach 12.21%. Using NaCt ( ψ<500 μm) as porogen, or the introduction of PVA could improve the performances of porous scaffold evidently. Conclusion Recombinant spider silk protein-PVA composite porous scaffolds will have a prospective application in tissue engineering.

  5. Study on Preparation Conditions of Sea Cucumber Polysaccharide with Low Protein by Enzymolysis%酶解法制备低蛋白含量海参多糖条件的研究

    Institute of Scientific and Technical Information of China (English)

    宿玮; 常耀光; 张翠玉; 崔宏博; 薛长湖

    2011-01-01

    In this research, using general low-value sea cucumber as raw material, enzymolysis was combined with ultrafiltration to prepare low-protein sea cucumber polysaccharide. Response surface methodology was adopted to study and optimize the parameters affecting protein content on the basis of single factor experiments. A second order quadratic e-quation was established. Results indicated that the optimal levels for achieving polysaccharide with the lowest protein content were obtained, including pH7.0, temperature of 51℃, substrate concentration of 4.82%, the ratio of enzyme to substrate of 4.08% and time of 12 h. The protein content of polysaccharide was 2.41%. Therefore, it could provide theoretical guidance for industrialized preparing sea cucumber polysaccharide with low protein. And it would benefit deep processing of low-value sea cucumber.%以国产大宗低值海参——海地瓜为原料,以酶解结合超滤技术制备海参多糖.通过单因素试验及响应面试验研究酶解过程各因素对海参多糖蛋白含量的影响,并对酶解条件进行优化.试验结果表明:成功建立了预测海参多糖中蛋白含量的数学模型;复合蛋白酶酶解的最优条件是:pH 7.0、温度51℃、底物质量分数4.82%、酶与底物的质量比4.08%、时间12h.经优化,制备了低蛋白含量海参多糖,多糖蛋白含量由19.91%降至2.41%.

  6. 菠萝蛋白酶水解泥鳅蛋白制备ACE抑制肽的研究%Preparation of ACE Inhibitory Peptides by Bromelain Hydrolysis of Loach(Misgurnus anguillicaudatus) Protein

    Institute of Scientific and Technical Information of China (English)

    姚东瑞; 盘赛昆; 周鸣谦; 王淑军; 胡金玲

    2012-01-01

    为了探讨利用泥鳅蛋白制备功能性肽的可能性,采用高效液相色谱法测定泥鳅肉水解物对血管紧张素转换酶(ACE)的抑制作用,从胰蛋白酶、胃蛋白酶、菠萝蛋白酶、复合风味蛋白酶、复合蛋白酶5种酶中筛选出菠萝蛋白酶作为酶解泥鳅肉制备具有降血压活性水解物的适宜水解酶。在单因素试验的基础上,采用L9(34)正交试验设计对该酶的酶解条件进行优化。结果表明最佳水解条件为:温度55℃,固液比1:3,pH6.5,加酶量1000U/g pro,水解时间90min。在该条件下,水解物的ACE抑制率IC50值为0.0184mg/mL,ACE抑制肽的相对分子质量主要集中在924左右。%The present deals with the enzymatic preparation of angiotensin-converting enzyme(ACE) inhibitory active peptides from loach(Misgurnus anguillicaudatus) protein.An L9(34) orthogonal array design methods based on single factor experiments was used to optimize the hydrolysis conditions for achieving maximum ACE inhibitory activity of loach protein hydrolysates.HPLC was used to determine ACE inhibitory activity of loach protein hydrolysates.Bromelain was found to be more suitable to prepare ACE inhibitory active peptides than trypsin,pepsin,flavourzyme and protamex.The optimal hydrolysis conditions were temperature of 55 ℃,solid-to-liquid ratio of 1:3,pH of 6.5,bromelain dose of 1000 U/g protein and hydrolysis duration of 90 min.The IC50 of the loach protein hydrolysate obtained under these conditions was 0.0184 mg/mL and the relative molecular weight distribution of ACE inhibitory peptides in it was mainly concentrated around 924.

  7. Preparation and Characterization of Poly(L-lactic acid)/Rapeseed Protein Blend Nanofiber%聚L-乳酸/菜籽蛋白共混纳米纤维毡的制备与表征

    Institute of Scientific and Technical Information of China (English)

    姜绍通; 刘涛涛; 姜苏薇; 江勤; 王华林

    2011-01-01

    以聚L-乳酸、菜籽蛋白为原料,高压静电纺丝制备聚L-乳酸(PLLA)/菜籽蛋白共混复合纳米纤维毡,考察了不同电压、极距和三氟乙酸添加量对纳米纤维形态及直径的影响,采用傅里叶变换红外光谱(FT-IR)、扫描电镜(SEM)和X-射线衍射(XRD)对相关产物进行结构表征.结果表明:复合纤维中PLLA与菜籽蛋白之间以氢键结合,PLLA的结晶性能降低;PLLA纺丝溶液中,菜籽蛋白的三氟乙酸溶液的适量引入可显著提高纺丝速率.在PLLA质量浓度为24%的氯仿溶液中,6.5%菜籽蛋白的三氟乙酸溶液加入量为0.25 mL,电压16 kV,极距10 cm的条件下,可快速制备平均直径622 am的PLLA/菜籽蛋白复合纳米纤维毡,纺丝速率达到5.2 mg/min.%The rapeseed protein/PLLA composite nanofiber felt was prepared via electrospinning. The effects of voltages, tip to collector distance and trifluoroaeetic acid on the morphology and structure of the nanofibers were investigated by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR),X-ray diffraction(XRD). Results indicated that there were hydrogen bonds between rapeseed protein and PLLA in the composite fibers, and the crystal structure of PLLA was destroyed seriously. The addition of rapeseed protein/trifluoroaeetie acid solution results in a higher spinning speed, and the finer fibers would be also obtained. The rapeseed protein/PLLA composite fiber felt (about 5.2 mg/min) with average diameter of 622 nm could be prepared quickly at the following optimal process parameters: the concentration of PLLA was 24 %(contained 1 g PLLA), the volume of rapeseed protein/trifluoroacetic acid solution was 0.25 mL, the applied voltage was 16 kV, and the tip-to-collector distance was 10 cm.

  8. 响应面法超声波辅助提取核桃蛋白工艺优化%Optimization of the preparation of walnut protein with ultrasonic-assisted extraction method

    Institute of Scientific and Technical Information of China (English)

    敬思群

    2012-01-01

    Walnut protein was obstained by ultrasonic-assisted extraction method with the defatted walnut meal as raw material. The preparation process of walnut protein was optimized according to Box- Behnken central composition design principle on foundation of single factor experiment, the method of response surface analysis with 3 factors and 3 levels combined with Minitab15.0-statistical data analysis software was adopted with the leaching rate of walnut protein as the response value. The effect of extraction time, temperature, liquid to solid ratio and pH on the leaching rate of walnut protein were studied in this paper. According to the above results, the optimum preparation conditions were obtained as follows: extraction time 19 min, reaction time 90 min, temperature 46 %, ratio of solvent: meal at 20:1 and pHS.6. Under these conditions, the leaching rate of walnut protein reached 18.66%.%以脱脂核桃粕为原料,利用超声波辅助提取制备核桃蛋白。在单因素实验的基础上,根据Box-Behnken的中心组合实验设计原理,运用Minitab15.0数据统计分析软件,采用3因素3水平的响应面分析法,以核桃蛋白浸出率为响应值,研究了超声时间、超声温度、液料比和pH对核桃蛋白浸出率的影响,并优化了提取工艺。确定了超声辅助提取核桃蛋白的最佳工艺条件为:超声时间为19 min,超声温度为46℃,液料比为20:1,pH为8.6,在此条件下核桃蛋白的浸出率达到68.98%。

  9. Expression of mouse calreticuin protein and preparation of its polyclonal antibodies%小鼠钙网蛋白的原核表达和多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    汪龚泽; 刘朝奇; 杨建林; 聂纪芹

    2011-01-01

    In order to investigate the biological properties of calreticulin(CRT), a protein present in the mammalian cells with highly conservative and multiple biological properties, a short fragment of mouse CRT was amplified through RT-PCR method and then was cloned into prokaryotic expression vector pDET28a( + ). The recombinant protein CRT was induced by IPTG in E.coli BL2KDE3) and the expressed protein was detected by Western blotting. The purified protein was used to immune mouse to prepare polyclonal antibody and the specificity and titer of the antibody were detected by ELISA, Western blotting and FCM. It was demonstrated that the recombinant prokaryotic expression plasmid pET28a( + ) /CRT was successfully con structed and the CRT protein was expressed in E. Coli BL2KDE3) with high efficiency. Western blot assay showed that this re combinant protein was characterized with its antibody. Mouse immunized with the purified protein produced high titer of anti body. Western blot assay displayed that the CRT protein was highly expressed in some of eukaryotic cells and could specifically combine with the antibody. FCM assay displayed that the antibody could also specifically combine with the membrane extracel lular region of CRT. It is evident that the preparation of recombinant CRT and its polyclonal antibodies have a strong specificity to match with the CRT protein from mouse and human.%钙网蛋白(calreticulin,CRT)是存在于哺乳动物细胞具有高度保守性和多种生物学活性的蛋白,为了更好的研究其生物学活性,本课题组通过PCR方法扩增CRT截短基因,将其克隆到原核表达载体PET-28a(+)中,经大肠杆菌表达并纯化CRT蛋白.以纯化的CRT蛋白抗原免疫BALB/c小鼠,制备多克隆的CRT血清.进一步采用Western blot、ELISA、流式细胞术等技术对制备的抗体进行初步鉴定.结果显示:原核表达重组质粒在大肠杆菌中能高效表达CRT蛋白;获得多抗血清应用Western blot鉴定几种

  10. 活性蛋白肉在速冻调理食品中的应用技术研究%Study on application of active protein meat alternatives in quick-frozen prepared food

    Institute of Scientific and Technical Information of China (English)

    尚丹; 史九根

    2015-01-01

    将大豆分离蛋白制备成蛋白肉并在各类含馅类速冻调理食品中使用。首先是利用大豆分离蛋白和胶凝剂制备活性蛋白肉,以蛋白肉为原料添加在各种调理食品中。以速冻饺子、鸡肉丸为例,代替10%、15%、20%、30%猪肉的蛋白肉,结果显示,在猪肉饺子中添加10%的活性蛋白肉产品口感更好,咀嚼感更强;另活性蛋白肉的添加有效吸收了饺子馅料在包制、成型和储存过程中析出的水分等液体汤料,从而降低了饺子皮的破皮率,提高饺子出品率;在鸡肉丸中加入5%、10%、15%、20%的蛋白肉,添加的鸡胸肉比例相应减少,结果显示,鸡肉丸中加入5%的活性蛋白肉和不添加蛋白肉的产品口感和质构接近,但在煮制过程中,添加活性蛋白肉的鸡肉丸出油少,保油效果更好。%Soy protein isolate was made into protein meat alternatives. And they were used in vari⁃ous quick-frozen food containing stuffing. Firstly soy protein isolate and gelling agent were used to pre⁃pare active protein meat alternatives. Protein meat alternatives were taken as material to add in all kinds of prepared food. Quick -frozen dumplings and chicken meatballs were taken as examples, replacing 10%, 15%, 20% and 30% pork with protein meat alternatives. The results showed that when the pork dumplings added with 10% active protein meat alternatives, the taste and crunch would be better. Be⁃sides the addition of active protein meat alternatives could efficiently absorb the water and soup separating from stuffing during making, shaping and storage process. Therefore the broken rate of dumpling wrapper decreased, and the output rate increased. The chicken meatballs were added with 5%, 10%, 15% and 20% protein meat alternatives, respectively. The addition proportion of chicken breast reduced corre⁃spondingly. The results showed that the chicken meatballs added with 5% protein meat

  11. High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization

    Directory of Open Access Journals (Sweden)

    Tanaka Shigeyasu

    2009-06-01

    Full Text Available Abstract Background Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR that displayed human (prorenin receptor (hPRR connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC. Results A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR. A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i., but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i.. Conclusion The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.

  12. Preparation and characterization of rosemary incorporated fish protein edible films%迷迭香添加鱼肉蛋白可食膜的制备与特性

    Institute of Scientific and Technical Information of China (English)

    翁武银; 陈亨莉; 刘光明; 苏文金; 曹敏杰

    2011-01-01

    Edible fish protein films based on silver carp meat were successfully prepared.The effect of rosemary antioxidant incorporation of film-forming solutions on the properties of edible fish protein films was investigated.As a result,white and transparent protein films could be produced using silver carp meat.The mechanical properties and water vapor permeability(WVP)of fish protein films were dependent mainly on the content of fish myofibrillar proteins while no obvious variation was observed between different fish species.The effect of incorporation of rosemary extract on the mechanical properties and WVP of films were not significant.However,the addition of rosemary extract led to the formation of yellow protein films with increased UV obstructing ability as well as antioxidant capacity.Eel meats packed with protein films were stored for 12h at 37℃ in the dark.Compared with the samples without packaging,the increase of eel meat POV and TBARS was markedly inhibited during the storage at 37℃.The inhibitory effect was more significant by adding rosemary extract into films.%以鲢鱼肉为主要原料制备蛋白可食膜,测定了迷迭香抗氧化剂添加对膜理化性质和抗氧化性能的影响。结果表明,利用鲢鱼肉可以制备成无色透明的蛋白可食膜,鱼肉蛋白膜的机械性质和水蒸气透过性(WVP)主要由肌原纤维蛋白含量决定,在鱼种间的差异并不显著。添加迷迭香抗氧化剂对鱼肉蛋白膜的机械性质和WVP影响不显著,但对膜的阻隔紫外线能力和抗氧化性能有提高作用。将蛋白膜包装的鳗鱼肉在37℃下放置12h,与未包装样品相比,鳗鱼肉的POV和TBARS的增加明显得到抑制,而添加迷迭香会进一步提高其抑制效果。

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

    Science.gov (United States)

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

    2007-05-16

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

  14. SDF-1γ/rhGM-CSF融合蛋白的制备及其趋化作用%Preparation of SDF-1γ/rhGM-CSF fusion protein and its chemotactic effect

    Institute of Scientific and Technical Information of China (English)

    居小萍; 徐新颜; 张晓青; 刘永明; 于春山; 曹洋森; 卢明智; 陈樱

    2011-01-01

    Objective:To prepare the fusion protein of SDF-1 and rhGM-CSF (SDF-17/rhGM-CSF) by genetic engineering technology, and investigate its hematopoietic and immune promotion functions in tumor patients. Methods; The expression vector for SDF-1γ/rhGM-CSF fusion protein, pPIC9k-SDFl-rhGM-CSFl, was constructed and the protein expression was induced by yeast transfection. SDF-1γ/rhGM-CSF fusion protein was further identified by Western blotting analysis. Colony-formation assay and chemoattract assay were used to study the roles of the prepared fusion protein in stimulating bone marrow cell colony-formation and in chemoattracting immature dendritic cells. Results; SDF-1 y/ rhGM-CSF fusion gene vector, pPIC9k-SDF1-rhGM-CSF1, was successfully constructed and expressed high level of SDF-1 y/rhGM-CSF fusion gene. The molecular weight of the expressed protein was about 25 000 and was recognized by GM-CSF specific antibody. The fusion protein had a stronger effect in stimulating bone marrow cell colony-formation than GM-CSF ( P < 0.05) and in chemoattracting immature dendritic cells than SDF-1 (P<0.05). Conclusion; SDF-1 -γ/rhGM-CSF fusion protein can promote bone marrow cell colony-formation and chemoattraction of immature dendritic cells, which might be used for promoting hematopoiesis and immune function of tumor patients after chemotherapy.%目的:利用基因工程技术制备SDF-1与hGM-CSF的融合蛋白(SDF-1γ/rhGM-CSF),研究该融合蛋白对肿瘤患者造血和免疫功能的增强作用.方法:构建表达SDF-1 γ/rhGM-CSF融合蛋白的pPIC9k-SDF1-rhGM-CSF1质粒,转染酵母菌,诱导SDF-1γ/rhGM-CSF融合蛋白的表达,Western blotting鉴定SDF-1γ/rhGM-CSF融合蛋白的表达.集落形成实验观察SDF-1γ/rhGM -CSF对骨髓细胞集落形成的影响,趋化实验检测其对未成熟树突状细胞(dendritic cell,DC)的趋化作用.结果:成功构建pPIC9k-SDF1 -rhGM-CSF1质粒,高表达SDF-1γ/rhGM-CSF融合蛋白,分子量约为25000,并可被GM

  15. 大豆蛋白胶麻杆刨花板制备工艺的研究%Study on preparation of soy protein adhesive-based hemp stalk particle board

    Institute of Scientific and Technical Information of China (English)

    庞媛; 杨光; 杨波; 翟艳

    2011-01-01

    为了开发环境友好型刨花板,探讨利用大豆蛋白胶黏剂压制麻杆刨花板的制备工艺,分析热压温度、热压时间、施胶量和密度对麻杆刨花板性能的影响.结果表明,大豆蛋白胶可以用于麻杆刨花板的制造,其最佳工艺参数为:热压温度180℃,热压时间25 min,施胶量18%,密度0.80 g/cm3.在此条件下,压制的板材的性能超过GB/T 4897.4-2003的要求.%Preparation of hemp stalk particle board derived from soy protein-based adhesive were investigated and the effects of hot pressing temperature, hot pressing time, soy protein-based adhesive content, and density of hemp particle on the particle board physical and mechanical properties were analyzed. The results showed that soy protein-based adhesive can be used in hemp stalk particle board production and the optimal parameters were hot pressing temperature 180℃ , hot pressing time 25 min, soy protein-based adhesive content 18% ,density of hemp particle board 0. 80 g/cm3. Under above conditions,the particle-board has mechanical properties exceeding the Chinese National GB/T 4897. 4-2003 standard requirements.

  16. 利用马铃薯淀粉生产的废水及废渣发酵制备蛋白饲料%The Preparation of Fermented Protein Feed from Waste Water and Wastes of Potate Starch Processing

    Institute of Scientific and Technical Information of China (English)

    陈辉; 李虹; 冯雷; 张露; 宋国勇

    2011-01-01

    Microorganism fermentation method was used to produce high value-added protein feed from waste-water and wastes of potato starch production. This can lower COD content in waste-water and comprehensive utilized the potato starch processing wastes. A yeast strain was screened in our laboratory, and the optimum preparation conditions of protein feed was determined as: 20% wastes in waste-water(W/V) , natural pH, 28℃, inoculation amount 10% , cultivation time 72 h. In the optimum condition, protein content of the protein feed production was 37.40% and waste-water COD decreased 72.29%.%以马铃薯淀粉生产的废水及废渣为原料,采用微生物发酵的方法,制备高附加值的蛋白饲料,同时降低废水的COD值,达到综合利用的目的。经试验筛选得到1株酵母,并确定了蛋白饲料的制各条件:废水中薯渣添加量20%、pH自然、温度28℃、接种量10%、发酵时间72h。在适宜条件下,制备的蛋白饲料蛋白含量达37.40%,废水COD降低72.29%。

  17. Cloning, Prokaryotic Expression of Echinococcus granulosus Heat Shock Protein 70 and Preparation of It's Antiserum%细粒棘球绦虫Hsp70基因的克隆、表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    赵莉; 薛晶; 石保新; 陈皓斐; 张文宝; 马正海; 张壮志; 张旭; 古努尔·吐尔逊; 米晓云; 金映红

    2012-01-01

    [Objective] The objective of the experiment is to express and purify E. granulosus (Eg) heat shock protein 70 (EgHsp70) in E. coli and prepare the antibody against E. granulosus. [ Methods] EgHsp70 gene was amplified by PCR and cloned into prokaryotic expression vector pMAL-p2x, and the recombinant plasmid was transformed into E. coil BL-21. The soluble expression conditions of fusion protein were optimized by induction with different concentrations, of IPTG different temperatures and cultivation times. The expressed fusion protein was purified by Mal-tag Magnetic Beads. To prepare the anti-serum, New Zealand white rabbits were immunized with purified EgHsp70 protein via hypodermic and volar. Western blot was used to determine the serum's specificity against EgHsp70 and native proteins. The serum titers were analyzed by ELISA. [Results] Full-length of EgHsp70 gene had an open reading frame of 765 bps encoding a protein mass of 68.6 kD. Restriction endonuclease analysis and DNA sequencing showed that EgHsp70 was cloned into the plasmid pMAL-p2x. Based on the optimization experiments, it was concluded that the best soluble expression conditions for the EgHsp70 protein are using 0.3 mmol· L-1 IPTG when bacterial cells growing to OD600 0.6 and induced for 4 h at 30℃. ELISA and Western blotting showed that the titers of the anti-serum were above 1 : 256 000, and the anti-serum could specifically bind with EgHsp70 protein and native proteins. [Conclusion] The EgHsp70 fusion protein was obtained by expressing in E.coli and purifying, and the antibody against EgHsp70 was prepared with the fusion protein immunized New Zealand white rabbits. This work will provide an antigen and detection antibody for further study on the EgHsp70 function. The protein is immunogenic and can be a vaccine candidate against Echinococcus infection.%[目的]克隆细粒棘球绦虫(Echinococcus granulosus,E.g)热休克蛋白家族基因Hsp70,在原核细胞中表达、纯化Hsp70蛋白并制

  18. Difluoromethane preparation

    NARCIS (Netherlands)

    Wiersma, A.; Sandt, E.J.A.; Van Bekkum, H.; Makkee, M.; Moulijn, J.A.

    1996-01-01

    Abstract of NL 9401574 (A) The invention relates to a method for preparing difluoromethane, wherein dichlorodifluoromethane or monochlorodifluoromethane is brought into contact with hydrogen in the presence of palladium on activated carbon, wherein the loading of the palladium on the activated c

  19. Preparation and identification of water-soluble calcium-binding protein from grape (Vitis vinifera L.) seeds%葡萄籽中水溶性钙结合蛋白的分离和鉴定

    Institute of Scientific and Technical Information of China (English)

    吕晨艳; 赵广华

    2015-01-01

    grape seed protein by the ammonium sulfate sediment was approximately 3-fold larger than that by the traditional method, demonstrating that the ammonium sulfate sediment was a better way to isolate mineral-containing protein as compared to the traditional method. A high yield of calcium by the ammonium sulfate sediment could be derived from its mild condition, whereas acid and alkaline used in the alkali extraction and acid precipitation possibly inhibits the binding of calcium ions with grape seed protein. The following FT-IR (Fourier transform infrared spectroscopy) study showed that the prominent band of apo grape seed protein attributed to random coils turns (1 666 cm−1) was shifted to lower wave number (1 660 cm−1) with a marked decreased in intensity upon calcium binding with the protein and indicated that the binding of calcium to the protein stabilizes the secondary structure of WGSP by changing state of C=O. Moreover, the abundant amino acid residues were found in WGSP to be glutamic and aspartic acids, which accounted for about 26.7% and 9.0% of the total amino acid, respectively, and these amino acids might be beneficial for calcium binding. This study could provide a foundation for the preparation of mineral-containing protein in food industry. This method may have a potential use in food industry for isolation of mineral-containing protein from other sources.%为了开发植物源葡萄籽补钙制剂,该研究通过亚细胞定位试验表明,葡萄籽的胚乳中含有大量的钙元素。通过电泳分析发现,葡萄籽的水溶性蛋白包括2种主要成分,其中一种是11 S球蛋白(蛋白质B),也是最主要的钙结合蛋白,另一种是表观分子量为670 kDa的蛋白质A。在蛋白质组成相同的情况下,用传统的碱溶酸沉法来分离葡萄籽蛋白会导致大量的钙流失。但用30%~50%硫酸铵沉淀法得到的蛋白质得率是(22.5±0.02)g/kg,蛋白质中钙质量分数(3.47%)

  20. Research progress on preparing functional polypeptide from whey protein hydrolysis%乳清蛋白水解制备功能性多肽的研究概况

    Institute of Scientific and Technical Information of China (English)

    李晓东; 蒋琛; 宋惠敏

    2014-01-01

    . In this experiment, whey protein was as raw material, hypocholestero-lemic peptides were separated and prepared from its hydrolysates. In this subject, by-product of cheese was full used, it not only reduced waste of resources and environmental pollution, but also provided a theoretical basis for the commercial development of hypocholesterolemic functional products. This paper reviews the whey protein source of functional polypeptides preparation, separation and purification technology and research status of active peptides.

  1. Self-assembled PEG-b-PDPA-b-PGEM copolymer nanoparticles as protein antigen delivery vehicles to dendritic cells: preparation, characterization and cellular uptake

    Science.gov (United States)

    Li, Pan; Zhou, Junhui; Huang, Pingsheng; Zhang, Chuangnian; Wang, Weiwei; Li, Chen; Kong, Deling

    2017-01-01

    Antigen uptake by dendritic cells (DCs) is a key step for initiating antigen-specific T cell immunity. In the present study, novel synthetic polymeric nanoparticles were prepared as antigen delivery vehicles to improve the antigen uptake by DCs. Well-defined cationic and acid-responsive copolymers, monomethoxy poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate)-block-poly(2-(guanidyl) ethyl methacrylate) (mPEG-b-PDPA-b-PGEM, PEDG) were synthesized by reversible addition-fragmentation chain transfer polymerization of 2-(diisopropylamino)ethyl methacrylate) and N-(tert-butoxycarbonyl) amino ethyl methacrylate monomers, followed by deprotection of tert-butyl protective groups and guanidinylation of obtained primary amines. 1H NMR, 13C NMR and GPC results indicated the successful synthesis of well-defined PEDG copolymers. PEDG copolymers could self-assemble into nanoparticles in aqueous solution, which were of cationic surface charges and showed acid-triggered disassembly contributed by PGEM and PDPA moieties, respectively. Significantly, PEDG nanoparticles could effectively condense with negatively charged model antigen ovalbumin (OVA) to form OVA/PEDG nanoparticle formulations with no influence on its secondary and tertiary structures demonstrating by far-UV circular dichroism and UV–vis spectra. In vitro antigen cellular uptake by bone marrow DCs (BMDCs) indicated using PEDG nanoparticles as antigen delivery vehicles could significantly improve the antigen uptake efficiency of OVA compared with free OVA or the commercialized Alum adjuvant. Moreover, as the surface cationic charges of OVA/PEDG nanoparticle formulations reduced, the uptake efficiency decreased correspondingly. Collectively, our work suggests that guanidinylated, cationic and acid-responsive PEDG nanoparticles represent a new kind of promising antigen delivery vehicle to DCs and hold great potential to serve as immunoadjuvants in the development of vaccines. PMID:28149525

  2. Preparation of Wild Hazelnut Protein Compound Yogurt Beverage%野生榛子复合蛋白酸乳饮料的研制

    Institute of Scientific and Technical Information of China (English)

    李延辉; 郑凤荣; 刘艳霞; 刘超

    2016-01-01

    The fermentation processing of hazelnut protein compound yogurt beverage made from hazelnut and fresh milk was studied by single factors tests and orthogonal tests. Taking milk amount as standards , the optimal fermentation procssing was as follows:starter culture 4%, including Bifidobacteria, Lactobacillus Bulgaricus and S.thermophilus, hazelnut seriflux 30%, sugar 9%. After 3.5 h fermented, the product was rich in nutrition and good flavor.%以鲜牛奶和野生榛仁乳为主要原料,通过单因素试验和正交试验研究开发新型蛋白酸乳饮料制品。试验结果表明,以牛乳质量为计算基准,添加其质量4%的双歧杆菌、保加利亚乳杆菌和嗜热链球菌混合发酵剂,30%打浆后的榛仁浆液,9%的绵白糖,发酵3.5 h后可制得质味俱佳的榛仁蛋白饮料。

  3. Photoactivation of alkyl C-H and silanization: a simple and general route to prepare high-density primary amines on inert polymer surfaces for protein immobilization.

    Science.gov (United States)

    Gan, Shenghua; Yang, Peng; Yang, Wantai

    2009-05-11

    Surface modification through implanting functional groups has been demonstrated to be extremely important to biomedical applications. The usage of organic polymer phase is often required to achieve satisfactory results. However, organic surfaces usually have poor chemical reactivity toward other reactants and target biomolecules because these surfaces usually only consist of simple alkyl (C-H) and/or alkyl ether (ROR') structures. For the first time, we here report the potential to perform silanization techniques on alkyl polymer surface, which provide a simple, fast, inexpensive, and general method to decorate versatile functional groups at the molecular level. As an example, high-density primary amines could be obtained on a model polymer, polypropylene substrate, through the reaction between amine-capped silane, 3-aminopropyltriethoxysilane (APTES) and hydroxylated polypropylene surface. A model protein, immunoglobulin (IgG), could be effectively immobilized on the surface after transforming amines to aldehydes by the aldehyde-amine condensation reaction between glutaraldehyde (GA) and amines. The routes we report here could directly make use of the benefits from well-developed silane chemistry, and hereby are capable of grafting any functionalities on inert alkyl surfaces via changing the terminal groups in silanes, which should instantly stimulate the development of many realms such as microarrays, immunoassays, biosensors, filtrations, and microseparation.

  4. Preparation of gluten free bread enriched with green mussel (Perna canaliculus) protein hydrolysates and characterization of peptides responsible for mussel flavour.

    Science.gov (United States)

    Vijaykrishnaraj, M; Roopa, B S; Prabhasankar, P

    2016-11-15

    Green mussel protein hydrolysates (GMPH) utilization for the enrichment of gluten-free bread followed by characterization of flavour peptides using chromatography and electronic nose techniques have been done. The degree of hydrolysis was carried out in each protease digest, and the higher degree of hydrolysis was observed in pepsin digestion. Gluten-free (GF) bread was formulated by using buckwheat flour (BWF), rice flour (RF) and chickpea flour (CPF) (70:20:10) and GMPH were added in the range of 0-20% in the GF bread for enrichment with GMPH. Radar plot of the electronic nose analysis showed that the sensors P30/2, T30/1 and T70/2 had a higher response to the GF bread and GMPH. Consequently, the peptide sequence was obtained manually by ESI-MS spectra of GMPH (KGYSSYICDK) and F-II (SSYCIVKICDK). Flavour quality was 97% discriminately comparable to the GMPH and F-II fractions. Mussel flavoured GF bread can be included in the celiac diet.

  5. Purification and characterization of Moschatin, a novel type I ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells.

    Science.gov (United States)

    Xia, Heng Chuan; Li, Feng; Li, Zhen; Zhang, Zu Chuan

    2003-10-01

    A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of approximately 29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

  6. Purification and characterization of Moschatin, a novel type Ⅰ ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata),and preparation of its immunotoxin against human melanoma cells

    Institute of Scientific and Technical Information of China (English)

    CHAO TONG; HENG YU FAN; DA YUAN CHEN; XIANG FEN SONG; HEIDE SCHATTEN; QING YUAN SUN

    2003-01-01

    A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of~29 kD. It is a rRNA Nglycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

  7. Expression, Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody Against NrfA%大肠杆菌NrfA蛋白表达、纯化及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    何婷婷; 龚钢明; 高然

    2012-01-01

    [ Objective] This study aimed to clone the E. Coli NrfA gene and construct the pET-28a( + )-NrfA prokaryotic expression vector for preparation of polyclonal antibody against E. Coli NrfA. [ Method] E. Coli NrfA gene was cloned from the E. Coli genome DNA by PCR and inserted into the vector pET-28a( + )to construct prokaryotic expression vector pET-28a( + )-NrfA. E. Coli NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by immunizing rabbit with routine method. The specificity and titer of polyclonal antibody were confirmed by ELISA and Western Blotting. [ Result] The constructed prokaryotic expression vector pET-28a( + )-NrfA was induced by IPTG, the recombinant NrfA protein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained after immunization and purification was about 1 =204 900. Western Blotting analysis indicated that the obtained polyclonal antibody against E. Coli NrfA protein had high titer and high specificity. [Conclusion] E. Coli NrfA gene was cloned and the prokaryotic expression vector pET-28a( + )-NrfA was constructed successfully, and the polyclonal antibody with high titer and high specificity was prepared, which had laid the foundation for the study of NrfA in different strains of bacteria.%[目的]克隆大肠杆菌NrfA基因,构建pET-28a(+ )-NrfA表达载体,制备相应的多克隆抗体并对其进行鉴定.[方法]以大肠杆菌基因组DNA为模板,PCR扩增得到NrfA基因编码区,构建pET-28a(+ )-NrfA表达载体;经IPTG诱导表达并纯化重组蛋白;再免疫新西兰雄兔,制备多克隆抗体;用ELISA方法检测抗体的效价,Western Blotting检测抗体的特异性.[结果]构建的表达载体pET-28a(+)-NrfA在大肠杆菌中诱导后可高效表达NrfA蛋白;免疫获得的多克隆抗体用ELISA检测,其效价为1∶204 900;经Western Blotting分析,抗体的特异性较好.[结论]成功克隆大肠杆菌的NrfA基

  8. Preparation and immunogenicity characteristics analysis of a epitope fusion protein of Epstein-Bar virus encoded latent membrane protein 2A%EBV编码潜伏膜蛋白2A抗原表位的串联表达及其免疫原性分析

    Institute of Scientific and Technical Information of China (English)

    曹清; 唐小军; 李文杰; 熊四平; 毛园; 熊林; 刘玉; 王长军; 冯振卿

    2013-01-01

    Objective:To prepare a immunogenic epitope tandem of latent membrane protein 2A (LMP2A) of Epstein-Ban:virus and analyze its characteristics of immunology.Methods:LMP2A epitopes were analyzed by using DNAstar software,and two immunogenic epitopes were selected due to stronger immunogenicity.The genes of these two epitopes were integrated through gene synthesis,and then cloned into prokaryotic expression vector pET28a.The recombinant epitopes were expressed and purified by E.coli BL21,and then the protein was utilized to immunize mouse to prepare anti-epitope fusion protein polyclonal antibody after the analysis of SDS-PAGE and Western blot assay.The titer of the antibody was assessed by ELISA and the immunohistochemical experiment was performed to test the specificity of the polyclonal antibody for natural LAMP2A.Results:Through prokaryotic expression and purification,high purity fusion protein was obtained.By mouse immunization,anti-LMP2A polyclonal antibody of high titer and specificity was prepared,which can be applied to ELISA and immunohistochemical analysis.Conclusion:An epitope fusion protein,which shares the natural antigen immunogenicity,can be used to prepare the polyclonal antibody specific for LMP2A.This study has laid a solid foundation for screening whole humanized genetic engineering antibody by epitope fusion protein.%目的:制备具有免疫原性的EB病毒潜伏膜蛋白2A (latent membrane protein 2A,LMP2A)的表位串联蛋白并分析其免疫学特性.方法:用DNAstar软件分析LMP2A的抗原表位,将预测的免疫原性较强的两个表位通过基因合成串联在一起,克隆到原核表达载体pET28a中,经大肠杆菌BL21表达并纯化.制备的含LMP2A表位重组蛋白经SDS-PAGE、Western blot鉴定后,免疫小鼠制备多克隆抗体,以ELISA检测抗体的效价,免疫组织化学法检测该抗体对天然LMP2A的特异性.结果通过原核表达与纯化,获得高纯度的表位融合蛋白,经小鼠免疫

  9. 准备配种期雌性水貂适宜日粮蛋白质水平的研究%Evaluation of Different Dietary Protein Levels on Preparative Mating Minks

    Institute of Scientific and Technical Information of China (English)

    蒋清奎; 张志强; 李光玉; 高秀华; 邢秀梅; 杨福合

    2012-01-01

    X The experiment was conducted to evaluate the regularity of digestibility and metabolism of diets with different levels in female minks on preparative mating period. 180 healthy female minks of one year and a half old were randomly assigned into six groups with 30 replicates and each replicate had 1 mink. The treatments were individually fed with 28. 59% (group I),32.31%(group Ⅱ),36. 21%(group Ⅲ),40. 35%(group Ⅳ) protein levels in fresh feed diets and with 32. 66% (group V ) ,40.47% (groupⅥ) protein levels in mixed feed diets. The period of trial lasted for 51 days,including 7 days preset period and 44 days test period. The results showed that the food intake of the fresh feed diets groups was higher than that of the mixed feed diets group,some had significantly difference(P<0. 05), On the item of the digestibility of dry mater, protein and fat,some had significantly difference (P<0. 05). The nitrogen intake, fecal nitrogen, urine nitrogen increased with the protein level in different groups. There was no significantly difference in nitrogen retention, the biology value of protein, net protein usage ratio. However, all of the three indexes tended to decrease when the dietary protein level reached 36. 21%. Of all the indexes, the mixed feed diets group were significantly or extremely lower than the fresh feed diets group(P<0.05 or P< 0.01). In conclusion, the minks had almost equally best nutrient digestibility and availability when the dietary protein level reached 32. 31% and 36. 21% , but in consideration of the feed expense and the particular characteristics of the preparative mating minks, 32. 31% protein level of diet would be the best choice in the preparative mating period, and the mixed feed diets were not recommended for this particular period.%本试验以准备配种期日粮蛋白质水平对水貂营养物质消化率及氮代谢的影响为研究目的.选择经产适龄母貂180只,随机分成6组,每组30个重复,每个重复1只水貂.6

  10. Study on Effect of Ventilation Tube Spacing on Thermal Performance of Cold Storage Floor Antifreezing Ventilation System%通风管间距对冷库地坪通风防冻系统传热性能影响的研究

    Institute of Scientific and Technical Information of China (English)

    贾景福; 郝满晋; 李建华

    2009-01-01

    The research aimed to investigate the effect of ventilation tube spacing on thermal performance of cold storage floor antifreezing ventilation system. A steady three-dimensional mathematical model of heat transfer was set up. The heat-transfer model was simplified reasonably. The definite conditions of the heat-transfer model were confirmed according to the heat-transfer process. The temperature field of this system were simulated and calculated by Phoenics under different kinds of working conditions, such as without circulating the ventilation system, with different tube spacing. The results showed that the effect of using mechanical antifreezing ventilation to cold storage floor was better. They also indicated the effect of ventilation tube spacing was greater on thermal performance of cold storage floor antifreezing ventilation system.%为了研究通风管间距对冷库地坪通风防冻系统传热性能的影响程度,建立了三维稳态传热数学模型,对传热模型进行了合理的简化,根据系统的传热过程确定了传热模型的定解条件,利用Phoenics软件对通风系统未启动和不同通风管间距下的传热模型分别进行了数值模拟分析.结果表明,对冷库地坪采取机械通风防冻的效果良好,通风管间距对冷库地坪通风防冻系统的传热性能有较大影响.

  11. Preparo de hidrolisados protéicos para a análise de aminoácidos Preparation of protein hydrolysates for amino acid analysis

    Directory of Open Access Journals (Sweden)

    Carlos Roberto Bernardi

    2003-12-01

    Full Text Available Amostras de milho, farelo de soja e hidrolisado ácido de caseína foram submetidas à hidrólise ácida em estufa (110 ± 5°C, durante 22horas com ácido clorídrico 6 N, em ampolas de vidro seladas sob vácuo. Os hidrolisados foram preparados por quatro métodos: 1 evaporação em evaporador rotativo sob vácuo; 2 evaporação em câmaras a vácuo em presença de pastilhas de NaOH; 3 evaporação em bloco de aquecimento sob fluxo de argônio; 4 neutralização com solução padrão de citrato/NaOH. Os aminoácidos foram separados e quantificados em analisador específico Beckman System 6300 com derivatização pós-coluna por ninidrina. O método de neutralização dos reagentes da hidrólise apresentou os melhores resultados considerando todos os aminoácidos. Além disso, foi mais rápido e prático no preparo de um grande número de amostras. Quanto aos métodos de evaporação, o uso de evaporador rotativo apresentou perdas significativas apenas para treonina e serina na amostra farelo de soja; sob fluxo de argônio observou-se baixos teores para treonina, fenilalanina e lisina; em câmara sob vácuo verificou-se perdas significativas para treonina, serina e tirosina.Samples of corn, soybean corn and casein (acid hydrolysate were submitted to acid hydrolysis in oven (110 ± 5°C, for 22 hours with 6 N HCl in sealed ampoules under vacuum. Four methods of hydrolysate preparation were used: 1 evaporation to dryness in rotary evaporator under vacuum, 2 evacuated desiccator under vacuum and NaOH, 3 flushing the open vial with argon and 4 neutralisation with standard citrate/NaOH solution. Amino acids were isolated and quantified in specific analyzer Beckman System 6300 with ninhidrin post-column derivatization. The neutralization method presented the best result considering all analysed amino acids. Besides, it was more practical for rapid processing of a large number of samples. With regard to the evaporation methods in the rotary evaporator

  12. How Prepared is Prepared Enough?

    Science.gov (United States)

    Porter-Levy; Macleod; Rickert

    1996-10-01

    A 17-year-old female was in the final stage in treatment of right unilateral cleft lip and palate. She had undergone a number of previous surgeries. Hearing and speech were good on evaluation, and her social and family situation were deemed excellent. After preparatory orthodontics she underwent a Lefort I maxillary advancement. Surgery was successful and she was admitted into postoperative recovery. However, the lack of adequate preoperative preparation caused traumatic reaction from the patient and her parents: anxiety over appearance, crying, refusal of oral fluids and oral care, refusal of analgesia, and refusal to mobilize. The patience and persistence of hospital staff slowly overcame all adversities and the patient moved on to full and successful recovery, but this case prompted changes in preoperative procedures and involvement of patients and their families in postoperative meal selection, planing, and preparation.

  13. 钝顶节旋藻(Arthrospira platensis)2-DE分析蛋白制备方法改进及试用于螺旋手性差异蛋白研究%AN IMPROVED METHOD OF 2-DE ANALYSIS PROTEIN PREPARATION AND USING FOR INVESTIGATION OF HELICAL-HANDED DIFFERENTIAL PROTEINS IN ARTHROSPIRA PLATENSIS

    Institute of Scientific and Technical Information of China (English)

    王景梅; 汪志平; 于金鑫; 刘新颖; 邵斌; 蓝瑾瑾; 马丽芳; 陈子元

    2013-01-01

    Based on the techniques of cell wall breaking by freeze-thaw and proteins extracting step-by-step, protein preparation method for 2-DE analysis of Arthrospira platensis was improved, and then was applied to investigation of helical chirality-related proteins. The results indicated that: (1) After Arthrospira cells were completely ruptured by five cycles of freeze-thaw, about 87% of water-soluble proteins could be extracted by Tris-HCl extractant for three times, and then water-insoluble proteins were extracted by SDS (twelve sodium dodecyl sulfate) extractant from the precipitate. (2) The above water-soluble and water-insoluble proteins were analyzed by two-dimensional gel electrophoresis (2-DE) after they were purified by the method of TCA/acetone. Owing to effective separation and high quality, the amount of detected protein spots in the water-soluble and water-insoluble protein patterns was more than 500 and 760 respectively, and their matching rate was only 7%. (3) By this method, 13 candidate proteins including five new were identified as related to helical chirality in Arthrospira, and their function were involved in photosynthesis, energy metabolism, exocytosis, environmental adaptation, etc. (4) Compared to the existing protein preparation methods for 2-DE analysis of Arthrospira, the method of "breaking cell wall by freeze-thaw and then extracting step-by-step" presented in this paper, has the advantages of simplification in operation and instruments, low cost and more information, and so on.%采用冻融破壁和蛋白质分步提取等技术,对钝顶节旋藻2-DE分析蛋白制备方法作了改进,并试用于螺旋手性差异表达蛋白研究,结果表明:(1)钝顶节旋藻细胞反复冻融5次完全破碎,用Tris-HC1提取液提取3次可提得近87%的水溶性蛋白,再用SDS(十二烷基磺酸钠)提取液可从沉淀中提得水不溶性蛋白;(2)将上述水溶性和水不溶性蛋白分别用TCA/丙酮法纯化后作双向电泳(2-DE)

  14. Preparation and Identification of the Polyclonal Antibody Against Plant Sweet Protein Mabinlin Ⅱ%植物甜蛋白马宾灵(MabinlinⅡ)多克隆抗体的制备与鉴定

    Institute of Scientific and Technical Information of China (English)

    顾文亮; 夏启玉; 姚晶; 胡新文; 郭建春

    2012-01-01

    马宾灵(MabinlinⅡ)是中国所特有的植物马槟榔种子中的甜味蛋白,将其作为新型甜味剂有着广阔的市场前景.为了给重组马宾灵的基因工程应用提供可靠的蛋白质检测与鉴定的抗体,通过离子交换法从马槟榔(Capparis masaikai Lévl)种子中分离纯化马宾灵,将其作为抗原免疫新西兰大白兔,收集免疫后血清经抗原亲和纯化制备马宾灵多克隆抗体.结果表明,制备的马宾灵多克隆抗体经ELISA检测效价比达到1:256000,Western-blot检测表明其具有良好的反应性和特异性.本研究制备的马宾灵多克隆抗体可用于马宾灵在不同生物反应器中表达的检测,为马宾灵的基因工程研究提供灵敏可靠的免疫学鉴定.%The plant sweet protein Mabinlin Ⅱ is found rich in mature seeds of Capparis masaikai Levl which is particular to China and may have wide application prospects in food sweeteners industry. In this study, in order to provide a reliable testing antibody for engineering applications of recombinant Mabinlin Ⅱ, Mabinlin Ⅱ was separated and purified from the mature seeds of Capparis masaikai Levl by ion exchange chromatography. The purified protein was used as antigen to immunize New Zealand rabbits to prepare polyclonal antibody against the Mabinlin Ⅱ protein. The result of Enzyme-Linked Immunosorbnent Assay (ELISA) showed that the titer of the polyclonal antibody to purified Mabinlin Ⅱ was 1:256000, and the polyclonal antibody against the purified Mabinlin E also had highly reactivity and specialty in western blot analysis. The polyclonal antibody against Mabinlin II could be used for detecting the protein amounts of Mabinlin Ⅱ expressed in various bioreactors, which may serve sensitive and reliable methods for immunology detection in genetic engineering research of Mabinlin Ⅱ .

  15. Preparation of antioxidant peptides of whey protein from the buffalo milk%水牛奶乳清蛋白制备抗氧化活性肽工艺的研究

    Institute of Scientific and Technical Information of China (English)

    闭秋华; 孙宁; 白文娟; 陈文硕; 王娇; 李全阳

    2012-01-01

    实验是以水牛奶为原料,分离纯化后得到乳清蛋白。利用碱性蛋白酶、中性蛋白酶、胰蛋白酶、木瓜蛋白酶和胃蛋白酶5种不同的蛋白酶对水牛奶乳清蛋白酶解以制备抗氧化活性多肽。酶筛选结果显示,中性蛋白酶是最适宜酶解水牛奶乳清蛋白制备抗氧化活性肽,其酶解液的还原能力和DPPH自由基清除率较其他4种酶高。探讨酶解反应时pH、温度、时间、酶浓度对酶解反应的水解度、酶解液的还原能力和DPPH自由基的清除率的影响,在单因素试验基础上,采用响应面法对酶解工艺进行优化。结果表明,中性蛋白酶酶解乳清蛋白的最佳工艺参数为:pH为7.4,温度为50.5℃,酶与底物浓度比为2.1%,酶解时间5.0h,此时2mg/mL酶解物的DPPH自由基清除率为32.58%。实测结果与预测值吻合效果良好。%The whey protein was separated from the buffalo milk. The whey protein of the buffalo milk was hydrolyzed with neutrase to prepare antioxidant peptide. Studying the effects of pH, temperature, time and enzyme-to-substrate ratio on degree of hydrolysis, DPPH radical-scavenging capacity and reducing power with neutrase. It was found that the hydrolysates with neutrase had the best effect of reducing ability and scavenging to DPPH free radical. Hydrolysis conditions for preparing protein hydrolysates from whey protein was used single factor experiments and response surface methodology (RSM) to optimize the enzymatic processes. An enzyme to substrate level of 2.1%, temperature of 50.5 %, a hydrolysis time of 5.0 h and the pH of 7.4 were found to be the optimum conditions to obtain a higher DPPH radical- scavenging capacity of hydrolysis, which the DPPH radical-scavenging capacity was up to 32.58%.Therefore, there was a good accordance between the predicted and observed values.

  16. 复合蛋白酶法制备玉米胚蛋白多肽及其抗氧化活性研究%Preparation of corn germ protein polypeptide by compound protease hydrolysis and its antioxidant activity

    Institute of Scientific and Technical Information of China (English)

    李艳娟; 李书国

    2015-01-01

    The corn germ protein polypeptides (CGPP)were prepared from protein of corn germ by com-pound enzyme (alkaline proteases:papain=1∶1 ).The reducibility of polypeptides was selected as a detec-ting index and the conditions of enzymatic hydrolysis were optimized by response-surface method designed by Box-Behnken on the basis of single factor experiment.The optimum enzymolysis conditions of the corn germ protein were determined:protease dosage 9 200 IU/g,substrate concentration 1 1% and hydrolysis time 188 min.The reducibility of polypeptides of corn germ protein was 0.229 under the optimal condi-tions.The polypeptide was added to the noodles to check its ability of oxidation resistance,anti-brown-ing,compared with the effect of ascorbic acid.The results showed that polypeptides of corn germ protein can effectively anti-browning in the storage of fresh noodles.When the additive amount was 0.9%,the effect of anti-browning was the same as ascorbic acid.TPA test results showed that adding 0.9% corn germ protein polypeptides into the noodles can improve its hardness and flexibility to a certain extent.%利用复合酶(碱性蛋白酶∶木瓜蛋白酶=1∶1)酶解玉米胚蛋白制备蛋白多肽,在单因素实验的基础上,采用Box-Behnken设计响应面法,以蛋白多肽的还原力为检测指标,优化酶解条件。确定玉米胚蛋白的最佳酶解条件为:加酶量9200 IU/g,底物浓度11%,酶解时间188 min,在此条件下测定的还原力为0.229。将玉米胚蛋白多肽添加到面条中,考察其在面条中的抗氧化防褐变能力,并与抗坏血酸的添加效果进行对比。结果表明,玉米胚蛋白多肽粉添加到面条中具有防褐变的作用,添加量为0.9%时对鲜面条褐变速率的抑制作用最强,与添加抗坏血酸的效果相当。此外质构仪分析(TPA)实验结果表明,在面条中添加0.9%的玉米胚蛋白多肽粉,对面条的硬度、弹性有一定程度的改善作用。

  17. Preparation of a poly(3'-azido-3'-deoxythymidine-co-propargyl methacrylate-co-pentaerythritol triacrylate) monolithic column by in situ polymerization and a click reaction for capillary liquid chromatography of small molecules and proteins.

    Science.gov (United States)

    Lin, Zian; Yu, Ruifang; Hu, Wenli; Zheng, Jiangnan; Tong, Ping; Zhao, Hongzhi; Cai, Zongwei

    2015-07-01

    Combining free radical polymerization with click chemistry via a copper-mediated azide/alkyne cycloaddition (CuAAC) reaction in a "one-pot" process, a facile approach was developed for the preparation of a poly(3'-azido-3'-deoxythymidine-co-propargyl methacrylate-co-pentaerythritol triacrylate) (AZT-co-PMA-co-PETA) monolithic column. The resulting poly(AZT-co-PMA-co-PETA) monolith showed a relatively homogeneous monolithic structure, good permeability and mechanical stability. Different ratios of monomers and porogens were used for optimizing the properties of a monolithic column. A series of alkylbenzenes, amides, anilines, and benzoic acids were used to evaluate the chromatographic properties of the polymer monolith in terms of hydrophobic, hydrophilic and cation-exchange interactions, and the results showed that the poly(AZT-co-PMA-co-PETA) monolith exhibited more flexible adjustment in chromatographic selectivity than that of the parent poly(PMA-co-PETA) and AZT-modified poly(PMA-co-PETA) monoliths. Column efficiencies for toluene, DMF, and formamide with 35,000-48,000 theoretical plates per m could be obtained at a linear velocity of 0.17 mm s(-1). The run-to-run, column-to-column, and batch-to-batch repeatabilities of the retention factors were less than 4.2%. In addition, the proposed monolith was also applied to efficient separation of sulfonamides, nucleobases and nucleosides, anesthetics and proteins for demonstrating its potential.

  18. Preparation and scaling up of a low phenylalanine enzymatic hydrolysate of bovine whey proteins Preparação e escalonamento de um hidrolisado enzimático de proteínas do soro de leite bovino

    Directory of Open Access Journals (Sweden)

    Marilisa Guimarães Lara

    2005-12-01

    Full Text Available We describe the preparation of pancreatic enzymes hydrolysate of milk whey proteins containing low levels of aromatic amino acids. Pancreatin and trypsin/chymotrypsin (6.3% w/w protein when used to hydrolyze whey proteins for 27 h at 37±2 ºC, released 74% of the Phe, 100% of the Tyr and 100% of the Trp as free amino acids. Most of the free aromatic amino acids present in 2 kg hydrolysate were separated from the remaining peptides and other amino acids by gel filtration on a 15 liter Sephadex G-25 column eluted with 5% acetic acid at 60 liters h-1 at 25ºC. The product, recovered in 37% yield, contained 0.70 mmol Phe, 0.41 mmol Tyr, and Foi descrita a preparação de um hidrolisado de proteínas do soro de leite bovino com enzimas pancreáticas, contendo baixos níveis de aminoácidos aromáticos. Quando utilizadas pancreatina e tripsina/quimotripsina, por 27h a 37±2ºC, foram liberados 74% de Phe, 100% de Tyr e 100% de Trp como aminoácidos livres. A maioria dos aminoácidos aromáticos livres, presentes em dois quilos de hidrolisado (15 litros, foi separada dos peptídeos e outros aminoácidos remanescentes por filtração em coluna de gel de Sephadex G-25C eluída com ácido acético 5%, fluxo de 60 litros por hora a 25ºC. O produto, recuperado com 37% de rendimento, continha 0,70 mmol de Phe, 0,41 mmol de Tyr e <0,01 mmol de Trp/100 mmol de aminoácidos recuperados. A composição em aminoácidos do hidrolisado foi similar às proteínas do soro com as quais foi preparado. Após adição de aminoácidos aromáticos apropriados, ele pode ser usado como fonte de nitrogênio para pacientes com fenilcetonuria ou tirosinemia.

  19. Prokaryotic Expression and Antiserum Preparation of the Coat Protein of Cymbidium Mosaic Virus%建兰花叶病毒CP基因的原核表达及抗血清制备

    Institute of Scientific and Technical Information of China (English)

    罗金水

    2009-01-01

    通过间接酶联免疫检测和电镜观察对从福建省漳州市采集的卡特兰病样进行检测,证明样品感染了建兰花叶病毒.设计一对特异性引物,扩增并克隆病毒分离物的外壳蛋白基因,随后将目的基因插入pET-29a(+)中构建相应的原核表达载体.目的蛋白经诱导表达及纯化后免疫家兔并获得了特异性抗血清.Westem blot检测结果表明.抗血清与诱导表达的CyMV外壳蛋白发生特异性反应.间接酶联免疫法检测结果表明,抗血清可检测病汁液的最低稀释度达1:51 200,最佳工作浓度为1:1000,病汁液灵敏度为0.39 mg/mL,而与TMV等11种同源或异源病毒均无明显的血清学交叉反应.%Cymbidium mosaic virus (CyMV) is one of the most important and worldwide viruses attacking orchids. This virus causes the symptoms of mosaic,chlorosis,necrosis and malformation in the orchids,and has a high economic impact to the orchid industry. Cattleya plants contracted with a disease were collected as samples from Zhangzhou,Fujian,and were identified to be infected with Cymbidium mosaic virus by using ID -ELISA and electronic microscopy assay. One pair of specific primers was designed for amplification of the coat protein(CP) gene from the samples infected with CyMV. The open reading frame encoding CP of CyMV isolate obtained from Zhangzhou,Fujian is 672 bp,encoding a 23.6 ku protein with 223 aa. The expected CP gene was then inserted into the pET-29a(+)vector for prokaryotic expression. And the aimed protein was purified and used to immune the rabbit for antiserum preparation. According the result of ID-ELISA analysis,specific rabbit anti-CyMV serum was prepared with a high titre of 1:51 200,a working concentration of 1:1 000 and sap sensitivity of 0.39 mg/mL. Western blot analysis confirmed that the antiserum reacted strongly and specifically to the CP of CyMV. There were no cross reactions between the antiserums and 11 species of homologous or heterologous

  20. 双酶水解蚕蛹蛋白制备氨基酸饮料的研究%Double Enzyme Hydrolysis Silkworm Chrysalis Protein Preparation Amino Acid Drink Research

    Institute of Scientific and Technical Information of China (English)

    于小磊; 耿丽晶; 郭雪松; 岳昊博

    2013-01-01

    为了充分利用蚕蛹资源,研究双酶水解蚕蛹蛋白的最佳条件,考察影响酶解效果的主要因素。确定了新鲜蚕蛹经漂烫、提脂、酶解、调配等工序制成蛋白饮料的工艺条件,消除了蚕蛹蛋白的褐变和凝沉现象。采用L9(33)正交试验设计方法,分别对酶解蚕蛹蛋白的温度、酶解时间、酶用量进行实验与结果分析,以确定双酶水解蚕蛹蛋白的最适条件。结果表明:最佳酶解条件为胰蛋白酶:pH 7.0,温度为45℃,酶解时间5 h,酶用量为1 g。木瓜蛋白酶:pH 6.0,温度为70℃,酶解时间4 h,酶用量为1 g。结论胰蛋白酶加入到木瓜蛋白酶酶解蚕蛹蛋白过程中,可使酶解液中氨态氮含量有明显的提高。经检验产品中细菌总数、大肠菌群、致病菌等指标符合相关食品卫生标准。氨基酸饮料中总糖(以蔗糖计)含量为14%,酸度(以柠檬酸计)含量为0.68 g/L,其产品符合饮料的理化检验及卫生指标。%In order to fully utilize the pupa resources, this article had studied the dual-pupa protein hydrolysis of the optimal conditions,determined the impact of the main factors enzyme effect. A fresh study by blanching chrysalis, to fat, enzyme, the deployment of processes such as protein drinks made from the process, eliminate the chrysalis of the Browning and condensate Shen phenomenon. By three factors and three levels orthogonal L9(33) Test methods, respectively, the pupa protein enzyme temperature, hydrolysis time, the amount of experiments and analysis, the double-pupa protein hydrolysis the optimum conditions. The results showed that:the best conditions for the enzyme trypsin: pH value of 7.0, the temperature of 45℃, hydrolysis time 5h, the amount of 1 g. Papain: pH value of 6.0, the temperature of 70℃, hydrolysis time 4 h, the amount of 1 g. Conclusion trypsin into the pupa protein enzyme papain process, will enable enzyme solution of ammonia

  1. Comparison of Functional Properties of Soybean Protein Isolates Prepared by Different Methods%不同方法制备的大豆分离蛋白功能性质的比较研究

    Institute of Scientific and Technical Information of China (English)

    郑二丽; 杨晓泉; 吴娜娜

    2012-01-01

    采用普通的碱溶酸沉方法、Samoto法、钙离子沉淀法制备出几种大豆分离蛋白,并对其产率、总脂含量及溶解性、乳化性等物化性质和功能性质进行系统比较。结果表明:钙离子沉淀法制备的大豆分离蛋白(Less-LPSPI)的产率是碱溶酸沉大豆分离蛋白(APP)产率的65%,但高于由Samoto法提取的7S与11S两种大豆分离蛋白产率之和;Less-LP SPI的总脂含量比APP降低了45%,由Samoto法提取的亲脂性蛋白(LP)中总脂含量达到7.48%,而7S与11S蛋白中脂含量分别为1.45%、2.36%。在功能性质上,LP的溶解性、乳化活性均比较差,7S、11S质量比1:2混合蛋白的溶解性最好,而Less-LP SPI在pH≤10时溶解性低于APP;乳化性方面,Less-LP SPI的乳化活性稍低于7S、11S质量比1:2的混合蛋白及APP,但乳化稳定性远高于后者。总体上,Less-LP SPI脂含量少、乳化稳定性好,方法简单,且可提高其货架期。%Several soybean protein isolates(SPI) were prepared by acid precipitation,Samoto method and calcium-precipitation method,respectively.The characteristics including protein yield,total lipid content,solubility and emulsification of these SPIs were investigated.Our results showed that the yield of calcium-precipitation protein(Less-LP SPI) was lower than that of acid precipitation protein(APP),but higher than 7S,11S extracted by Samoto fractionation procedure.The lipid content of Less-LP SPI was decreased by 45% when compared with that of APP.The lipid content of lipophilic protein(LP) obtained from Samoto fractionation was 7.48%,but the lipid contents in 7S and 11S were 1.45% and 2.36%,respectively.The Samoto LP-rich fraction had inferior solubility and emulsifying properties.Less-LP SPI had lower solubility(pH ≤ 10) and emulsifying activity than APP,while Less-LP SPI had superior emulsion stability.In summary,Less-LP SPI has lower lipid and better emulsion stability and therefore can improve the shelf

  2. Racemic protein crystallography.

    Science.gov (United States)

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  3. Preparation of Garlic Oil Microcapsules by Soybean Protein Isolate-Chitosan Coacervation%大豆分离蛋白-壳聚糖复凝聚法制备大蒜油微胶囊的工艺研究

    Institute of Scientific and Technical Information of China (English)

    黄国清; 肖军霞; 仇宏伟

    2014-01-01

    研究以大豆分离蛋白和壳聚糖为复合壁材,以大蒜油为芯材,探讨了采用复凝聚法制备大蒜油微胶囊的最佳工艺。结果表明:大蒜油与复合壁材按质量比1∶2混合,在45℃,6000 r/min下乳化5 min后,调节乳状液的pH 值至6.5,于100 r/min搅拌反应10 min,再经18.75 U/g SPI的谷氨酰胺转氨酶固化1 h,大蒜油微胶囊的包埋效率和产率最高分别达到了69.20%和64.77%。在此条件下制备的大蒜油微胶囊具有典型的蒜香味,且大蒜油的刺激性气味有所降低。%The preparation of garlic oil microcapsules through complex coacervation by using soybean protein and chitosan as the complex wall material and garlic oil as the core material is studied.After the garlic oil is mixed with the complex wall material in the mass ratio of 1∶2 and emulsified at 6000 r/min and 45 ℃ for 5 min,the emulsion pH is adjusted to 6.5 and stirred at 100 r/min for 10 min.Then transglutaminase is added in the dosage of 18.75 U/g SPI to cross-link the microcap-sules for 1 h.Under such conditions,the microencapsulation efficiency and yield reach 69.20% and 64.77% respectively.The garlic oil prepared under the optimized conditions possesses the typical fla-vour of garlic oil with reduced pungent odor.

  4. Novel dimeric β-helical model of an ice nucleation protein with bridged active sites

    Directory of Open Access Journals (Sweden)

    Walker Virginia K

    2011-09-01

    Full Text Available Abstract Background Ice nucleation proteins (INPs allow water to freeze at high subzero temperatures. Due to their large size (>120 kDa, membrane association, and tendency to aggregate, an experimentally-determined tertiary structure of an INP has yet to be reported. How they function at the molecular level therefore remains unknown. Results Here we have predicted a novel β-helical fold for the INP produced by the bacterium Pseudomonas borealis. The protein uses internal serine and glutamine ladders for stabilization and is predicted to dimerize via the burying of a solvent-exposed tyrosine ladder to make an intimate hydrophobic contact along the dimerization interface. The manner in which PbINP dimerizes also allows for its multimerization, which could explain the aggregation-dependence of INP activity. Both sides of the PbINP structure have tandem arrays of amino acids that can organize waters into the ice-like clathrate structures seen on antifreeze proteins. Conclusions Dimerization dramatically increases the 'ice-active' surface area of the protein by doubling its width, increasing its length, and presenting identical ice-forming surfaces on both sides of the protein. We suggest that this allows sufficient anchored clathrate waters to align on the INP surface to nucleate freezing. As PbINP is highly similar to all known bacterial INPs, we predict its fold and mechanism of action will apply to these other INPs.

  5. 香石竹斑驳病毒外壳蛋白基因的原核表达及抗血清制备%Prokaryotic Expression and Antiserum Preparation of the Coat Protein Gene of Carnation mottle virus

    Institute of Scientific and Technical Information of China (English)

    李晓鹏; 王连春; 彭杰军; 姬盼; 李准; 孔宝华; 陈海如

    2013-01-01

    香石竹斑驳病毒(Carnation mottle virus,CarMV)是侵染香石竹的主要病毒.本研究从已鉴定的CarMV毒源植物中提取总RNA,克隆外壳蛋白(cp)基因后构建pET30a-CP重组质粒,并转入BL21 (DE3) pLysS进行原核表达.通过对诱导温度、初菌浓度、IPTG的浓度等表达条件进行优化,目的蛋白在37℃,IPTG终浓度为1.0 mmol/L的条件下,诱导表达6h后获得最高表达量.采用割胶的方法纯化cp蛋白,纯化后的目的蛋白免疫小鼠获得了特异性抗血清.经Western-blot检测,结果表明所制备的抗血清与诱导表达的重组蛋白及接种CarMV的香石竹样品均发生特异性结合.利用所制备抗血清对昆明地区的48个香石竹样品进行抽检,结果表明香石竹样品的带毒率为79.2%.本研究为大规模抗血清生产、检测应用和深入研究该病毒cp基因与寄主的互作奠定基础.%Carnation mottle virus (CarMV) is one of the important viruses infecting carnation. In this study, RNA was extracted from CarMV infected carnation, and coat protein (cp) gene was cloned into pET30a ( + ) and transformed into BL21 (DE3) pLysS. The optimal conditionsor the highest expres-sion of coat protein was expressing at 37℃ for 6 h and inducing with 1. 0 mmol/L IPTG. The expressed protein was purified with gradient SDS-PAGE and the antiserum against the protein was raised in Kun-ming mice. The result of Western-blot analysis confirmed that the antiserum reacted strongly and spe-cifically recombinant CarMV cp and CarMV infected samples. Forty-eight carnation samples in Kun-ming were tested with the antibody made in this experiment, of which 79. 2% of the samples were in-fected by CarMV. This study provides a good basis for massive CarMV cp antiserum preparation CarMV detection and interaction studies between CarMV and host plants.

  6. 芹菜过敏原蛋白Api g1.02的克隆、表达及单抗制备%Gene Cloning, Protein Expression and Monoclonal Antibody Preparation of Celery Allergen Protein Api g1.02

    Institute of Scientific and Technical Information of China (English)

    王海艳; 袁飞; 吴亚君; 杨海荣; 李刚; 陈颖

    2012-01-01

    RNA was extracted from celery, the celery allergen protein Api gl.02 gene was amplified by HT-PCR and cloned into the pET-32a expression vector. The lecombinant plasmid pET-32a- Api g 1.02 was transformed into E coli BL21 (DE3) pLys for expression after being identified with PCR, enzyme digestion and sequencing. The transformed bacteria were cultivated and induced with 1 mmol/L isoproyhhio-B-D-galactoside (IPTG), the bacteria sediment was collected after exposure to IPTG for 2, 3, 4, 5, 6, 7h and analyzed by SDS-PAGE. Monoclonal antibody was prepared after the expressed protein being purified and was analysed by western blot. The results showed the celery allergen protein Api gl.02 was expressed efficiently in fusion protein form, the molecular weight of the fusion protein was 35 ku, the expression level of the fusion protein was highest after exposure to IPTG for 6 h and accounted for approximately 40% of the total cellular proteins; The western blot analysis showed the recombinant protein had a good immunogenicity. This result is a better basic for immunological detection research on celery allergen protein.%提取芹菜总RNA,反转录PCR (RT-PCR)扩增出过敏原蛋白Api g1.02基因,经PCR、酶切和序列测定后,得到重组质粒pET-32a- Api g1.02.将重组质粒转化到BL21 (DE3)pLys感受态细胞中,用1mmol/L异丙基硫代-B-D-半乳糖苷(IPTG)对转化菌进行诱导表达及SDS-PAGE分析后,将表达蛋白提取纯化,制备单克隆抗体并进行Western blot分析.结果表明芹菜过敏原蛋白Api g1.02在体外获得了高效表达,表达融合蛋白分子质量约35 ku,诱导6h后表达量最高,占菌体总蛋白的40%左右;制备的单克隆抗体能与表达蛋白发生免疫印迹反应,说明所表达的蛋白具有良好的免疫原性.该研究结果为建立芹菜过敏原蛋白的免疫学检测方法奠定了基础.

  7. 大豆分离蛋白-水溶性大豆多糖可食性复合膜的制备与性质%Preparation and characterization of soy protein isolates and soluble soybean polysaccharides composite film

    Institute of Scientific and Technical Information of China (English)

    赵欣; 管骁

    2013-01-01

    Soy protein isolates (SPI) and soluble soybean polysaccharides (SSPS) were used as the main raw material to prepare edible composite film.The properties of the film were studies.The ratio of SPI to SSPS,the a ddition amount of glycerin and sodium alginate and calcium ion concentration were chosen as the effect factors.Single factor experiment and orthogonal tests were conducted to explore the optimal formula of SPI and SSPS composite film.The properties of the composite film were evaluated by water soluble,water vapor permeability,tensile strength,and breaking elongation rate.The results showed that the optimal formula of SPI and SSPS composite film was:SPI to SSPS ratio 1∶ 7,glycerin addition amount 2%,sodium alginate 4%,and calcium ion concentration 1.0mol/L.Under the optimal process conditions,the comprehensive performance of SPI and SSPS compound film had highest score of 67.79.%以大豆分离蛋白(soy protein isolates,SPI)和水溶性大豆多糖(soluble soybean polysaccharides,SSPS)为主要原料进行了可食性复合膜的制备与性质研究.综合考虑SPI与SSPS的比例、甘油、海藻酸钠添加量及钙离子浓度等影响因素,通过单因素与正交实验对成膜配方进行研究,得到了复合膜的最佳配比,并从水溶性、水蒸气透过性、抗拉伸强度、断裂延伸率等方面对膜的性质进行了综合评价.结果显示:在SPI∶ SSPS质量比为1∶7,甘油添加量2%,海藻酸钠添加量4%,Ca2浓度为1.0mol/L的条件下,复合膜的综合性能评分最高,为67.8.

  8. 大豆分离蛋白/聚乙烯醇塑料的制备及性能研究%Preparation and Properties of Soy Protein Isolate/PVA Plastics

    Institute of Scientific and Technical Information of China (English)

    刘亮; 张丽叶

    2011-01-01

    Polymer blends were prepared via thermo-pressing mixtures of soy protein isolate(SPI) and poly (vinyl alcohol) (PVA) using glycerol as a plasticizer. The structure, morphology, and properties of the blends were investigated using X-ray diffraction, dynamic mechanical analyzer, differential scanning calorimetry analyzer, universal electronic tensile testing machine, and scanning electron microscopy. Phase separation was observed between glycerol and SPI forming glycerol-rich and protein-rich domains. The addition of PVA significantly damaged the crystalline structure of SPI in glycerol-rich domain, and increased its glass transition temperature. When the content of PVA was 1 phr, the tensile strength of SPI/PVA increased by 41.5 %, and the 24 h water absorption of SPI/PVA decreased from 134.86 % to 77.38 %, based on neat SP1.%将聚乙烯醇(PVA)按不同比例与大豆分离蛋白(SPI)混合,采用丙三醇作为增塑剂,经模压成型制备SPI/PVA塑料,采用X射线衍射仪、动态热机械分析仪、差示扫描量热仪、万能电子拉力试验机、扫描电子显微镜等研究了SPI/PVA塑料的结构、形态和性能。结果表明,丙三醇增塑的SPI会出现微相分离,即出现富丙三醇微区和富蛋白微区,而PVA的加入主要破坏了SPI在富丙三醇微区的晶体结构,并使富丙三醇微区的玻璃化转变温度向高温方向偏移。PVA的加入还明显提高了SPI/PVA塑料的拉伸强度,当PVA含量为1份时,其拉伸

  9. 冰亲和吸附装置对胶原抗冻肽的分离纯化%Purification of antifreeze collagen peptides by ice affinity adsorption protocol

    Institute of Scientific and Technical Information of China (English)

    阮功成; 曹慧; 徐斐; 于劲松

    2015-01-01

    抗冻肽是一类可以非依数性降低体系冰点的多肽,在生命体内具有非常重要的生理作用.研究搭建了冰亲和吸附装置,并以微生物保护活性为指标,优化了其对猪皮胶原酶解复合物中胶原抗冻肽的吸附条件,同时对分离产物的性质进行了研究.结果表明:在胶原酶解复合物浓度为1 mg/mL,吸附时间为10h,吸附温度为-5℃,吸附次数为2次的条件下,所获得的胶原抗冻肽对微生物的低温保护活性最强.经过冰亲和吸附后,胶原抗冻肽的主要洗脱峰之间的分离效果明显增加,其胶原抗冻肽的分子质量主要分布小于1 000 Da、且富含甘氨酸、脯氨酸和羟脯氨酸.%Antifreeze collagen peptides is a family of peptides which can lowering the freezing point of the system non-colligatively.This plays an important role in protecting organisms from freezing injury and damage.In this study, we studied the ice affinity adsorption equipment, used micro-organisms protection as the index to optimize the condition of purifying pig skin collagen peptides by enzymatic hydrolysis.The optimum conditions were: crude mixture concentration of 1 mg/mL, at-5 ℃ for 10 hour, adsorption twice.After ice affinity adsorption, collagen peptides peaks had better separation effect, the molecular weight mainly distributed within the range of 1000 Da, and major collagen amino acids were Proline and Hydroxyproline.

  10. Batter and method for preparing a pasta

    NARCIS (Netherlands)

    Wind, P.; Linden, van der E.

    2011-01-01

    This invention describes a batter that is suitable for preparing a pasta. The batter comprises water, a starch and a protein, whereby the weight ratio between the protein and the total amount of starch in the batter is represented by the symbol y and whereby the weight percentage of the total amount

  11. Recombinant human brain myelin basic protein and its antibody preparation%重组人脑髓鞘碱性蛋白及其抗体的制备与研究

    Institute of Scientific and Technical Information of China (English)

    刘戟; 王若菡; 刘鱼; 陈俊杰

    2006-01-01

    BACKGROUND: Central nervous system (CNS) myelin is made up of 70% lipids and 30% proteins with human brain myelin basic protein (hMBP) constituting 1/3 of the proteins. MBP is a family of proteinswith four isoforms only in human brain. OBJECTIVE: To investigate the expression of MBP and its immunological function. DESIGN: Single sample study. SETTING: Department of Biochemistry and Molecule Biology, Huaxi College of Priclinical Medicine and Forensic Medicine, Sichuan University. MATERIALS: This experiment was conducted at Department of Bio chemistry and Molecule Biology, Huaxi College of Priclinical Medicine and Forensic Medicine, Sichuan University from August 2003 to March 2004. Relative molecular weight was 215000 hMBP cDNA clone pGEMP,prokaryotic expression recombinant vector pGEX-5T, T4 DNA Ligase,calf intestine alkaline phosphates, X-gal, IPTG, 123 Ladder (BRL), nitrocellu lose ,4-chloro-1-Naphthol;MBP and Anti- MBP ELISA kit. INTERVENTIONS: ①hBMP gene cDNA clone segment was digested with EcoR1 and BamH1; ②Construction and transformation of the recombinant expression vector p5TMP; ③ The growth and induced expression of the recombinant expression vector transformed bacteria; ④ Preparation of the antibody of recombinant hMBP. MAIN OUTCOME MEASURES: ① Detection and identification of the expressed protein product; ② Detection and identification of hMBP anti body. RESULTS: ① Screening and identification of recombinant MBP: The 4 999 bp pGEX-5T DNA and 557 bp MBP inserted fragment were obtained, indicating that the white colonies contained recombinant expression plasmid of exogenous DNA; ② A dense band disappeared from the control plasmid while a new special polypeptide band with apparent molecular weight 42 000 was detected in recombinant cell lysate; ③After 5 subcutaneous injections, antibody was obtained at 1:16 titer. The specificity of the antibody to MBP was confirmed.CONCLUSION: Compared with the original expression vector, the new

  12. Preparation and Characterization of the Monoclonal Antibodies Against p27 Protein of Avian Leukosis Virus%禽白血病病毒 p27蛋白单克隆抗体的制备及鉴定

    Institute of Scientific and Technical Information of China (English)

    谢华丽; 邵华斌; 杨峻; 程国富; 胡薛英; 谷长勤; 张万坡; 罗青平

    2013-01-01

    The purified recombinant protein p27 of avian leukosis virus as an immunogen was used to immu-niz Balb/c mice.By cell culture and monoclonal antibody of dilution method,mAb named 2F3,5C2 and 5C7 were prepared against avian leukosis virus p27 protein by fusing myeloma cell line SP2/0 with spleen lymphocytes of immunized Balb/c mice.By biological characteristic analysis,the number of three strain hybridoma cell chromosomes was about 90.Then immunological identifications were conducted by means of ELISA/Western blot and IFA,the three mAb against p27 protein of ALV possessed high antibody ti-ters and good specificity.The result showed that the three McAbs had good reactogenicity.These three McAbs can be used as a valuable tool for avian leukosis virus epitope analysis,the detection of the virus and Kit research.%以纯化的禽白血病病毒 p27重组蛋白作为免疫原,按常规方法免疫 Balb/c 小鼠,取其脾细胞与 SP2/0细胞融合,通过细胞培养和有限稀释法制备单克隆抗体,最终获得了3株能稳定分泌禽白血病病毒 p27蛋白单克隆抗体的细胞株2F3、5C2和5C7。经生物学特性分析,3株杂交瘤细胞染色体数均为90条左右,通过 ELISA、Western blot 和间接免疫荧光等方法对所获得的3株单克隆抗体进行了鉴定,证明3株单克隆抗体细胞株所分泌的抗体效价高,而且均能够和禽白血病病毒 p27蛋白特异性发生反应。说明该3株单克隆抗体均具有与禽白血病病毒 p27蛋白发生反应的反应原性。3株单克隆抗体的成功获得为禽白血病病毒抗原表位的分析,病毒的检测以及试剂盒的研发奠定了基础。

  13. Optimizing expression and antibody preparation of recombinant Streptococcus mutans surface protein%重组变异链球菌表面蛋白的可溶性表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    金洁; 樊明文; 李宇红

    2012-01-01

    Objective The soluble protein lecombinant Streptococcus mutans surface protein (rPAc) was expressed in Escherichia coli(E.coli) after the optimization of inducing conditions. The antisenim against rPAc was obtained by immunizing mice with the purified rPAc. Methods The soluble expression of rPAc in Exoli was further optimized by means of different culture conditions. Polyclonal antibody was made by immunizing mice with purified rPAc. Western blot and enzyme linked immunosorbent assay (ELISA) were carried out to identify the immunocompetence of the antibody. Results The highest soluble expression level of rPAc was obtained at Luria-Bertani (LB) medium (pH=7.2) when optical density (OD600cm) was 0.6 after being induced at 30℃ for 4 h and the concentration of isopropyl (S-D-l-thigalactopyrano