Ice Shaping Properties, Similar to That of Antifreeze Proteins, of a Zirconium Acetate Complex

Science.gov (United States)

Deville, Sylvain; Viazzi, Céline; Leloup, Jérôme; Lasalle, Audrey; Guizard, Christian; Maire, Eric; Adrien, Jérôme; Gremillard, Laurent

2011-01-01

The control of the growth morphologies of ice crystals is a critical issue in fields as diverse as biomineralization, medicine, biology, civil or food engineering. Such control can be achieved through the ice-shaping properties of specific compounds. The development of synthetic ice-shaping compounds is inspired by the natural occurrence of such properties exhibited by antifreeze proteins. We reveal how a particular zirconium acetate complex is exhibiting ice-shaping properties very similar to that of antifreeze proteins, albeit being a radically different compound. We use these properties as a bioinspired approach to template unique faceted pores in cellular materials. These results suggest that ice-structuring properties are not exclusive to long organic molecules and should broaden the field of investigations and applications of such substances. PMID:22028886

Ice shaping properties, similar to that of antifreeze proteins, of a zirconium acetate complex.

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Sylvain Deville

Full Text Available The control of the growth morphologies of ice crystals is a critical issue in fields as diverse as biomineralization, medicine, biology, civil or food engineering. Such control can be achieved through the ice-shaping properties of specific compounds. The development of synthetic ice-shaping compounds is inspired by the natural occurrence of such properties exhibited by antifreeze proteins. We reveal how a particular zirconium acetate complex is exhibiting ice-shaping properties very similar to that of antifreeze proteins, albeit being a radically different compound. We use these properties as a bioinspired approach to template unique faceted pores in cellular materials. These results suggest that ice-structuring properties are not exclusive to long organic molecules and should broaden the field of investigations and applications of such substances.

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

Science.gov (United States)

Duman, John G

2015-06-01

Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization - a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones - and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants. © 2015. Published by The Company of Biologists Ltd.

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

International Nuclear Information System (INIS)

Hakim, Aaron; Thakral, Durga; Zhu, Darren F.; Nguyen, Jennifer B.

2012-01-01

A novel hyperactive antifreeze protein from R. inquisitor (RiAFP) has been overexpressed, purified and crystallized. A complete native X-ray diffraction data set was recorded to 1.3 Å resolution. Antifreeze proteins (AFPs) are a specialized evolutionary adaptation of a variety of bacteria, fish, arthropods and other organisms to inhibit ice-crystal growth for survival in harsh subzero environments. The recently reported novel hyperactive AFP from Rhagium inquisitor (RiAFP) is the second distinct type of AFP in beetles and its structure could reveal important molecular insights into the evolution of AFPs. For this purpose, RiAFP was overexpressed in Escherichia coli, purified and crystallized at 293 K using a combination of 23% PEG 3350 and 0.2 M ammonium sulfate as a precipitant. X-ray diffraction data were collected to 1.3 Å resolution using a synchrotron-radiation source. The crystals belonged to the trigonal space group P3 1 21 (or P3 2 21), with unit-cell parameters a = b = 46.46, c = 193.21 Å

Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers.

Science.gov (United States)

Graham, Ben; Bailey, Trisha L; Healey, Joseph R J; Marcellini, Moreno; Deville, Sylvain; Gibson, Matthew I

2017-12-11

Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large volumes of solvent "antifreezes". Herein, we report polyproline as a minimum (bio)synthetic mimic of antifreeze proteins that is accessible by solution, solid-phase, and recombinant methods. We demonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein

International Nuclear Information System (INIS)

Petit-Haertlein, Isabelle; Blakeley, Matthew P.; Howard, Eduardo; Hazemann, Isabelle; Mitschler, Andre; Haertlein, Michael; Podjarny, Alberto

2009-01-01

Perdeuterated type III antifreeze protein has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.85 Å resolution from a 0.13 mm 3 crystal. The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D 2 O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 Å were obtained using a ‘radically small’ perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35 mm, corresponding to a volume of 0.13 mm 3

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...... of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs....... 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...

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.

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

International Nuclear Information System (INIS)

Yang, Q.; Feng, H.

2013-01-01

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

The mysteries of memory effect and its elimination with antifreeze proteins

Energy Technology Data Exchange (ETDEWEB)

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

2008-07-01

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

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...

Janus effect of antifreeze proteins on ice nucleation.

Science.gov (United States)

Liu, Kai; Wang, Chunlei; Ma, Ji; Shi, Guosheng; Yao, Xi; Fang, Haiping; Song, Yanlin; Wang, Jianjun

2016-12-20

The mechanism of ice nucleation at the molecular level remains largely unknown. Nature endows antifreeze proteins (AFPs) with the unique capability of controlling ice formation. However, the effect of AFPs on ice nucleation has been under debate. Here we report the observation of both depression and promotion effects of AFPs on ice nucleation via selectively binding the ice-binding face (IBF) and the non-ice-binding face (NIBF) of AFPs to solid substrates. Freezing temperature and delay time assays show that ice nucleation is depressed with the NIBF exposed to liquid water, whereas ice nucleation is facilitated with the IBF exposed to liquid water. The generality of this Janus effect is verified by investigating three representative AFPs. Molecular dynamics simulation analysis shows that the Janus effect can be established by the distinct structures of the hydration layer around IBF and NIBF. Our work greatly enhances the understanding of the mechanism of AFPs at the molecular level and brings insights to the fundamentals of heterogeneous ice nucleation.

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

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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.

Solution Structure of an Antifreeze Protein CfAFP-501 from Choristoneura fumiferana

International Nuclear Information System (INIS)

Li Congmin; Guo Xianrong; Jia Zongchao; Xia Bin; Jin Changwen

2005-01-01

Antifreeze proteins (AFPs) are widely employed by various organisms as part of their overwintering survival strategy. AFPs have the unique ability to suppress the freezing point of aqueous solution and inhibit ice recrystallization through binding to the ice seed crystals and restricting their growth. The solution structure of CfAFP-501 from spruce budworm has been determined by NMR spectroscopy. Our result demonstrates that CfAFP-501 retains its rigid and highly regular structure in solution. Overall, the solution structure is similar to the crystal structure except the N- and C-terminal regions. NMR spin-relaxation experiments further indicate the overall rigidity of the protein and identify a collection of residues with greater flexibilities. Furthermore, Pro91 shows a cis conformation in solution instead of the trans conformation determined in the crystal structure

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

International Nuclear Information System (INIS)

Ahn, Mija; Murugan, Ravichandran N.; Bang, Jeong Kyu; Kim, Hak Jun; Shin, Song Yub; Kim, Eunjung; Lee, Jun Hyuck

2012-01-01

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

Seasonal changes in antifreeze protein gene transcription and water content of beetle Microdera punctipennis (Coleoptera, Tenebrionidae) from Gurbantonggut desert in Central Asia.

Science.gov (United States)

Hou, F; Ma, J; Liu, X; Wang, Y; Liu, X N; Zhang, F C

2010-01-01

Desert beetle Microdera punctipennis (Coleoptera: Tenebriondae) is a special species in Gurbantonggut Desert in Central Asia. To investigate the possible strategy it employs for cold survival, seasonal changes in supercooling point (SCP), body water content, haemolymph osmolality and antifreeze protein gene (Mpafp) expression were measured over 13 months. Our results show SCPs in M. punctipennis adults changed from -8.0°C in summer to -18.7°C in winter. During winter, adults endured modest water loss; total water decreased from 65.4 percent in summer to 55.9% in winter. Mpafp mRNAs level increased by 13.1 fold from summer to early winter, and haemolymph osmolality increased accordingly from 550 mOsm to 1486 mOsm. Correlation coefficient of Mpafp mRNAs level and SCP indicates that Mpafp mRNA explained 65.3 percent of the variation in SCPs. The correlation between Mpafp mRNA level and total water reflected an indirect influence of antifreeze protein on water content via reducing SCP.

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.

Hyperactive antifreeze proteins from longhorn beetles: some structural insights.

Science.gov (United States)

Kristiansen, Erlend; Wilkens, Casper; Vincents, Bjarne; Friis, Dennis; Lorentzen, Anders Blomkild; Jenssen, Håvard; Løbner-Olesen, Anders; Ramløv, Hans

2012-11-01

This study reports on structural characteristics of hyperactive antifreeze proteins (AFPs) from two species of longhorn beetles. In Rhagium mordax, eight unique mRNAs coding for five different mature AFPs were identified from cold-hardy individuals. These AFPs are apparently homologues to a previously characterized AFP from the closely related species Rhagium inquisitor, and consist of six identifiable repeats of a putative ice binding motif TxTxTxT spaced irregularly apart by segments varying in length from 13 to 20 residues. Circular dichroism spectra show that the AFPs from both species have a high content of β-sheet and low levels of α-helix and random coil. Theoretical predictions of residue-specific secondary structure locate these β-sheets within the putative ice-binding motifs and the central parts of the segments separating them, consistent with an overall β-helical structure with the ice-binding motifs stacked in a β-sheet on one side of the coil. Molecular dynamics models based on these findings show that these AFPs would be energetically stable in a β-helical conformation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Antifreeze life cycle assessment (LCA

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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.

Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein.

Science.gov (United States)

Ideta, Atsushi; Aoyagi, Yoshito; Tsuchiya, Kanami; Nakamura, Yuuki; Hayama, Kou; Shirasawa, Atsushi; Sakaguchi, Kenichiro; Tominaga, Naomi; Nishimiya, Yoshiyuki; Tsuda, Sakae

2015-01-01

Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24-48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP-embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.

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

2015-10-08

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.

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.

Efficiency of Composite Binders with Antifreezing Agents

Science.gov (United States)

Ogurtsova, Y. N.; Zhernovsky, I. V.; Botsman, L. N.

2017-11-01

One of the non-heating methods of cold-weather concreting is using concretes hardening at negative temperatures. This method consists in using chemical additives which reduce the freezing temperature of the liquid phase and provide for concrete hardening at negative temperatures. The non-heating cold-weather concreting, due to antifreezing agents, allows saving heat and electric energy at the more flexible work performance technology. At selecting the antifreezing components, the possibility of concreting at temperatures up to minus 20 °C and combination with a plasticizer contained in the composite binder were taken into account. The optimal proportions of antifreezing and complex agents produced by MC-Bauchemie Russia for fine-grained concretes were determined. So, the introduction of antifreezing and complex agents allows obtaining a structure of composite characteristic for cement stone in the conditions of below zero temperatures at using different binders; the hydration of such composite proceeded naturally. Low-water-demand binders (LWDB) based composites are characterized by a higher density and homogeneity due to a high dispersity of a binder and its complicated surface providing for a lot of crystallization centers. LWDB contains small pores keeping water in a liquid form and promoting a more complete hydration process.

Anti-freezing of air-cooled heat exchanger by switching off sectors

International Nuclear Information System (INIS)

Wang, Weijia; Kong, Yanqiang; Huang, Xianwei; Yang, Lijun; Du, Xiaoze; Yang, Yongping

2017-01-01

Highlights: • The anti-freezing of air-cooled heat exchanger by switching off sectors is studied. • The water side heat loads of various sectors are compared for different cases. • Anti-freezing turbine back pressure is proposed and obtained for various cases. • As wind speed increases, the energy efficiency can be clearly improved by sector off. • By switching frontal sector off, anti-freezing operation is most energy efficient. - Abstract: With the air side huge heat transfer surface, the air-cooled heat exchanger will take a serious freezing risk in cold winter. Therefore, it is of benefit to the safe operation of natural draft dry cooling system to propose the anti-freezing measures. In this work, the flow and heat transfer models of the cooling air coupling with the circulating water, are developed and numerically simulated for the anti-freezing by switching various sectors off. The local thermo-flow fields of cooling air are presented, and the water side heat loads of various sectors are compared for various cases. The anti-freezing turbine back pressure is proposed and obtained for the energy efficiency analysis. The results show that the sector switching off approach can effectively prevent the air-cooled heat exchanger from freezing and improve the energy efficiency of the cooling system, especially at high wind speeds. Moreover, with the frontal sector switching off, the most energy efficient anti-freezing operation of natural draft dry cooling system can be achieved.

The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein

Science.gov (United States)

Daley, Margaret E.; Sykes, Brian D.

2003-01-01

Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the β-helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the 3Jαβ 1H-1H scalar coupling constants, the χ1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand. PMID:12824479

Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy

International Nuclear Information System (INIS)

Daley, Margaret E.; Sykes, Brian D.

2004-01-01

The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance 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 1 H- 13 C NOE were determined in this study. The CαH relaxation measurements were compared to the previously measured 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 χ 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

Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

Science.gov (United States)

Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

2014-01-01

Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

Bacterial Ice Crystal Controlling Proteins

Science.gov (United States)

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

2014-01-01

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

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.

Mimicking the properties of antifreeze glycoproteins: synthesis and characterization of a model system for ice nucleation and antifreeze studies.

Science.gov (United States)

Hederos, Markus; Konradsson, Peter; Borgh, Annika; Liedberg, Bo

2005-08-25

Synthesis of beta-D-Gal-(1 --> 3)-beta-D-GalNAc coupled to HOC2H4NHCOC15H30SH is described. This compound was coadsorbed at various proportions with C2H5OC2H4NHCOC15H30SH to form statistically mixed self-assembled monolayers (SAMs) on gold in an attempt to mimic the properties of the active domain in antifreeze glycoproteins (AFGPs). The monolayers were characterized by null ellipsometry, contact angle goniometry, X-ray photoelectron spectroscopy, and infrared reflection-absorption spectroscopy. The disaccharide compound adsorbed preferentially, and SAMs prepared at a solution molar ratio >0.3 displayed total wetting. The mixed SAMs showed well-organized alkyl chains up to a disaccharide surface fraction of 0.8. The amount of gauche conformers in the alkyls increased rapidly above this point, and the monolayers became disordered and less densely packed. Furthermore, the generated mixed SAMs were subjected to water vapor at constant relative humidity and the subsequent ice crystallization on a cooled substrate was monitored via an optical microscope. Interestingly, rapid crystallization occurred within a narrow range of temperatures on mixed SAMs with a high disaccharide content, surface fraction >0.3. The reported crystallization temperatures and the ice layer topography were compared with results obtained for a much simpler reference system composed of -OH/-CH3 terminated n-alkanethiols in order to account for changes in topography of the water/ice layer with surface energy. Although preliminary, the obtained results can be useful in the search for the molecular mechanism behind the antifreeze activity of AFGPs.

ANTIFREEZE PROTEINS IN PLANTS: AN OVERVIEW WITH AN INSIGHT INTO THE DETECTION TECHNIQUES INCLUDING NANOBIOTECHNOLOGY

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Bhavana Sharma

2014-08-01

Full Text Available Antifreeze proteins (AFPs are a class of polypeptides which enables various organisms to survive subzero temperatures and have been found in vertebrates, invertebrates, plants, fungi and lichens. AFPs possess the characteristic thermal hysteresis (TH and ice recrystallization inhibition (IRI properties which allow them to adsorb the surface of ice crystals and inhibit their growth and recrystallization. AFPs are also known as ice restructuring proteins due to their ability to modify ice crystal morphology which leads to formation of hexagonal shape ice crystals in the presence of AFPs and disc shape AFPs in its absence. AFPs have various applications in medical, agricultural, industrial and biotechnological field. This review provides an overview of the AFPs, their TH and IRI properties and potential biotechnological applications of AFPs. Various conventional detection methods like Capillary assay and Differential Scanning Calorimetry (DSC with their advantages and disadvantages are discussed in detail along with the commonly used Splat assay and Nanoliter osmometer. Moreover, a novel, high-throughput and efficient nanobiotechnological method for AFP detection is also discussed. The method is based on colorimetric detection of freeze-labile gold nanoparticles and can provide an alternative to overcome the limitations of conventional methods by providing quick and easy way to screen AFPs in multiple systems simultaneously

Potential Antifreeze Compounds in Present-Day Martian Seepage Groundwater

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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.

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...

Modified Antifreeze Liquids for Use on Surfaces

Science.gov (United States)

Lynn, R. O.

1983-01-01

Report presents results of evaluation of two antifreeze liquids, dimethyl sulfoxide and ethylene glycol and five viscosity modifiers: gelatin, gum tragacanth, starch, agarose powder and citrus pectin. Purpose of evaluation to find best way of dealing with frost formation on Space Shuttle.

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.

Calorimetric determination of inhibition of ice crystal growth by antifreeze protein in hydroxyethyl starch solutions.

Science.gov (United States)

Hansen, T N; Carpenter, J F

1993-01-01

Differential scanning calorimetry and cryomicroscopy were used to investigate the effects of type I antifreeze protein (AFP) from winter flounder on 58% solutions of hydroxyethyl starch. The glass, devitrification, and melt transitions noted during rewarming were unaffected by 100 micrograms/ml AFP. Isothermal annealing experiments were undertaken to detect the effects of AFP-induced inhibition of ice crystal growth using calorimetry. A premelt endothermic peak was detected during warming after the annealing procedure. Increasing the duration or the temperature of the annealing for the temperature range from -28 and -18 degrees C resulted in a gradual increase in the enthalpy of the premelt endotherm. This transition was unaffected by 100 micrograms/ml AFP. Annealing between -18 and -10 degrees C resulted in a gradual decrease in the premelt peak enthalpy. This process was inhibited by 100 micrograms/ml AFP. Cryomicroscopic examination of the samples revealed that AFP inhibited ice recrystallization during isothermal annealing at -10 degrees C. Annealing at lower temperatures resulted in minimal ice recrystallization and no visible effect of AFP. Thus, the 100 micrograms/ml AFP to have a detectable influence on thermal events in the calorimeter, conditions must be used that result in significant ice growth without AFP and visible inhibition of this process by AFP. Images FIGURE 8 PMID:7690257

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. © 2016 The Protein Society.

Synthesis of Structures Related to Antifreeze Glycoproteins

OpenAIRE

Fyrner, Timmy

2005-01-01

In this thesis, synthesis of structures related to antifreeze glycoproteins (AFGPs) are presented. Synthetic routes to a protected carbohydrate derivative, 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside, and a tBu-Ala-Thr-Ala-Fmoc tripeptide, are described. These compounds are meant to be used in the assembly of AFGPs and analogues thereof. A Gal-GlcN disaccharide was synthesized via glycosylation between the donor, bromo-2-O-benzo...

Plant ice-binding (antifreeze) proteins

Science.gov (United States)

Proteins that determine the temperature at which ice crystals will form in water-based solutions in cells and tissues, that bind to growing ice crystals, thus affecting their size, and that impact ice re-crystallization have been widely-documented and studied in many plant, bacterial, fungal, insect...

Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection

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Nusrat Yasmin

2017-01-01

Full Text Available A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+ and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

Protein-Based Nanoparticle Preparation via Nanoprecipitation Method

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Mohamad Tarhini

2018-03-01

Full Text Available Nanoparticles are nowadays largely investigated in the field of drug delivery. Among nanoparticles, protein-based particles are of paramount importance since they are natural, biodegradable, biocompatible, and nontoxic. There are several methods to prepare proteins containing nanoparticles, but only a few studies have been dedicated to the preparation of protein- based nanoparticles. Then, the aim of this work was to report on the preparation of bovine serum albumin (BSA-based nanoparticles using a well-defined nanoprecipitation process. Special attention has been dedicated to a systematic study in order to understand separately the effect of each operating parameter of the method (such as protein concentration, solvent/non-solvent volume ratio, non-solvent injection rate, ionic strength of the buffer solution, pH, and cross-linking on the colloidal properties of the obtained nanoparticles. In addition, the mixing processes (batch or drop-wise were also investigated. Using a well-defined formulation, submicron protein-based nanoparticles have been obtained. All prepared particles have been characterized in terms of size, size distribution, morphology, and electrokinetic properties. In addition, the stability of nanoparticles was investigated using Ultraviolet (UV scan and electrophoresis, and the optimal conditions for preparing BSA nanoparticles by the nanoprecipitation method were concluded.

In silico characterization of antifreeze proteins using computational ...

Indian Academy of Sciences (India)

WINTEC

GRAVY, Grand Average Hydropathy. structure of protein (3D coordinates data). The 3D structure of AFPs Q01758 and P05140 were gener- ated by homology modelling using Esypred34 server. The similar 3D structures (for the AFPs Q01758 and. P05140 sequences) in the Protein Data bank. (www.rscb.org) were identified ...

Preparation of GST Fusion Proteins.

Science.gov (United States)

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-04-01

INTRODUCTIONThis protocol describes the preparation of glutathione-S-transferase (GST) fusion proteins, which have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

Studies on new antifreeze protein from the psychrophilic diatom ...

African Journals Online (AJOL)

Administrator

2011-09-12

Sep 12, 2011 ... 27-kDa protein modified with His-tag. According to bioinformatics data, a comparison ... for plant biotechnology application on tomato. MATERIALS AND METHODS. The ESTs library was ... specific expression in tomato as previously described (Deng, 2003). Sequence analysis and prediction of protein ...

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

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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

Preparing and evaluating delivery systems for proteins

DEFF Research Database (Denmark)

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

2006-01-01

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

Experimental studies of thermal preparation of internal combustion engine

Science.gov (United States)

Karnaukhov, N. N.; Merdanov, Sh M.; V, Konev V.; Borodin, D. M.

2018-05-01

In conditions of autonomous functioning of road construction machines, it becomes necessary to use its internal sources. This can be done by using a heat recovery system of an internal combustion engine (ICE). For this purpose, it is proposed to use heat accumulators that accumulate heat of the internal combustion engine during the operation of the machine. Experimental studies have been carried out to evaluate the efficiency of using the proposed pre-start thermal preparation system, which combines a regular system based on liquid diesel fuel heaters and an ICE heat recovery system. As a result, the stages of operation of the preheating thermal preparation system, mathematical models and the dependence of the temperature change of the antifreeze at the exit from the internal combustion engine on the warm-up time are determined.

Characterization and Preparation of Broken Rice Proteins Modified by Proteases

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Lixia Hou

2010-01-01

Full Text Available Broken rice is an underutilized by-product of milling. Proteins prepared from broken rice by treatments with alkaline protease and papain have been characterized with regard to nutritional and functional properties. The protein content and the protein recovery were 56.45 and 75.45 % for alkaline protease treatment, and 65.45 and 46.32 % for papain treatment, respectively. Protease treatment increased the lysine and valine content, leading to a more balanced amino acid profile. Broken rice proteins had high emulsifying capacity, 58.3–71.6 % at neutral pH, and adequate water holding capacity, ranging from 1.96 to 2.93 g/g of proteins. At pH=7.0, the broken rice protein had the highest water holding capacity and the best interfacial activities (emulsifying capacity, emulsifying stability, foaming capacity and foaming stability, which may be the result of the higher solubility at pH=7.0. The interfacial activities increased with the increase in the mass fraction of broken rice proteins. The proteins prepared by the papain treatment had higher water holding capacity (p>0.05, emulsifying capacity (p0.05 than alkaline protease treatment at the same pH or mass fraction. To test the fortification of food products with broken rice proteins, pork sausages containing the proteins were prepared. Higher yield of the sausages was obtained with the increased content of broken rice proteins, in the range of 2.0–9.0 %. The results indicate that broken rice proteins have potential to be used as the protein fortification ingredient for food products.

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

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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.

QUALITY AND NUTRITIONAL VALUE OF WHEAT BREAD WITH A PREPARATION OF OAT PROTEINS

OpenAIRE

Renata Sabat; KrzysztofBuksa; Barbara Mickowska; Rafał Ziobro; Halina Gambuś; Dorota Pastuszka

2012-01-01

The aim of this study was to investigate possibilities and advisability of the use of oats insoluble protein preparation for the production of wheat bread, in order to increase the amount of protein and biological value of protein in this kind of bakery. Research material consisted of the preparation of insoluble oats protein, wheat flour and wheat bread made with the share of oat protein: 5%, 7.5% and 10%, by weight of wheat flour. AOAC methods (2006) were used to determine protein, β-D-gluc...

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

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. © 2015 Published by Elsevier Ltd.

Preparation of Modified Films with Protein from Grouper Fish

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M. A. Valdivia-López

2016-01-01

Full Text Available 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.

Preparation and characterization of magnetic polymer nanospheres with high protein binding capacity

International Nuclear Information System (INIS)

Liu Xianqiao; Guan Yueping; Liu Huizhou; Ma Zhiya; Yang Yu; Wu Xiaobing

2005-01-01

A novel magnetic support with high protein binding capacity was prepared by mini-emulsion polymerization. The magnetic poly(methacrylate-divinylbenzene) nanospheres prepared are 390 nm in diameter with narrow size distribution and star-like external morphology which leads to a large increase in specific surface area. Experimental results indicate that the maximum protein binding capacity is 316 mg bovine hemoglobin (BHb)/g support

EFFECT OF ADDITION OF PROTEIN PREPARATIONS ON THE QUALITY OF EXTRUDED MAIZE EXTRUDATES

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Elżbieta Rytel

2013-02-01

Full Text Available The method of extrusion enables enrichment of snacking products with protein preparation simultaneously providing a high quality of end products. Maize semolina with particle size of 500-1250 μm was used as raw material and as additives soybean protein isolate, distillery yeast Safethanol 3035 and laboratory obtained potato protein preparation. Snacks were determined for contents of dry matter, protein, fat as well as for texture, volume weight, bulk density and sensory traits. The application of 3% and 6% additions of protein preparations in extruded snacks production was found to exert a beneficial effect on their chemical composition without deteriorating sensory characteristics. The higher, 6%, addition of proteins to extrudates turned out to significantly reduce content of fat (by 18% and ash (by 50%, and to increase total protein content by 26%, on average, in the products examined as compared to the samples free of additives. The addition of potato protein to extrudates, especially at the higher dose (6%, significantly improved their consistency and texture, simultaneously diminishing the expansion ratio of ready products. The higher (6% addition of yeast protein applied in the production of extrudates resulted in slight deterioration of their taste and aroma, yet had a positive effect on the structure and expansion ratio of the ready products. The extrudates produced with the addition of soybean protein were characterized by a good expansion ratio, uniform structure, irrespective of preparation dose and simultaneously demonstrated lower bulk mass as compared to the other products obtained in the experiment.

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

Science.gov (United States)

Ouyang, Ruizhuo; Lei, Jianping; Ju, Huangxian

2010-05-01

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 × 1018 g - 1, which well matched their maximum binding capacity toward bovine Hb. Both the simple method for preparation of MIPs and the magnetic nanospheres showed good application potential in fast separation, effective concentration and selective biosensing of large protein molecules.

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.

Preparation and mechanical properties of edible rapeseed protein films.

Science.gov (United States)

Jang, Sung-Ae; Lim, Geum-Ok; Song, Kyung Bin

2011-03-01

Edible films were manufactured from rapeseed oil extraction residues. To prepare rapeseed protein (RP) films, various concentrations of plasticizers and emulsifiers were incorporated into the preparation of a film-forming solution. The optimal conditions for the preparation of the RP film were 2% sorbitol/0.5% sucrose as plasticizer and 1.5% polysorbate 20 as an emulsifier. In addition, RP blend films were prepared. Gelidium corneum or gelatin was added to improve the physical properties of the RP film, and the highest tensile strength value of the films was 53.45 MPa for the 3% RP/4% gelatin film. Our results suggest that the RP-gelatin blend film is suitable for applications in food packaging. Edible RP films prepared in the present investigation can be applied in food packaging.

Preparation and characterization of alginate microspheres for sustained protein delivery within tissue scaffolds

International Nuclear Information System (INIS)

Zhai Peng; Chen, X B; Schreyer, David J

2013-01-01

Tissue engineering scaffolds are designed not only to provide structural support for the repair of damaged tissue, but can also serve the function of bioactive protein delivery. Here we present a study on the preparation and characterization of protein-loaded microspheres, either alone or incorporated into mock tissue scaffolds, for sustained protein delivery. Alginate microspheres were prepared by a novel, small-scale water-in-oil emulsion technique and loaded with fluorescently labeled immunoglobulin G (IgG). Microsphere size appears to be influenced by the magnitude and distribution of force generated by mechanical stirring during emulsion. Protein release studies show that sustained IgG release from microspheres could be achieved and that application of a secondary coating of chitosan could further slow the rate of protein release. Preservation of bioactivity of released IgG protein was confirmed using an immunohistochemical assay. When IgG-loaded microspheres were incorporated into mock scaffolds, initial protein release was diminished and the overall time course of release was extended. The present study demonstrates that protein-loaded microspheres can be prepared with a controlled release profile and preserved biological activity, and can be incorporated into scaffolds to achieve sustained and prolonged protein delivery in a tissue engineering application. (paper)

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes.

Science.gov (United States)

Yamaguchi, K; Asakawa, H

1988-07-01

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.

Effects of antifreezes and bundled material on the stability and optical limiting in aqueous suspensions of carbon nanotubes

Energy Technology Data Exchange (ETDEWEB)

Vlasov, Andrey Yu.; Venediktova, Anastasia V.; Sokolova, Ekaterina P. [Department of Chemistry, St. Petersburg State University, Universitetsky Pr. 26, St. Petersburg 198504 (Russian Federation); Videnichev, Dmitry A. [S.I. Vavilov State Optical Institute, Birzhevaya line 12, St. Petersburg 199034 (Russian Federation); St. Petersburg National Research University ITMO, Kronverksky pr. 49, St. Petersburg 197101 (Russian Federation); Lasers and Optical Systems JSC, Birzhevaya line 12, St. Petersburg 199034 (Russian Federation); Kislyakov, Ivan M. [S.I. Vavilov State Optical Institute, Birzhevaya line 12, St. Petersburg 199034 (Russian Federation); St. Petersburg National Research University ITMO, Kronverksky pr. 49, St. Petersburg 197101 (Russian Federation); Obraztsova, Elena D. [A.M. Prokhorov Institute of General Physics, Russian Academy of Sciences, Vavilov Str. 38, Moscow 119991 (Russian Federation)

2012-12-15

This work gives data on the stability of dispersions of single wall carbon nanotubes stabilized by sodium dodecylbenzenesulfonate in binary polar solvents ''water + antifreeze'' (glycerol, polyethyleneglycole) with eutectic compositions. The absorption spectra of the suspensions demonstrate no changes during 1-year storage with temperature spanning from -40 to +40 C. The systems provide relevant optical power limiting properties, the one with glycerol showing good resistance to optical bleaching effects. We also demonstrate that aqueous dispersions of nanotubes exhibit considerable enhancement of optical limiting parameters alongside an increase of the bundled material populace. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

Science.gov (United States)

Katsura, Kazushige; Matsuda, Takayoshi; Tomabechi, Yuri; Yonemochi, Mayumi; Hanada, Kazuharu; Ohsawa, Noboru; Sakamoto, Kensaku; Takemoto, Chie; Shirouzu, Mikako

2017-11-01

Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

DEFF Research Database (Denmark)

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

into automated robotic high-throughput screens, which allows subsequent protein quantification. In this integrated solution, samples are directly forwarded to automated cell lysate preparation and preparation of dilution series, including reformatting to a protein spotter-compatible format after the high......-throughput screening. Tracking of huge sample numbers and data analysis from a high-content screen to RPPAs is accomplished via MIRACLE, a custom made software suite developed by us. To this end, we demonstrate that the RPPAs generated in this manner deliver reliable protein readouts and that GAPDH and TFR levels can...

Sulfonate-terminated carbosilane dendron-coated nanotubes: a greener point of view in protein sample preparation.

Science.gov (United States)

González-García, Estefanía; Gutiérrez Ulloa, Carlos E; de la Mata, Francisco Javier; Marina, María Luisa; García, María Concepción

2017-09-01

Reduction or removal of solvents and reagents in protein sample preparation is a requirement. Dendrimers can strongly interact with proteins and have great potential as a greener alternative to conventional methods used in protein sample preparation. This work proposes the use of single-walled carbon nanotubes (SWCNTs) functionalized with carbosilane dendrons with sulfonate groups for protein sample preparation and shows the successful application of the proposed methodology to extract proteins from a complex matrix. SEM images of nanotubes and mixtures of nanotubes and proteins were taken. Moreover, intrinsic fluorescence intensity of proteins was monitored to observe the most significant interactions at increasing dendron generations under neutral and basic pHs. Different conditions for the disruption of interactions between proteins and nanotubes after protein extraction and different concentrations of the disrupting reagent and the nanotube were also tried. Compatibility of extraction and disrupting conditions with the enzymatic digestion of proteins for obtaining bioactive peptides was also studied. Finally, sulfonate-terminated carbosilane dendron-coated SWCNTs enabled the extraction of proteins from a complex sample without using non-environmentally friendly solvents that were required so far. Graphical Abstract Green protein extraction from a complex sample employing carbosilane dendron coated nanotubes.

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

Preparation of recombinant coat protein of Prunus necrotic ringspot virus.

Science.gov (United States)

Petrzik, K; Mráz, I; Kubelková, D

2001-02-01

The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

Preparation of encapsulated proteins dissolved in low viscosity fluids

International Nuclear Information System (INIS)

Ehrhardt, Mark R.; Flynn, Peter F.; Wand, A. Joshua

1999-01-01

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

QUALITY AND NUTRITIONAL VALUE OF WHEAT BREAD WITH A PREPARATION OF OAT PROTEINS

Directory of Open Access Journals (Sweden)

Renata Sabat

2012-02-01

Full Text Available The aim of this study was to investigate possibilities and advisability of the use of oats insoluble protein preparation for the production of wheat bread, in order to increase the amount of protein and biological value of protein in this kind of bakery. Research material consisted of the preparation of insoluble oats protein, wheat flour and wheat bread made with the share of oat protein: 5%, 7.5% and 10%, by weight of wheat flour. AOAC methods (2006 were used to determine protein, β-D-glucan and dietary fiber in raw materials and final products. Amino acid composition was measured with the help of amino acid analyzer AAA 400 and used to calculate chemical score (CS and the integrated index of essential amino acids (EAAI, according to FAO/WHO/UNU, 2007. Quality of breads was evaluated by their volume, baking yield and total baking loss, and organoleptic assessment. Bread crumb texture profile was analyzed by texture analyzer TA.XT Plus.

Protein preparations in the development of media fragrances in technology of food products

OpenAIRE

I. N. Tolpigina; I. E. Martemianova; L. V. Antipova; I. V. Polenov

2013-01-01

Investigated the sorption properties of plant and animal proteins, common on the Russian market. Installed the recommended dosage of the preparations of animal protein and vegetable origin, as well as the biological value.

Protein preparations in the development of media fragrances in technology of food products

Directory of Open Access Journals (Sweden)

I. N. Tolpigina

2013-01-01

Full Text Available Investigated the sorption properties of plant and animal proteins, common on the Russian market. Installed the recommended dosage of the preparations of animal protein and vegetable origin, as well as the biological value.

Statistical optimization of the growth factors for Chaetoceros neogracile using fractional factorial design and central composite design.

Science.gov (United States)

Jeong, Sung-Eun; Park, Jae-Kweon; Kim, Jeong-Dong; Chang, In-Jeong; Hong, Seong-Joo; Kang, Sung-Ho; Lee, Choul-Gyun

2008-12-01

Statistical experimental designs; involving (i) a fractional factorial design (FFD) and (ii) a central composite design (CCD) were applied to optimize the culture medium constituents for production of a unique antifreeze protein by the Antartic microalgae Chaetoceros neogracile. The results of the FFD suggested that NaCl, KCl, MgCl2, and Na2SiO3 were significant variables that highly influenced the growth rate and biomass production. The optimum culture medium for the production of an antifreeze protein from C. neogracile was found to be Kalleampersandrsquor;s artificial seawater, pH of 7.0ampersandplusmn;0.5, consisting of 28.566 g/l of NaCl, 3.887 g/l of MgCl2, 1.787 g/l of MgSO4, 1.308 g/l of CaSO4, 0.832 g/l of K2SO4, 0.124 g/l of CaCO3, 0.103 g/l of KBr, 0.0288 g/l of SrSO4, and 0.0282 g/l of H3BO3. The antifreeze activity significantly increased after cells were treated with cold shock (at -5oC) for 14 h. To the best of our knowledge, this is the first report demonstrating an antifreeze-like protein of C. neogracile.

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

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)

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

Directory of Open Access Journals (Sweden)

Nitzan Krinsky

Full Text Available 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.

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

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. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

[Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

Science.gov (United States)

Song, Xiaoli; Liu, Xiaoyun; Cai, Lei; Wu, Jianwei; Wang, Jihua

2015-12-01

In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

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.

Antioxidative properties of milk protein preparations fermented by Polish strains of Lactobacillus helveticus.

Science.gov (United States)

Skrzypczak, Katarzyna W; Gustaw, Waldemar Z; Jabłońska-Ryś, Ewa D; Michalak-Majewska, Monika; Sławińska, Aneta; Radzki, Wojciech P; Gustaw, Klaudia M; Waśko, Adam D

2017-01-01

The increasing significance of food products containing substances with antioxidative activi- ties is currently being observed. This is mainly due to the fact that pathogenic changes underlying some diseases are related to the carcinogenic effects of free radicals. Antioxidative compounds play an important role in supporting and enhancing the body’s defense mechanisms, which is useful in preventing some civili- zation diseases. Unfortunately, it has been already proved that some synthetic antioxidants pose a potential risk in vivo. Therefore, antioxidant compounds derived from a natural source are extremely valuable. Milk is a source of biologically active precursors, which when enclosed in structural protein sequences are inactive. The hydrolysis process, involving bacterial proteolytic enzymes, might release biopeptides that act in various ways, including having antioxidant properties. The objective of this study was to determine the antioxidant properties of milk protein preparations fermented by Polish strains of L. helveticus. The research also focused on evaluating the dynamics of milk acidification by these strains and analyzing the textural properties of the skim milk fermented products obtained. The research studied Polish strains of L. helveticus: B734, 141, T80 and T105, which have not yet been used industrially. The antioxidant properties of 1% (w/v) solutions of milk protein preparations (skim milk powder, caseinoglycomacropeptide and α-lactoalbumin) fermented by these strains were determined by neutralizing the free radicals with 2,2-diphenyl-1-picrylhydrazyl (DPPH˙). Moreover, solutions of skim milk powder (SMP) fermented by the microorganisms being tested were analyzed on gel electrophoresis (SDS-PAGE). The dynamics of milk acidification by these microorganisms was also analyzed L. helveticus strains were used to prepare fermented regenerated skim milk products that were subjected to texture profile analysis (TPA) performed using a TA-XT2i

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.

Preparation of mesoporous silica microparticles by sol-gel/emulsion route for protein release.

Science.gov (United States)

Vlasenkova, Mariya I; Dolinina, Ekaterina S; Parfenyuk, Elena V

2018-04-06

Encapsulation of therapeutic proteins into particles from appropriate material can improve both stability and delivery of the drugs, and the obtained particles can serve as a platform for development of their new oral formulations. The main goal of this work was development of sol-gel/emulsion method for preparation of silica microcapsules capable of controlled release of encapsulated protein without loss of its native structure. For this purpose, the reported in literature direct sol-gel/W/O/W emulsion method of protein encapsulation was used with some modifications, because the original method did not allow to prepare silica microcapsules capable for protein release. The particles were synthesized using sodium silicate and tetraethoxysilane as silica precursors and different compositions of oil phase. In vitro kinetics of bovine serum albumin (BSA) release in buffer (pH 7.4) was studied by Fourier transform infrared (FTIR) and fluorescence spectrometry, respectively. Structural state of encapsulated BSA and after release was evaluated. It was found that the synthesis conditions influenced substantially the porous structure of the unloaded silica particles, release properties of the BSA-loaded silica particles and structural state of the encapsulated and released protein. The modified synthesis conditions made it possible to obtain the silica particles capable of controlled release of the protein during a week without loss of the protein native structure.

Ice cream structure modification by ice-binding proteins.

Science.gov (United States)

Kaleda, Aleksei; Tsanev, Robert; Klesment, Tiina; Vilu, Raivo; Laos, Katrin

2018-04-25

Ice-binding proteins (IBPs), also known as antifreeze proteins, were added to ice cream to investigate their effect on structure and texture. Ice recrystallization inhibition was assessed in the ice cream mixes using a novel accelerated microscope assay and the ice cream microstructure was studied using an ice crystal dispersion method. It was found that adding recombinantly produced fish type III IBPs at a concentration 3 mg·L -1 made ice cream hard and crystalline with improved shape preservation during melting. Ice creams made with IBPs (both from winter rye, and type III IBP) had aggregates of ice crystals that entrapped pockets of the ice cream mixture in a rigid network. Larger individual ice crystals and no entrapment in control ice creams was observed. Based on these results a model of ice crystals aggregates formation in the presence of IBPs was proposed. Copyright © 2017 Elsevier Ltd. All rights reserved.

A comparison of tissue preparation methods for protein extraction of cocoa (Theobroma cacao L. pod

Directory of Open Access Journals (Sweden)

Ascensión Martínez-Márquez

2017-04-01

Full Text Available Cocoa, Theobroma cacao L. is one of the main tropical industrial crops. Cocoa has a very high level of interfering substances, such as polysaccharides and phenolic compounds that could prevent the isolation of suitable protein. Efficient methods of protein extraction are a priority to successfully apply proteomic analyses. We compared and evaluated two methods (A and B of tissue preparation for total protein extract by phenol/SDS extraction protocol. The difference in the application of the two methods was that extensively washed dry powder of pod tissue were made in Method A, whereas that crude extract were prepared Method B. Extracted proteins were examined using one-dimensional electrophoresis (1-D. Results show that each extraction method isolated a unique subset of cocoa pod proteome. Principal component analysis showed little variation in the data obtained using Method A, while that in Methods B showed no low reproducibility, thus demonstrating that Method A is a reliable for preparing cocoa pod proteins. The protocol is expected to be applicable to other recalcitrant plant tissues and to be of interest to laboratories involved in plant proteomics analyses. A combination of extraction approaches is recommended for increasing proteome coverage when using gel-based isolation techniques.

Physicochemical properties and storage stability of soybean protein nanoemulsions prepared by ultra-high pressure homogenization.

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Xu, Jing; Mukherjee, Dipaloke; Chang, Sam K C

2018-02-01

This study investigated the effects of the ultrahigh pressure homogenization (pressure, protein concentration, oil phase fraction, pH, temperature, and ionic strength) and storage on the properties of nanoemulsions (100-500nm range), which were stabilized by laboratory-prepared soybean protein isolate (SPI), β-conglycinin (7S) and glycinin (11S). The nanoemulsions made with SPI, 7S and 11S proteins exhibited considerable stability over various ionic strengths (0-500mM NaCl), pH (7), thermal treatments (30-60°C) and storage (0-45days). The far-UV spectra of SPI, 7S, 11S dispersions, and SPI-, 7S-, 11S protein-stabilized nanoemulsions were analyzed for the protein structural changes following lipid removal. The ultra-high pressure homogenization changed the secondary structure of SPI, 7S, 11S proteins in the nanoemulsions, and enhanced their stability. This study demonstrated that SPI, 7S, and 11S proteins can be used as effective emulsifiers in nanoemulsions prepared by ultra-high pressure homogenization. Copyright © 2017. Published by Elsevier Ltd.

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

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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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

International Nuclear Information System (INIS)

Tada, Masahito; Shinohara, Yoshinori; Kato, Ichiro; Hiraga, Koichi; Aizawa, Tomoyasu; Demura, Makoto; Mori, Yoshihiro; Shinoda, Hiroyuki; Mizuguchi, Mineyuki; Kawano, Keiichi

2006-01-01

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

Methods for the preparation of protein-oligonucleotide-lipid constructs.

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Takasaki, Jennifer; Raney, Sameersingh G; Chikh, Ghania; Sekirov, Laura; Brodsky, Irina; Tam, Ying; Ansell, Steven M

2006-01-01

A mixture of ionizable cationic lipids, steric barrier lipids, and colipids is used to encapsulate oligonucleotide DNA in lipidic particles called SALP. This material is under development as an adjuvant for vaccines. Previously we have shown that coupling the antigen directly to the surface of SALP can lead to enhanced immunological responses in vivo. Two different methods for preparing ovalbumin-SALP were assessed in this work. Originally the conjugates were prepared by treating SALP containing a maleimide-derivatized lipid with thiolated ovalbumin, a method we refer to as active coupling. This reaction was found to be difficult to control and generally resulted in low coupling efficiencies. The issues relating to this approach were characterized. We have recently developed alternative techniques based on first coupling ovalbumin to a micelle and then incubating the resultant product with SALP, methods we refer to as passive coupling. We have shown that this method allows accurate control of the levels of protein associated SALP and does not suffer from surface saturation effects seen with the active coupling method that places maximum limits on the amount of protein that can be coupled to the SALP surface. The products from the passive coupling protocol are shown to have activity comparable to those derived from the active coupling protocol in investigations of in vivo immune responses.

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.

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

International Nuclear Information System (INIS)

Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O.; Tumpey, Terrence M.; Pushko, Peter

2016-01-01

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.

Microspheres for protein delivery prepared from amphiphilic multiblock copolymers. 1. Influence of preparation techniques on particle characteristics and protein delivery.

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Bezemer, J M; Radersma, R; Grijpma, D W; Dijkstra, P J; van Blitterswijk, C A; Feijen, J

2000-07-03

The entrapment of lysozyme in amphiphilic multiblock copolymer microspheres by emulsification and subsequent solvent removal processes was studied. The copolymers are composed of hydrophilic poly(ethylene glycol) (PEG) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks. Direct solvent extraction from a water-in-oil (w/o) emulsion in ethanol or methanol did not result in the formation of microspheres, due to massive polymer precipitation caused by rapid solvent extraction in these non-solvents. In a second process, microspheres were first prepared by a water-in-oil-in-water (w/o/w) emulsion system with 4% poly(vinyl alcohol) (PVA) as stabilizer in the external phase, followed by extraction of the remaining solvent. As non-solvents ethanol, methanol and mixtures of methanol and water were employed. However, the use of alcohols in the extraction medium resulted in microspheres which gave an incomplete lysozyme release at a non-constant rate. Complete lysozyme release was obtained from microspheres prepared by an emulsification-solvent evaporation method in PBS containing poly(vinyl pyrrolidone) (PVP) or PVA as stabilizer. PVA was most effective in stabilizing the w/o/w emulsion. Perfectly spherical microspheres were produced, with high protein entrapment efficiencies. These microspheres released lysozyme at an almost constant rate for approximately 28 days. The reproducibility of the w/o/w emulsion process was demonstrated by comparing particle characteristics and release profiles of three batches, prepared under similar conditions.

Legume Protein Isolates for Stable Acidic Emulsions Prepared by Premix Membrane Emulsification

NARCIS (Netherlands)

Ladjal Ettoumi, Yakoub; Berton-Carabin, Claire; Chibane, Mohamed; Schroën, Karin

2017-01-01

Proteins originating from dry legumes are not that much used in food formulations, yet, they are interesting components from a sustainability point of view, and could have interesting functional properties, e.g. for emulsion preparation. Therefore, this work focuses on the potential of the water

Preparation of fluorescent tocopherols for use in protein binding and localization with the alpha-tocopherol transfer protein.

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Nava, Phillip; Cecchini, Matt; Chirico, Sara; Gordon, Heather; Morley, Samantha; Manor, Danny; Atkinson, Jeffrey

2006-06-01

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin alpha-tocopherol were prepared incorporating fluorophores at the terminus of omega-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of alpha-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein's binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (alpha-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2R,4'R,8'R-alpha-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of alpha-TTP-mediated inter-membrane transfer of alpha-tocopherol and to investigate the mechanism of alpha-TTP function at membranes of different composition.

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

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

2016-07-15

The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury. Copyright © 2016 Elsevier Inc. All rights reserved.

[PREPARATION OF HUMAN TISSUE PROTEIN EXTRACTS ENRICHED WITH THE SPHINGOMYELIN SYNTHASE 1].

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Sudarkina, O Yu; Dergunova, L V

2015-01-01

Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.

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

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Ace Baehaki

2015-12-01

Full Text Available The objective of this research was to make a protein hydrolysates from catfish (Pangasius pangasius enzymatically using papain enzyme and analyzed the antioxidant activity of protein hydrolysates produced. The research used the method completely randomized design with two replications the treatment were the difference concentration of the papain enzyme (0%, 1%, 2%, 3%, 4%, 5%, and 6%. The parameters of research were antioxidative activity using DPPH (2,2-difenil-1–pikrilhidrazil, protein content, and molecular weight using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. The results showed that catfish protein hydrolysates prepared by papain enzyme has antioxidative activity. The highest degree of hydrolysis was 71.98% at enzyme concentration of 6%. Based on the DPPH scavenging method catfish protein hydrolysates has the antioxidative activity with the value 37.85-67.62%. The protein content of catfish protein hydrolysates were 20.86-54.47 mg/ml. The molecular weight of catfish protein hydrolyzates were 11.90-65.20 kDa.

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

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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.

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

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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...

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

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Zheng Xiaojuan

2009-10-01

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

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

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

2011-01-01

We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5–12 new techniques learned, which are transferrable to graduate research in academia and industry. PMID:21691428

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

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Kuffel, Anna; Zielkiewicz, Jan

2016-02-14

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

Label-Free Quantitative Analysis of Mitochondrial Proteomes Using the Multienzyme Digestion-Filter Aided Sample Preparation (MED-FASP) and "Total Protein Approach".

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Wiśniewski, Jacek R

2017-01-01

Determination of proteome composition and measuring of changes in protein titers provide important information with a substantial value for studying mitochondria.This chapter describes a workflow for the quantitative analysis of mitochondrial proteome with a focus on sample preparation and quantitative analysis of the data. The workflow involves the multienzyme digestion-filter aided sample preparation (MED-FASP) protocol enabling efficient extraction of proteins and high rate of protein-to-peptide conversion. Consecutive protein digestion with Lys C and trypsin enables generation of peptide fractions with minimal overlap, largely increases the number of identified proteins, and extends their sequence coverage. Abundances of proteins identified by multiple peptides can be assessed by the "Total Protein Approach."

Preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient.

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Bermejo, Marie Kristel; Milenkovic, Marija; Salahpour, Ali; Ramsey, Amy J

2014-09-03

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960's, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

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...

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

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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...

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

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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.

The effect of calcium on the composition and physical properties of whey protein particles prepared using emulsification.

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Westerik, Nieke; Scholten, Elke; Corredig, Milena

2015-06-15

Protein microparticles were formed through emulsification of 25% (w/w) whey protein isolate (WPI) solutions containing various concentrations of calcium (0.0-400.0mM) in an oil phase stabilized by polyglycerol polyricinoleate (PGPR). The emulsions were heated (at 80°C) and the microparticles subsequently re-dispersed in an aqueous phase. Light microscopy and scanning electron microscopy (SEM) images revealed that control particles and those prepared with 7.4mM calcium were spherical and smooth. Particles prepared with 15.0mM calcium gained an irregular, cauliflower-like structure, and at concentrations larger than 30.0mM, shells formed and the particles were no longer spherical. These results describe, for the first time, the potential of modulating the properties of dense whey protein particles by using calcium, and may be used as structuring agents for the design of functional food matrices with increased protein and calcium content. Copyright © 2015. Published by Elsevier Ltd.

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

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

2016-03-17

An integrated sample preparation method, termed "imFASP", which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50-500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Aqueous geochemistry in icy world interiors: Equilibrium fluid, rock, and gas compositions, and fate of antifreezes and radionuclides

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Neveu, Marc; Desch, Steven J.; Castillo-Rogez, Julie C.

2017-09-01

The geophysical evolution of many icy moons and dwarf planets seems to have provided opportunities for interaction between liquid water and rock (silicate and organic solids). Here, we explore two ways by which water-rock interaction can feed back on geophysical evolution: the production or consumption of antifreeze compounds, which affect the persistence and abundance of cold liquid; and the potential leaching into the fluid of lithophile radionuclides, affecting the distribution of a long-term heat source. We compile, validate, and use a numerical model, implemented with the PHREEQC code, of the interaction of chondritic rock with pure water and with C, N, S-bearing cometary fluid, thought to be the materials initially accreted by icy worlds, and describe the resulting equilibrium fluid and rock assemblages at temperatures, pressures, and water-to-rock ratios of 0-200 ° C, 1-1000 bar, and 0.1-10 by mass, respectively. Our findings suggest that water-rock interaction can strongly alter the nature and amount of antifreezes, resulting in solutions rich in reduced nitrogen and carbon, and sometimes dissolved H2, with additional sodium, calcium, chlorine, and/or oxidized carbon. Such fluids can remain partially liquid down to 176 K if NH3 is present. The prominence of Cl in solution seems to hinge on its primordial supply in ices, which is unconstrained by the meteoritical record. Equilibrium assemblages, rich in serpentine and saponite clays, retain thorium and uranium radionuclides unless U-Cl or U-HCO3 complexing, which was not modeled, significantly enhances U solubility. However, the radionuclide 40 K can be leached at high water:rock ratio and/or low temperature at which K is exchanged with ammonium in minerals. We recommend the inclusion of these effects in future models of the geophysical evolution of ocean-bearing icy worlds. Our simulation products match observations of chloride salts on Europa and Enceladus; CI chondrites mineralogies; the observation of

S100B Protein concentration in milk-formulas for preterm and term infants. Correlation with industrial preparation procedures.

Science.gov (United States)

Nigro, Francesco; Gagliardi, Luigi; Ciotti, Sabina; Galvano, Fabio; Pietri, Amedeo; Tina, Gabriella Lucia; Cavallaro, Daniela; La Fauci, Luca; Iacopino, Leonardo; Bognanno, Matteo; Li Volti, Giovanni; Scacco, Antonio; Michetti, Fabrizio; Gazzolo, Diego

2008-05-01

Human milk S100B protein possesses important neurotrophic properties. However, in some conditions human milk is substituted by milk formulas. The aims of the present study were: to assess S100B concentrations in milk formulas, to verify any differences in S100B levels between preterm and term infant formulas and to evaluate the impact of industrial preparation at predetermined phases on S100B content. Two different set of samples were tested: (i) commercial preterm (n = 36) and term (n = 36) infant milk formulas; ii) milk preterm (n = 10) and term infant (n = 10) formulas sampled at the following predetermined industrial preparation time points: skimmed cow milk (Time 0); after protein sources supplementation (Time 1); after pasteurization (Time 2); after spray-drying (Time 3). Our results showed that S100B concentration in preterm formulas were higher than in term ones (p 0.05) at Time 2, whereas a significant (p pasteurization but not spry-drying. New feeding strategies in preterm and term infants are therefore warranted in order to preserve S100B protein during industrial preparation.

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.

A novel approach to preparing magnetic protein microspheres with core-shell structure

Energy Technology Data Exchange (ETDEWEB)

Jiang Wei, E-mail: climentjw@126.co [National Special Superfine Powder Engineering Research Center, Nanjing University of Science and Technology, Nanjing 210094 (China); Sun Zhendong; Li Fengsheng [National Special Superfine Powder Engineering Research Center, Nanjing University of Science and Technology, Nanjing 210094 (China); Chen Kai; Liu Tianyu; Liu Jialing [Department of Physics, Nanjing University of Science and Technology, Nanjing 210094 (China); Zhou Tianle [Department of Materials Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Guo Rui [Department of Mechanical Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China)

2011-03-15

Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe{sub 3}O{sub 4} cores and coated with globular bovine serum albumin (BSA). Under an optimized condition, up to 57.8 wt% of approximately 10 nm superparamagnetic Fe{sub 3}O{sub 4} nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides of the abundant functional groups. The possible formation mechanism of magnetic microspheres was discussed in detail. - Research Highlights: Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe{sub 3}O{sub 4} cores and coated with globular bovine serum albumin (BSA). 57.8 wt% of approximately 10 nm superparamagnetic Fe{sub 3}O{sub 4} nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides the abundant functional groups.

A novel approach to preparing magnetic protein microspheres with core-shell structure

International Nuclear Information System (INIS)

Jiang Wei; Sun Zhendong; Li Fengsheng; Chen Kai; Liu Tianyu; Liu Jialing; Zhou Tianle; Guo Rui

2011-01-01

Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe 3 O 4 cores and coated with globular bovine serum albumin (BSA). Under an optimized condition, up to 57.8 wt% of approximately 10 nm superparamagnetic Fe 3 O 4 nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides of the abundant functional groups. The possible formation mechanism of magnetic microspheres was discussed in detail. - Research Highlights: → Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method.→ The microspheres are composed of the oleic acid and undecylenic acid modified Fe 3 O 4 cores and coated with globular bovine serum albumin (BSA).→ 57.8 wt% of approximately 10 nm superparamagnetic Fe 3 O 4 nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides the abundant functional groups.

77 FR 19000 - Foreign-Trade Zone 99-Wilmington, DE: Application for Manufacturing Authority; Fisker Automotive...

Science.gov (United States)

2012-03-29

..., anti-freeze, chemical preparations, tubing, flexible rubber tubes/hoses, self-adhesive plastic or... lenses, mirrors, car covers, fiberglass parts, heat deflectors, tube/pipe fittings, pins, hangers, body... and flanges, chain, heat exchangers, spray nozzles, universal joints, clutches, half/drive shafts...

AcEST: BP917335 [AcEST

Lifescience Database Archive (English)

Full Text Available _MOUSE DNA-binding protein SMUBP-2 OS=Mus musculus GN=Ighmbp2 PE=1 SV=1 Length = 993 Score = 42.4 bits (98),...THGEYTSAAE 635 >tr|Q9EQN5|Q9EQN5_RAT Antifreeze-enhancer binding protein AEP OS=Rattus norvegicus GN=Ighmbp2

Res Dec 07 Final 05-12-07

Indian Academy of Sciences (India)

client1

2007-12-05

Dec 5, 2007 ... glycerol and aminoacids such as alanine and proline. They are believed to stabilize cellular proteins and membranes at low temperature and hence called cryoprotectants. The cryoprotec- tive role of glycine betaine (GB) in bacteria is beginning to be understood. Antifreeze Proteins: An Overview. Birds ...

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

Science.gov (United States)

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

2016-06-01

Ice-binding proteins (IBPs), such as antifreeze proteins (AFPs) and ice-nucleating proteins (INPs), have been described in diverse cold-adapted organisms, and their potential applications in biotechnology have been recognized in various fields. Currently, both IBPs are being applied to biotechnological processes, primarily in medicine and the food industry. However, our knowledge regarding the diversity of bacterial IBPs is limited; few studies have purified and characterized AFPs and INPs from bacteria. Phenotypically verified IBPs have been described in members belonging to Gammaproteobacteria, Actinobacteria and Flavobacteriia classes, whereas putative IBPs have been found in Gammaproteobacteria, Alphaproteobacteria and Bacilli classes. Thus, the main goal of this minireview is to summarize the current information on bacterial IBPs and their application in biotechnology, emphasizing the potential application in less explored fields such as agriculture. Investigations have suggested the use of INP-producing bacteria antagonists and AFPs-producing bacteria (or their AFPs) as a very attractive strategy to prevent frost damages in crops. UniProt database analyses of reported IBPs (phenotypically verified) and putative IBPs also show the limited information available on bacterial IBPs and indicate that major studies are required. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Structure/Function Studies of Insect Antifreeze Proteins

National Research Council Canada - National Science Library

Duman, John

1997-01-01

...-x5-x6-Cys-X8-X9-Ala-X11-Th-X13 where X3 and X1 tend toward charged residues, X5 toward threonine or serine, X9 toward asparagine or aspartate, X6 toward asparagine or lysine, and X13 toward alanine...

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

Directory of Open Access Journals (Sweden)

Jiping Cai

2008-01-01

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

Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells.

Science.gov (United States)

Fischer, Roman; Kessler, Benedikt M

2015-04-01

We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists. © 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

UTILIZATION OF MEMBRANE MICROFILTRATION IN PREPARATION OF HYDROLYZED VEGETABLE PROTEIN FROM FERMENTED RED BEAN (Phaseolus vulgaris L. EXTRACT AS FORTIFICATION AGENT

Directory of Open Access Journals (Sweden)

Sri Moerniati

2010-06-01

Full Text Available Preparation of Hydrolyzed Vegetable Protein (HVP as savory flavor from fermented red bean broth through stirred membrane cell using micro filtration membrane with pore size of 0.45 µm was performed to get fortified agent utilized in preparation of beans sauce. The objective of this work was to study an effect of pressure and kind of red bean broth extract on content of total protein, soluble protein and dry solid in the retentate and permeate as hydrolyzed vegetable protein used for fortified agent of red bean sauces. Preparation process of hydrolyzed vegetable protein was done using fixed rotary speed of 400 rpm, pressure of 20, 25 and 30 psi at room temperature. To investigate the effect of pressure on this separation, the feed were red bean broth extract fermented for 6, 8, 10 and 12 weeks, respectively. Fermentation process were conducted using salt fermentation with inoculum of Rhizopus-C1, salt and red bean ratios of 30:10:60%. The analysis of flux and contents of total protein, dissolved protein and dry solid in the retentate and permeate was carried out, and the result of experiment showed that interaction of Red bean broth extract with 6, 8, 10 and 12 weeks of fermentation and operation condition of microfiltration membrane separation tends to affect on flux and content of total protein, dissolved protein and dry solid in retentate and permeate. Red bean broth extract for 6 weeks fermentation resulted higher protein content in permeate as hydrolyzed vegetable protein than in retentate. Permeate at pressure of 25 psi gives flux value of 0.0217 mL/cm2.minute and contents of total protein of 1.31 %, dissolved protein of 6.9 mg/g, and dry solid of 2.6%, while retentate as hydrolyzed vegetable protein or fortified agent indicate contents of total protein of 1.52%, dissolved protein of 4.15 mg/g, and dry solid of 3.64%. It was found that micro filtration process was able to increase dissolved protein content of about 3 times.   Keywords

Cloning and mRNA expression pattern analysis under low ...

African Journals Online (AJOL)

Jane

2011-07-13

Jul 13, 2011 ... This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of .... Company, RevertAidTM First Strand cDNA Synthesis Kit from .... different times of low temperature in root, stem and leaf of Dongmu-70 rye.

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

Preparation, aroma characteristics and volatile compounds of flavorings from enzymatic hydrolyzed rice bran protein concentrate.

Science.gov (United States)

Arsa, Supeeraya; Theerakulkait, Chockchai

2018-02-19

Rice bran is a by-product obtained from the rice milling industry. The aims of this research were to add value to rice bran by preparation of enzymatic hydrolyzed rice bran protein concentrate (HRPC) as a flavoring agent and the flavoring which was produced by HRPC has not been investigated. Different drying methods (freeze-drying and spray-drying) and fructose additions were studied for improvement of rice bran protein sensorial aroma characteristics. The most abundant amino acids in liquid HRPC (LH) were glutamic acid, arginine, aspartic acid and leucine. The intensity of desirable aromas, such as cereal-like, nut-like, milk-powder-like, sweet, and cocoa-like aroma, were higher in spray-dried HRPC powder (SHP) than in LH and freeze-dried HRPC. Volatile compounds, such as aldehydes, pyrazines and ketones, were significantly increased in HRPC powders in which fructose was added before spray-drying (SHP-F). Higher amounts of 2-methylbutanal, 3-methylbutanal, phenylacetaldehyde, 2,5-dimethylpyrazine, vanillin, 2-acetylpyrrole and maltol were detected in SHP-F. Moreover, these compounds had high odor active values, which accounted for the cocoa-like, sweet, nut-like, and milk-powder-like characteristics of SHP-F. These findings could lead to the creation of desirable aroma characteristics of rice bran protein concentrate by different preparation methods. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

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.

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

International Nuclear Information System (INIS)

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

2014-01-01

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. - 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

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

International Nuclear Information System (INIS)

Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio; Bermejo-Barrera, Pilar; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad; Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario

2007-01-01

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

Obtention and characterization of dried gels prepared with whey proteins, honey and hydrocolloids mixture.

Science.gov (United States)

Rodriguez, Ana C; Torrez Irigoyen, Martín R; Navarro, Alba S; Yamul, Diego K

2017-11-01

Large amounts of honey and liquid whey derived from the dairy industry are produced in Argentina. Honey is exported in bulk and whey is transformed into whey protein concentrates and isolates. The objective of this work was to investigate the effect of pH, composition and storage time on the properties of dried gels with honey, whey proteins and hydrocolloids. Color properties varied according to pH and composition. The fracture stress of dried gels prepared with corn starch was higher than that of gels prepared with guar gum in all conditions assayed. Young's modulus was higher at pH 7 for both compositions and increased with storage time. Rubbery characteristics were found in dried gels with guar gum, while both corn starch and guar gum made the microstructure rougher. Multivariate analysis showed that samples could be grouped by pH. Panelists preferred pH 7 products over acidic ones, and no significant differences in sensory properties were found using either corn starch or guar gum in the formulation. The results demonstrated that it is possible to generate a new product, which may open new applications for honey and whey in food formulations. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

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.

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

Science.gov (United States)

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

2013-06-19

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

Journal of Chemical Sciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Diabatic potential energy surfaces of H + CO .... to locate the minimum energy conformation using quantum chemical theory. ... Effect of electrostatic interactions on the formation of proton transfer pathways in human carbonic anhydrase II ... In silico characterization of antifreeze proteins using computational tools and servers.

Seed Protein Content and Consistency of Tofu Prepared with Different Magnesium Chloride Concentrations in Six Japanese Soybean Varieties

OpenAIRE

Toda, Kyoko; Ono, Tomotada; Kitamura, Keisuke; Hajika, Makita; Takahashi, Koji; Nakamura, Yoshiyuki

2003-01-01

The relationship between the protein content of soybean seeds and the consistency of tofu was examined for six Japanese soybean varieties, Enrei, Fukuyutaka, Sachiyutaka, Ayakogane, Hatayutaka and Tachinagaha. The seed protein content was estimated by determining the nitrogen content using the Dumas method. Tofu was prepared from a raw homogenate of water-soaked soybeans by heating and by the addition of MgCl_2 as a coagulant. The tofu consistency was evaluated by measuring the breaking stres...

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...

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

Energy Technology Data Exchange (ETDEWEB)

Torres-Lugo, Madeline [University of Puerto Rico, Mayagueez Campus, Department of Chemical Engineering (United States); Peppas, Nicholas A. [Purdue University, NSF Program on Therapeutic and Diagnostic Devices, School of Chemical Engineering (United States)], E-mail: peppas@ecn.purdue.edu

2002-04-15

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

Preparation of Cu{sup 2+}/NTA-derivatized branch polyglycerol magnetic nanoparticles for protein adsorption

Energy Technology Data Exchange (ETDEWEB)

ShiXing Wang, E-mail: wsxkm@sina.com; Sun Wentong [Yunnan Institute of Product Quality Supervision and Inspection (China); Zhou Yang, E-mail: zhouyang8250@sohu.co [Kunming University of Science and Technology, Faculty of Metallurgical and Energy Engineering (China)

2010-09-15

In this report, we described the preparation of Cu{sup 2+}/nitrilotriacetic acids (NTA)-derivatized branch polyglycerol magnetic nanoparticles for protein adsorption with avoidance of nonspecific interactions at the same time. Magnetic nanoparticles (MNPs) were synthesized by the coprecipitation method. The transmission electron microscopy results showed that the average diameter of MNPs was 15.8 {+-} 4.6 nm. X-ray photoelectron spectroscopy and Fourier Transform infrared measurements indicated that branch polyglycerols were grafted on MNPs via the ring-opening polymerization of glycidol and that Cu{sup 2+} ions had been successfully immobilized on the surface of MNPs. The protein immobilization effect was characterized by UV-Vis spectrum. The results proved that Cu{sup 2+}/NTA-derivatized branch polyglycerol magnetic nanoparticles effectively adsorbed bovine haemoglobin and rarely adsorbed lysozyme and {gamma}-globin.

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...

A novel approach to preparing magnetic protein microspheres with core-shell structure

Science.gov (United States)

Jiang, Wei; Sun, Zhendong; Li, Fengsheng; Chen, Kai; Liu, Tianyu; Liu, Jialing; Zhou, Tianle; Guo, Rui

2011-03-01

Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe 3O 4 cores and coated with globular bovine serum albumin (BSA). Under an optimized condition, up to 57.8 wt% of approximately 10 nm superparamagnetic Fe 3O 4 nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides of the abundant functional groups. The possible formation mechanism of magnetic microspheres was discussed in detail.

Food emulsion type oil in water prepared with high-protein from shrimp (Penaeus vannamei heads flour – SHF

Directory of Open Access Journals (Sweden)

Yuli Cano

2017-09-01

Full Text Available The use of flour from shrimp (Penaeus vannamei heads with a high content of protein (SHF to stabilize food emulsions type oil in water (o/w is an alternative to take advantage of the by-products of the shrimp industry. The aim of this work was to prepare food emulsion type oil in water (o/w using the SHF due to the high percentage in proteins; for this procedure a physicochemical and bromatological characterization of flour of shrimps (Penaeus vannamei heads has been done, in which a percentage of protein 51 %, moisture of 11,82 %, fat 8,52 % and 22,23 % of ash has been obtained. The base emulsions may be used in food products such as salad dressing, mayonnaise, spreads, dressings and other products. The different emulsions with adequate rheological and microstructural characteristics were prepared using different concentrations of palm oil (20, 30 and 40%w/w and different concentrate of SHF (0,5, 1 and 2 % w/w. Therefore, we have obtained a food emulsion stable type oil in water (O/W with 2 % w/w of SHF, which presented a behavior non-Newtonian fluid type shear-thinning and homogeneous distribution of droplets.

Protein fibrillization: preparation, mechanism and application

NARCIS (Netherlands)

Akkermans, C.

2008-01-01

The development of new functional ingredients is important for future food products. This PhD research aimed at the development of protein based structuring agents. Structuring agents are ingredrients that can be used to tailor the texture (and the mouth-feel) of products. Proteins were transferred

Fulltext PDF

Indian Academy of Sciences (India)

client1

12. Mayer-Jensen Shell Model and Magic Numbers. An Independent Nucleon Model with Spin-Orbit. Coupling. R Velusamy. 46. 25 Antifreeze Proteins of Bacteria. MK Chattopadhyay. Collimator. 31 Seven Different Proofs of the Irrationality of 12. VG Tikekar. Prism Table. Telescope. 40 Getting Sloshed in Outer Space.

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

Directory of Open Access Journals (Sweden)

Tchankouo-Nguetcheu Stephane

2012-11-01

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

Sample preparation guidelines for two-dimensional electrophoresis.

Science.gov (United States)

Posch, Anton

2014-12-01

Sample preparation is one of the key technologies for successful two-dimensional electrophoresis (2DE). Due to the great diversity of protein sample types and sources, no single sample preparation method works with all proteins; for any sample the optimum procedure must be determined empirically. This review is meant to provide a broad overview of the most important principles in sample preparation in order to avoid a multitude of possible pitfalls. Sample preparation protocols from the expert in the field were screened and evaluated. On the basis of these protocols and my own comprehensive practical experience important guidelines are given in this review. The presented guidelines will facilitate straightforward protocol development for researchers new to gel-based proteomics. In addition the available choices are rationalized in order to successfully prepare a protein sample for 2DE separations. The strategies described here are not limited to 2DE and can also be applied to other protein separation techniques.

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

International Nuclear Information System (INIS)

Lu Yan; Yan Changling; Gao Shuyan

2009-01-01

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

Proteomic tools for environmental microbiology--a roadmap from sample preparation to protein identification and quantification.

Science.gov (United States)

Wöhlbrand, Lars; Trautwein, Kathleen; Rabus, Ralf

2013-10-01

The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell. Within the past decade, proteomic methods became increasingly accessible to microbiologists mainly due to the robustness of analytical methods (e.g. 2DE), and affordability of mass spectrometers and their relative ease of use. This review provides an overview on the complex portfolio of state-of-the-art proteomics and highlights the basic principles of key methods, ranging from sample preparation of laboratory or environmental samples, via protein/peptide separation (gel-based or gel-free) and different types of mass spectrometric protein/peptide analyses, to protein identification and abundance determination. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Energy Technology Data Exchange (ETDEWEB)

Lu Yan, E-mail: yanlu2001@sohu.com [College of Chemistry and Environmental Science, Henan Normal University, 46 Jlanshe Road, Xinxiang 453007 (China); Yan Changling; Gao Shuyan [College of Chemistry and Environmental Science, Henan Normal University, 46 Jlanshe Road, Xinxiang 453007 (China)

2009-04-01

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

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)

High Tensile Strength of Engineered β-Solenoid Fibrils via Sonication and Pulling.

Science.gov (United States)

Peng, Zeyu; Parker, Amanda S; Peralta, Maria D R; Ravikumar, Krishnakumar M; Cox, Daniel L; Toney, Michael D

2017-11-07

We present estimates of ultimate tensile strength (UTS) for two engineered β-solenoid protein mutant fibril structures (spruce budworm and Rhagium inquisitor antifreeze proteins) derived from sonication-based measurements and from force pulling molecular dynamics simulations, both in water. Sonication experiments generate limiting scissioned fibrils with a well-defined length-to-width correlation for the mutant spruce budworm protein and the resultant UTS estimate is 0.66 ± 0.08 GPa. For fibrils formed from engineered R. inquisitor antifreeze protein, depending upon geometry, we estimate UTSs of 3.5 ± 3.2-5.5 ± 5.1 GPa for proteins with interfacial disulfide bonds, and 1.6 ± 1.5-2.5 ± 2.3 GPa for the reduced form. The large error bars for the R. inquisitor structures are intrinsic to the broad distribution of limiting scission lengths. Simulations provide pulling velocity-dependent UTSs increasing from 0.2 to 1 GPa in the available speed range, and 1.5 GPa extrapolated to the speeds expected in the sonication experiments. Simulations yield low-velocity values for the Young's modulus of 6.0 GPa. Without protein optimization, these mechanical parameters are similar to those of spider silk and Kevlar, but in contrast to spider silk, these proteins have a precisely known sequence-structure relationship. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Cloning and mRNA expression pattern analysis under low ...

African Journals Online (AJOL)

This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of Dong-mu 70 rye (Secale cereale) by designing special primers according to Genbank's EAPP gene sequence, and analyzing the influence of low temperature stress on the expression of mRNA with RT-PCR. The results indicated that the ...

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

/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is used as the first protein screening method in many laboratories because of its inherent simplicity, mass accuracy, sensitivity and relatively high sample throughput. We present a simplified sample preparation method for MALDI-MS that enables in-gel digestion...... 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...... protocol while being less labour intensive and more cost-effective due to minimal consumption of reagents, enzymes and consumables. Preliminary data obtained on a MALDI quadrupole-TOF tandem mass spectrometer demonstrated the utility of the on-probe digestion protocol for peptide mass mapping and peptide...

Preparation and characterization of baru (Dipteryx alata Vog) nut protein isolate and comparison of its physico-chemical properties with commercial animal and plant protein isolates.

Science.gov (United States)

Nunes, Ângela A; Favaro, Simone P; Miranda, Cesar H B; Neves, Valdir A

2017-01-01

The Brazilian leguminous tree locally known in the Cerrado Biome as baru (Dipteryx alata Vog), provides a healthy edible oil source. The proteinaceous cake remaining after oil extraction could be transformed into new products to foodstuff development, such as protein concentrates and isolates, adding value to the production chain. In this study, it is described the preparation and characterization of baru nut protein isolate (BPI) from deffated baru flour, and measurements of its functional, nutritional, and thermal properties, in comparison to the more common vegetable (soybeans) and animal (casein and albumin) protein sources of the food industry. BPI presented higher protein content than soybean, casein and albumin commercial protein isolates, despite losses of albumins and low molecular weight globulins during the isolation procedure. Thermodynamics studies suggested that BPI has a well-conserved protein arrangement and lower thermostability than the other protein sources. BPI showed high in vitro digestibility and suitable and desirable functional properties such as water and oil absorption capacity, emulsifying activity, and foam formation and stability at mild and neutral pH. BPI could be used either as a substitute ingredient in oily food formulations or in the development of new products of its own. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Preparation of a novel fluorescence probe of terbium-europium co-luminescence composite nanoparticles and its application in the determination of proteins

Energy Technology Data Exchange (ETDEWEB)

Gao Feng [College of Chemistry and Materials Science, Anhui Key Laboratory of Chemo/Biosensing, Anhui Normal University, Wuhu 241000 (China)], E-mail: summit8848cn@hotmail.com; Luo Fabao; Tang Lijuan; Dai Lu [College of Chemistry and Materials Science, Anhui Key Laboratory of Chemo/Biosensing, Anhui Normal University, Wuhu 241000 (China); Wang Lun [College of Chemistry and Materials Science, Anhui Key Laboratory of Chemo/Biosensing, Anhui Normal University, Wuhu 241000 (China)], E-mail: wanglun@mail.ahnu.edu.cn

2008-03-15

Terbium-europium Tb-Eu/acetylacetone(acac)/poly(acrylamide) (PAM) co-luminescence composite nanoparticles were successfully prepared using the ultrasonic approach. The as-prepared composite nanoparticles show the characteristic emission spectra of Tb{sup 3+}, located at 496 and 549 nm. Furthermore, the nanoparticles are water soluble, stable and have extremely narrow emission bands and high internal fluorescence quantum yield due to the co-luminescence effect. Further studies indicate that proteins can interact with the nanoparticles and induce the fluorescence quenching of the nanoparticles. Based on the fluorescence quenching of nanopaticles in the presence of proteins, a novel method for the sensitive determination of trace amounts of proteins was proposed. Under the optimal experimental conditions, the linear ranges of calibration curves are 0-3.5 {mu}g mL{sup -1} for human serum albumin (HSA) and 0-4.0 {mu}g mL{sup -1} for {gamma}-globulin ({gamma}-IgG), respectively. The limits of detection are 7.1 for HSA and 6.7ng mL{sup -1} for {gamma}-IgG, respectively. The method was applied to the quantification of proteins in synthetic samples and actual human serum samples with satisfactory results. This proposed method is sensitive, simple and has potential application in the clinical assay of proteins.

Preparation of C-terminally modified chemokines by expressed protein ligation.

Science.gov (United States)

Baumann, Lars; Steinhagen, Max; Beck-Sickinger, Annette G

2013-01-01

In order to link structural features on a molecular level to the function of chemokines, site-specific modification strategies are strongly required. These can be used to incorporate fluorescent dyes and/or physical probes to allow investigations in a wide range of biological and physical techniques, e.g., nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, fluorescence resonance energy transfer (FRET), or fluorescence correlation spectroscopy (FCS). Only a limited number of functional groups within the 20 canonical amino acids allow ligation strategies that can be helpful to introduce novel functionalities, which in turn expand the scope of chemoselective and orthogonal reactivity of (semi)synthetic chemokines. In the present chapter we mainly focus on the fabulous history of native chemical ligation (NCL) and provide a general protocol for the preparation of C-terminally modified SDF-1α including tips and tricks for practical work. We believe that this protocol can be easily adapted to other chemokines and many proteins in general.

Preparation of monoclonal antibodies against radiation-induced protein

International Nuclear Information System (INIS)

Nozawa, R.; Tanaka, A.; Watanabe, H.; Kitayama, S.

1992-01-01

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

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...

The impact of particle preparation methods and polymorphic stability of lipid excipients on protein distribution in microparticles

DEFF Research Database (Denmark)

Liu, Jingying; Christophersen, Philip C; Yang, Mingshi

2017-01-01

OBJECTIVE: The present study aimed at elucidating the influence of polymorphic stability of lipid excipients on the physicochemical characters of different solid lipid microparticles (SLM), with the focus on the alteration of protein distribution in SLM. METHODS: Labeled lysozyme was incorporated...... provides updated knowledge for rational development of lipid-based formulations for oral delivery of peptide or protein drugs.......OBJECTIVE: The present study aimed at elucidating the influence of polymorphic stability of lipid excipients on the physicochemical characters of different solid lipid microparticles (SLM), with the focus on the alteration of protein distribution in SLM. METHODS: Labeled lysozyme was incorporated...... into SLM prepared with different excipients, i.e. trimyristin (TG14), glyceryl distearate (GDS), and glyceryl monostearate (GMS), by water-oil-water (w/o/w) or solid-oil-water (s/o/w) method. The distribution of lysozyme in SLM and the release of the protein from SLM were evaluated by confocal laser...

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.

Urine sample preparation for proteomic analysis.

Science.gov (United States)

Olszowy, Pawel; Buszewski, Boguslaw

2014-10-01

Sample preparation for both environmental and more importantly biological matrices is a bottleneck of all kinds of analytical processes. In the case of proteomic analysis this element is even more important due to the amount of cross-reactions that should be taken into consideration. The incorporation of new post-translational modifications, protein hydrolysis, or even its degradation is possible as side effects of proteins sample processing. If protocols are evaluated appropriately, then identification of such proteins does not bring difficulties. However, if structural changes are provided without sufficient attention then protein sequence coverage will be reduced or even identification of such proteins could be impossible. This review summarizes obstacles and achievements in protein sample preparation of urine for proteome analysis using different tools for mass spectrometry analysis. The main aim is to present comprehensively the idea of urine application as a valuable matrix. This article is dedicated to sample preparation and application of urine mainly in novel cancer biomarkers discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of ice recrystallization inhibition activity of poly(vinyl alcohol) using a supramolecular trigger

OpenAIRE

Phillips, Daniel J.; Congdon, Thomas; Gibson, Matthew I.

2016-01-01

Antifreeze (glyco)proteins (AF(G)Ps) have potent ice recrystallisation inhibition (IRI) activity – a desirable phenomenon in applications such as cryopreservation, frozen food and more. In Nature AF(G)P activity is regulated by protein expression levels in response to an environmental stimulus; temperature. However, this level of regulation is not possible in synthetic systems. Here, a synthetic macromolecular mimic is introduced, using supramolecular assembly to regulate activity. Catechol-t...

Activation of Ice Recrystallization Inhibition Activity of Poly(vinyl alcohol) using a Supramolecular Trigger†

OpenAIRE

Phillips, Daniel J.; Congdon, Thomas R.; Gibson, Matthew I.

2016-01-01

Antifreeze (glyco)proteins (AF(G)Ps) have potent ice recrystallisation inhibition (IRI) activity – a desirable phenomenon in applications such as cryopreservation, frozen food and more. In Nature AF(G)P activity is regulated by protein expression levels in response to an environmental stimulus; temperature. However, this level of regulation is not possible in synthetic systems. Here, a synthetic macromolecular mimic is introduced, using supramolecular assembly to regulate ac...

A rare polyglycine type II-like helix motif in naturally occurring proteins.

Science.gov (United States)

Warkentin, Eberhard; Weidenweber, Sina; Schühle, Karola; Demmer, Ulrike; Heider, Johann; Ermler, Ulrich

2017-11-01

Common structural elements in proteins such as α-helices or β-sheets are characterized by uniformly repeating, energetically favorable main chain conformations which additionally exhibit a completely saturated hydrogen-bonding network of the main chain NH and CO groups. Although polyproline or polyglycine type II helices (PP II or PG II ) are frequently found in proteins, they are not considered as equivalent secondary structure elements because they do not form a similar self-contained hydrogen-bonding network of the main chain atoms. In this context our finding of an unusual motif of glycine-rich PG II -like helices in the structure of the acetophenone carboxylase core complex is of relevance. These PG II -like helices form hexagonal bundles which appear to fulfill the criterion of a (largely) saturated hydrogen-bonding network of the main-chain groups and therefore may be regarded in this sense as a new secondary structure element. It consists of a central PG II -like helix surrounded by six nearly parallel PG II -like helices in a hexagonal array, plus an additional PG II -like helix extending the array outwards. Very related structural elements have previously been found in synthetic polyglycine fibers. In both cases, all main chain NH and CO groups of the central PG II -helix are saturated by either intra- or intermolecular hydrogen-bonds, resulting in a self-contained hydrogen-bonding network. Similar, but incomplete PG II -helix patterns were also previously identified in a GTP-binding protein and an antifreeze protein. © 2017 Wiley Periodicals, Inc.

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

Before a novel protein can be used in foods, its potential allergenicity must be assessed. In this study, healthy volunteers consumed ice structuring protein (ISP) Type III preparation or a control material 5 days a week for a total of 8 weeks. General measures of health were recorded during...... 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...

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.

Effect of Different Tumbling Marination Methods and Time on the Water Status and Protein Properties of Prepared Pork Chops

Directory of Open Access Journals (Sweden)

Tian Gao

2015-07-01

Full Text Available The combined effect of tumbling marination methods (vacuum continuous tumbling marination, CT; vacuum intermittent tumbling marination, IT and effective tumbling time (4, 6, 8, and 10 h on the water status and protein properties of prepared pork chops was investigated. Results showed that regardless of tumbling time, CT method significantly decreased the muscle fiber diameter (MD and significantly increased the total moisture content, product yield, salt soluble proteins (SSP solubility, immobilized water component (p<0.05 compared with IT method. With the effective tumbling time increased from 4 h to 10 h, the fat content and the MD were significantly decreased (p<0.05, whereas the SSP solubility of prepared pork chops increased firstly and then decreased. Besides, an interactive effect between CT method and effective tumbling time was also observed for the chemical composition and proportion of immobilized water (p<0.05. These results demonstrated that CT method of 8 h was the most beneficial for improving the muscle structure and water distribution status, increasing the water-binding capacity and accelerating the marinade efficiency of pork chops; and thus, it should be chosen as the most optimal treatment method for the processing production of prepared pork chops.

Resonance – Journal of Science Education | News

Indian Academy of Sciences (India)

Maria Goeppert Mayer · Bikramjit Banerjee · More Details Fulltext PDF. pp 12-24 General Article. Mayer-Jensen Shell Model and Magic Numbers - An Independent Nucleon Model with Spin-Orbit Coupling · R Velusamy · More Details Fulltext PDF. pp 25-30 General Article. Antifreeze Proteins of Bacteria · M K Chattopadhyay.

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.

Modeling of Rocket Fuel Heating and Cooling Processes in the Interior Receptacle Space of Ground-Based Systems

Directory of Open Access Journals (Sweden)

K. I. Denisova

2016-01-01

Full Text Available The propellant to fill the fuel tanks of the spacecraft, upper stages, and space rockets on technical and ground-based launch sites before fueling should be prepared to ensure many of its parameters, including temperature, in appropriate condition. Preparation of fuel temperature is arranged through heating and cooling the rocket propellants (RP in the tanks of fueling equipment. Processes of RP temperature preparation are the most energy-intensive and timeconsuming ones, which require that a choice of sustainable technologies and modes of cooling (heating RP provided by the ground-based equipment has been made through modeling of the RP [1] temperature preparation processes at the stage of design and operation of the groundbased fueling equipment.The RP temperature preparation in the tanks of the ground-based systems can be provided through the heat-exchangers built-in the internal space and being external with respect to the tank in which antifreeze, air or liquid nitrogen may be used as the heat transfer media. The papers [1-12], which note a promising use of the liquid nitrogen to cool PR, present schematic diagrams and modeling systems for the RP temperature preparation in the fueling equipment of the ground-based systems.We consider the RP temperature preparation using heat exchangers to be placed directly in RP tanks. Feeding the liquid nitrogen into heat exchanger with the antifreeze provides the cooling mode of PR while a heated air fed there does that of heating. The paper gives the systems of equations and results of modeling the processes of RP temperature preparation, and its estimated efficiency.The systems of equations of cooling and heating RP are derived on the assumption that the heat exchange between the fuel and the antifreeze, as well as between the storage tank and the environment is quasi-stationary.The paper presents calculation results of the fuel temperature in the tank, and coolant temperature in the heat exchanger, as

Preparation and characterization of milk protein films and their application for packaging of Cheddar cheese

OpenAIRE

Wagh, Y. R.; Pushpadass, Heartwin A.; Emerald, F. Magdaline Eljeeva; Nath, B. Surendra

2013-01-01

Casein and whey protein concentrate (WPC) films, plasticized with glycerol and sorbitol independently, were prepared by casting. The film thickness, water vapour and oxygen permeation and tensile and moisture sorption properties of the films were determined. The tensile strength (TS), tensile strain (TE) and elastic modulus (EM) of the films ranged from 0.71 to 4.58 MPa, 19.22 to 66.63 % and 2.05 to 6.93 MPa, respectively. The film properties were influenced by the type of biopolymer (casein ...

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

International Nuclear Information System (INIS)

Baca, E.; Skarzynska, M.; Gawel, J.; Jaworski, S.

1996-01-01

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

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.

Application of gamma radiation and heat treatment for the preparation of fish protein concentrates from Bombay duck (Harpodon nehereus)

International Nuclear Information System (INIS)

Warrier, S.B.K.; Ninjoor, V.; Kumta, U.S.

1976-01-01

Limitations in the conventional procedures for the manufacture of fish protein concentrate (FPG), such as residual toxicity of the solvents and loss in functional properties necessitate the need for evolving newer methods. A simple method has been developed for the preparation of FPC from the muscle of Bombay duck employing radiation-heat combination treatments. This involves extraction of the muscle proteins in 0.01 M phosphate buffer containing 5 percent sodium chloride, pH 7.5, irradiation at a dose of 100-250 krad followed by heat treatment at 60 degC for 10 min. The procedure permits the precipitation of fibrillar proteins to the extent of 83 percent. There is no change in the digestibility of the precipitated proteins as revealed by the tryptic hydrolysis. These results point to the synergestic effect of radiation and heat on the precipitation of proteins - an observation that has high potential for scaling up as a new process. (author)

Chemical composition of protein concentrate prepared from Yellowfin tuna Thunnus albacares roe by cook-dried process

Directory of Open Access Journals (Sweden)

Hyun Ji Lee

2016-05-01

Full Text Available Abstract Roe is the term used to describe fish eggs (oocytes gathered in skeins and is one of the most valuable food products from fishery sources. Thus, means of processing are required to convert the underutilized yellowfin tuna roes (YTR into more marketable and acceptable forms as protein concentrate. Roe protein concentrates (RPCs were prepared by cooking condition (boil-dried concentrate, BDC and steam-dried concentrate, SDC, respectively and un-cooking condition (freeze-dried concentrate, FDC from yellowfin tuna roe. The yield of RPCs was in the range from 22.2 to 25.3 g/100 g of roe. RPCs contained protein (72.3–77.3 %, moisture (4.3–5.6 %, lipid (10.6–11.3 % and ash (4.3–5.7 % as the major constituents. The prominent amino acids of RPCs were aspartic acid, 8.7–9.2, glutamic acid, 13.1–13.2, and leucine, 8.5–8.6 g/100 g of protein. Major differences were not observed in each of the amino acid. K, S, Na, and P as minerals were the major elements in RPCs. No difference noted in sodium dodecyl sulfate polyacrylamide gel electrophoresis protein band (15–100 K possibly representing partial hydrolysis of myosin. Therefore, RPCs from YTR could be use potential protein ingredient for human food and animal feeds.

Fluorescent molecularly imprinted polymer thin films for specific protein detection prepared with dansyl ethylenediamine-conjugated O-acryloyl L-hydroxyproline.

Science.gov (United States)

Inoue, Yuki; Kuwahara, Atsushi; Ohmori, Kohei; Sunayama, Hirobumi; Ooya, Tooru; Takeuchi, Toshifumi

2013-10-15

Protein-imprinted polymers, capable of specific transduction of protein binding events into fluorescent signal change, were designed and synthesized by using dansyl ethylenediamine-conjugated O-acryloyl L-hydroxyproline (Hyp-En-Dans). Human serum albumin (HSA) was used as a model target protein and HSA-imprinted polymers (HSA-IP) were prepared on glass substrates. Specific fluorescence change was observed for HSA binding on the imprinted polymer thin film, whereas a weaker response was observed for other proteins, including bovine serum albumin, chymotrypsin, lysozyme, and avidin. The binding specificity was found to derive from the rigid structure of the hydrogen-bondable pyrrolidine moiety. Compared with SPR measurements, the non-specific binding caused by the polymer matrix and/or randomly located fluorescent monomer residues that did not compose specific binding sites did not contribute to the observed fluorescence change. These results revealed that the proposed protein-imprinting technique using Hyp-En-Dans could provide a highly selective protein-sensing platform, in which only specific binding events would be detected by fluorescent measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

Science.gov (United States)

Zhang, Zhenbin; Sun, Liangliang; Zhu, Guijie; Cox, Olivia F; Huber, Paul W; Dovichi, Norman J

2016-01-05

A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

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. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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...... flavourings as the culture is growing, and (5) injecting the liquid into pieces of meat....

Antifreeze proteins enable plants to survive in freezing conditions

Indian Academy of Sciences (India)

1. Acaenamagellanica. Prickly burr. Doucet et al. 2000. 2. Acer saccharoides. Maple. Doucet et al. 2000. 3. Agrostistenuis. Creeping bentgrass. Doucet et al. 2000. 4. Alliarapetiolata. Garlic mustard. Urrutia et al. 1992. 5. Ammopiptanthusmongolicus. Evergreen legume. Wang et al. 2003. 6. Aster cordifolius. Wood aster.

Metal binding proteins and antifreeze proteins in the beetle Tenebrio molitor: a study on possible competition for the semi-essential amino acid cysteine

OpenAIRE

Pedersen, Sindre Andre

2007-01-01

In their natural environment animals are confronted by both physical (eg. extreme temperatures, desiccation) and chemical stressors (e.g. pollutants). Stress may be defined as a condition that is evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche (van Straalen 2003). Various defence systems exist to cope with different forms of stress and restore homeostasis. Often, production of various proteins or enzymes are ...

Preparation of Low Allergenic Protein Concentrated Natural Rubber Latex Using Suitable Low Molecular Weight Cellulose Derivatives Induced by Gamma Irradiation

International Nuclear Information System (INIS)

Siri-Upathum, Chyagrit; Boonyawat, Jariya

2007-08-01

Full text: Low molecular weight carboxy methyl cellulose (CMC), hydroxyl ethyl cellulose (HEC), hydroxyl propyl cellulose (HPC) and methyl cellulose (MC) prepared by radiation-induced degradation were added into diluted natural concentrated latex prior to centrifuge for a purpose of reducing allergenic rubber protein in the latex. Optimum molecular weight (Mv) of CMC and HEC for such a purpose was found to be 17-18 kDa which decreased allergenic rubber protein (14-94 kDa) to an undetectable amount as determined by SDS PAGE method

Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry.

Science.gov (United States)

Nilsson, C L; Puchades, M; Westman, A; Blennow, K; Davidsson, P

1999-01-01

Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, beta2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition, >25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.

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

International Nuclear Information System (INIS)

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

2016-01-01

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"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.

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.

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,

Preparation of non-porous microspheres with high entrapment efficiency of proteins by a (water-in-oil)-in-oil emulsion technique.

Science.gov (United States)

Viswanathan, N B; Thomas, P A; Pandit, J K; Kulkarni, M G; Mashelkar, R A

1999-03-08

Emulsification-solvent removal methods have been widely used for encapsulating bioactive macromolecules like proteins and polypeptides in biodegradable polymers. We report, a (water-in-oil)-in-oil emulsion technique wherein proteins and polypeptides differing in molecular weight and shape were encapsulated in polymers of current biomedical interest. When an oil was used as the processing medium in combination with a carefully selected mixed solvent system such that a stable (w/o1/o2 emulsion is formed and solvents are removed by a combination of extraction and evaporation, the entrapment efficiency was high and the product nonporous. The entrapment efficiency of globular proteins exceeded 90% while that of fibrous proteins was around 70%. Fracture studies revealed that the polymer matrix was dense. The mechanism of entrapment involved solvent-induced precipitation of the protein as the microspheres were being formed. The principle of the method will find use in preparation of non-porous polymer microparticles with reduced burst effect.

Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein.

Science.gov (United States)

Heissel, Søren; Bunkenborg, Jakob; Kristiansen, Max Per; Holmbjerg, Anne Fich; Grimstrup, Marie; Mørtz, Ejvind; Kofoed, Thomas; Højrup, Peter

2018-07-01

Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Emission of ammonia from indoor concrete wall and assessment of human exposure.

Science.gov (United States)

Bai, Z; Dong, Y; Wang, Z; Zhu, T

2006-04-01

Addition of urea-based antifreeze admixtures during cement mixing can make it possible to produce concrete cement in construction of buildings in cold weather; this, however, has led to increasing indoor air pollution due to continuous transformation and emission from urea to gaseous ammonia in indoor concrete wall. It is believed that ammonia is harmful to human body and exposure to ammonia can cause some serious symptoms such as headaches, burns, and even permanent damage to the eyes and lungs. In order to understand the emission of ammonia from indoor concrete wall in civil building and assess the health risk of people living in these buildings, the experimental pieces of concrete wall were first prepared by concreting cement and urea-based antifreeze admixtures to simulate the indoor wall in civil building in this work. Then environmental chamber was adopted for studying the effect of temperature, relative humility and air exchange rate on emission of ammonia from experimental pieces of concrete wall. Also the field experiment was made at selected rooms in given civil buildings. Exposure and potential dose of adult and children exposed to indoor/outdoor ammonia in summer and in winter are calculated and evaluated by using Scenario Evaluation Approach. The results indicated that high air exchange rate leads to decreased ammonia concentration, and elevation of temperature causes increasing ammonia concentration and volatilizing rate in chamber. The complete emission of ammonia from the wall containing urea-based antifreeze admixtures needs more than 10 years in general. Ventilating or improving air exchange can play a significant role in reducing ammonia concentration in actual rooms in field experiments. Urea-based antifreeze admixtures in concrete wall can give rise to high exposure and potential dose, especially in summer. Generally, adults have a high potential dose than children, while children have personal average dose rate beyond adults in the same

Yolk-Shell Porous Microspheres of Calcium Phosphate Prepared by Using Calcium L-Lactate and Adenosine 5'-Triphosphate Disodium Salt: Application in Protein/Drug Delivery.

Science.gov (United States)

Ding, Guan-Jun; Zhu, Ying-Jie; Qi, Chao; Sun, Tuan-Wei; Wu, Jin; Chen, Feng

2015-06-26

A facile and environmentally friendly approach has been developed to prepare yolk-shell porous microspheres of calcium phosphate by using calcium L-lactate pentahydrate (CL) as the calcium source and adenosine 5'-triphosphate disodium salt (ATP) as the phosphate source through the microwave-assisted hydrothermal method. The effects of the concentration of CL, the microwave hydrothermal temperature, and the time on the morphology and crystal phase of the product are investigated. The possible formation mechanism of yolk-shell porous microspheres of calcium phosphate is proposed. Hemoglobin from bovine red cells (Hb) and ibuprofen (IBU) are used to explore the application potential of yolk-shell porous microspheres of calcium phosphate in protein/drug loading and delivery. The experimental results indicate that the as-prepared yolk-shell porous microspheres of calcium phosphate have relatively high protein/drug loading capacity, sustained protein/drug release, favorable pH-responsive release behavior, and a high biocompatibility in the cytotoxicity test. Therefore, the yolk-shell porous microspheres of calcium phosphate have promising applications in various biomedical fields such as protein/drug delivery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM.

Science.gov (United States)

Hauer, Florian; Gerle, Christoph; Fischer, Niels; Oshima, Atsunori; Shinzawa-Itoh, Kyoko; Shimada, Satoru; Yokoyama, Ken; Fujiyoshi, Yoshinori; Stark, Holger

2015-09-01

We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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

Science.gov (United States)

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

2016-08-05

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

Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity.

Science.gov (United States)

Zamani, Abbas; Benjakul, Soottawat

2016-02-01

Fish proteases, especially trypsin, could be used to prepare fish protein hydrolysates with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified by ammonium sulfate precipitation and soybean trypsin inhibitor (SBTI)-Sepharose 4B affinity chromatography. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (20, 30 and 40% DH) was prepared using the purified trypsin, and antioxidative activities (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activities, ferric-reducing antioxidant power and ferrous-chelating activity) of the hydrolysate were determined. Trypsin was purified 26.43-fold with a yield of 13.43%. The purified trypsin had a molecular weight (MW) of 23.5 kDa and optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was thermally stable up to 50 °C. Both SBTI (0.05 mmol L(-1)) and N-p-tosyl-L-lysine-chloromethylketone (5 mmol L(-1)) completely inhibited trypsin activity. Antioxidative activities of the hydrolysate from Indian mackerel protein isolate increased with increasing DH up to 40% (P unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyze a specific synthetic substrate and the response to specific trypsin inhibitors. The purified trypsin could hydrolyze Indian mackerel protein isolate, and the resulting hydrolysate exhibited antioxidative activity depending on its DH. © 2015 Society of Chemical Industry.

A Standardized and Reproducible Urine Preparation Protocol for Cancer Biomarkers Discovery

Directory of Open Access Journals (Sweden)

Julia Beretov

2014-01-01

Full Text Available A suitable and standardized protein purification technique is essential to maintain consistency and to allow data comparison between proteomic studies for urine biomarker discovery. Ultimately, efforts should be made to standardize urine preparation protocols. The aim of this study was to develop an optimal analytical protocol to achieve maximal protein yield and to ensure that this method was applicable to examine urine protein patterns that distinguish disease and disease-free states. In this pilot study, we compared seven different urine sample preparation methods to remove salts, and to precipitate and isolate urinary proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE profiles showed that the sequential preparation of urinary proteins by combining acetone and trichloroacetic acid (TCA alongside high speed centrifugation (HSC provided the best separation, and retained the most urinary proteins. Therefore, this approach is the preferred method for all further urine protein analysis.

Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn.

Science.gov (United States)

Tao, Dingyin; Sun, Liangliang; Zhu, Guijie; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2011-01-01

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation and characterization of protein isolate from Yellowfin tuna Thunnus albacares roe by isoelectric solubilization/precipitation process

Directory of Open Access Journals (Sweden)

Hyun Ji Lee

2016-05-01

Full Text Available Abstract Isoelectric solubilization/precipitation (ISP processing allows selective, pH-induced water solubility of proteins with concurrent separation of lipids and removal of materials not intended for human consumption such as bone, scales, skin, etc. Recovered proteins retain functional properties and nutritional value. Four roe protein isolates (RPIs from yellowfin tuna roe were prepared under different solubilization and precipitation condition (pH 11/4.5, pH 11/5.5, pH 12/4.5 and pH 12/5.5. RPIs contained 2.3–5.0 % moisture, 79.1–87.8 % protein, 5.6–7.4 % lipid and 3.0–3.8 % ash. Protein content of RPI-1 and RPI-2 precipitated at pH 4.5 and 5.5 after alkaline solubilization at pH 11, was higher than those of RPI-3 and RPI-4 after alkaline solubilization at pH 12 (P < 0.05. Lipid content (5.6–7.4 % of RPIs was lower than that of freeze-dried concentrate (10.6 %. And leucine and lysine of RPIs were the most abundant amino acids (8.8–9.4 and 8.5–8.9 g/100 g protein, respectively. S, Na, P, K as minerals were the major elements in RPIs. SDS-PAGE of RPIs showed bands at 100, 45, 25 and 15 K. Moisture and protein contents of process water as a 2’nd byproduct were 98.9–99.0 and 1.3–1.8 %, respectively. Therefore, yellowfin tuna roe isolate could be a promising source of valuable nutrients for human food and animal feeds.

Preparation and characterization of milk protein films and their application for packaging of Cheddar cheese.

Science.gov (United States)

Wagh, Y R; Pushpadass, Heartwin A; Emerald, F Magdaline Eljeeva; Nath, B Surendra

2014-12-01

Casein and whey protein concentrate (WPC) films, plasticized with glycerol and sorbitol independently, were prepared by casting. The film thickness, water vapour and oxygen permeation and tensile and moisture sorption properties of the films were determined. The tensile strength (TS), tensile strain (TE) and elastic modulus (EM) of the films ranged from 0.71 to 4.58 MPa, 19.22 to 66.63 % and 2.05 to 6.93 MPa, respectively. The film properties were influenced by the type of biopolymer (casein and whey protein concentrate), plasticizer and its concentration. Increasing the plasticizer concentration, increased the film thickness, TE and water vapour permeability (WVP), but decreased the TS and EM. As the concentration of plasticizer increased to the highest level, the film thickness increased from 0.168 to 0.305 mm for glycerol-plasticized films and from 0.251 to 0.326 mm for sorbitol-plasticized films. The film thickness increased because the amount of plasticizer in the film network increased and the amount of biopolymer remained same. Casein films showed superior tensile properties as compared to WPC films. The WVP of both casein and WPC films lied between 3.87 and 13.97 g.mm./(m(2).h.kPa). The moisture sorption isotherms of both films were typical of high-protein material, and were adequately described by the GAB model. The oxygen permeability of casein films was relatively lower than that of WPC films, regardless of the plasticizer used. The sensory data revealed that the organoleptic quality of Cheddar cheese was unaffected by milk-protein film packaging.

Efficient DNP NMR of Membrane Proteins: Sample Preparation Protocols, Sensitivity, and Radical Location

Science.gov (United States)

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

2016-01-01

Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~4 fold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes. PMID:26873390

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

Energy Technology Data Exchange (ETDEWEB)

Liao, Shu Y.; Lee, Myungwoon; Wang, Tuo [Massachusetts Institute of Technology, Department of Chemistry (United States); Sergeyev, Ivan V. [Bruker Biospin (United States); Hong, Mei, E-mail: meihong@mit.edu [Massachusetts Institute of Technology, Department of Chemistry (United States)

2016-03-15

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

Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins.

Science.gov (United States)

Schaffer, Miroslava; Mahamid, Julia; Engel, Benjamin D; Laugks, Tim; Baumeister, Wolfgang; Plitzko, Jürgen M

2017-02-01

While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

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...

Cloning-free template DNA preparation for cell-free protein synthesis via two-step PCR using versatile primer designs with short 3'-UTR.

Science.gov (United States)

Nomoto, Mika; Tada, Yasuomi

2018-01-01

Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

MANIFESTATION OF THE EFFECT OF CRYOSELECTION IN CARP OFFSPRINGS OBTAINED FROM DEFROSTED SPERM SUSPENSION WITH MODIFIED CRYOPROTECTIVE SOLUTIONS

Directory of Open Access Journals (Sweden)

V. Cherepnin

2016-09-01

Full Text Available Purpose. The purpose of these studies was to evaluate the effect of the cryoselection of carp sperm of multiple age groups depending on the quality of thawed sperm after modification of the composite cryoprotective medium, which was used for the dilution of native sperm before freezing. Methodology. Coenzyme B12 (cobamamide, blood plasma of Prussian carp (Carassius gibelio, which was subjected to natural cold-acclimation, and purified protein antifreeze tmAFP isolated from larval mealworm (Tenebrio molitor, which also was subjected to natural cold-acclimation, were sued as modifiers. Findings. The results of the study demonstrated that qualitative and quantitative characteristics of the defrosted sperm, results of the incubation, rearing of embryos and larvae of Nyvky scaled carp (NLC, as well as fish culture parameters of produced young-of-the-year depended from on the composition of cryoprotective medium. The best results were demonstrated for the experimental groups, obtained with the use of the cryoprotective solution supplemented with purified antifreeze protein tmAFP. The larvae obtained from the sperm cryopreserved with the addition of cryoprotective medium TmAFP had better resistance to dehydration, surpassing the experimental groups obtained from the modifiers of Prussian carp plasma and cobamamide. There is a consolidation in the action of the related extracellular cryoprotectors, which were isolated from the cold-resistant organisms. And carp produced with their used demonstrated better performance during their rearing. The fact can be established that the manifestation of cryoselective effect depends on the integrity of sperm hereditary material and does not depend on the effect of extremely low temperatures on the cell membranes. Originality. There were the first experiments, where Prussian carp plasma and antifreeze protein tmAFP were used as extracellular cryoprotectors. Practical value. The modifications of composite

Fluorine-18 labeling of proteins

International Nuclear Information System (INIS)

Kilbourn, M.R.; Dence, C.S.; Welch, M.J.; Mathias, C.J.

1987-01-01

Two fluorine-18-labeled reagents, methyl 3-[ 18 F]fluoro-5-nitrobenzimidate and 4-[ 18 F]fluorophenacyl bromide, have been prepared for covalent attachment of fluorine-18 to proteins. Both reagents can be prepared in moderate yields (30-50%, EOB) in synthesis times of 50-70 min. Reaction of these reagents with proteins (human serum albumin, human fibrinogen, and human immunoglobulin A) is pH independent, protein concentration dependent, and takes 5-60 min at mild pH (8.0) and temperature (25-37 degrees C), in yields up to 95% (corrected). The 18 F-labeled proteins are purified by size exclusion chromatography

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

Directory of Open Access Journals (Sweden)

Sara S. Duvenage

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. Navorsing het beduidende proteïen en minder ernstige

How to select BCAA preparations.

Science.gov (United States)

Kato, Akinobu; Suzuki, Kazuyuki

2004-12-01

In Japan, oral branched-chain amino acid (BCAA) preparations are used in nutritional therapy for correcting disorders of protein and amino acid metabolism in patients with liver cirrhosis. There are two forms of oral BCAA preparations: enteral nutrition products for liver failure (or elemental nutrition products for liver cirrhosis) and oral BCAA granular products. Granular products are indicated for patients with uncompensated liver cirrhosis who have no dietary restriction and hypoproteinemia. Enteral nutrition products are indicated for patients who have a history of hepatic encephalopathy and exhibit protein intolerance. In clinical practice, the existence of protein intolerance in patients with uncompensated liver cirrhosis should be determined based on a history of hepatic encephalopathy and blood ammonia concentration. When patients exhibit protein intolerance, they are given a low protein diet (approximately 0.5-1.0g/kg/day) with enteral nutrition products for liver failure. However, when patients consume adequate amounts of a well-balanced diet and ammonia concentration does not increase, it is possible to control their condition with granular products. However, when patients cannot achieve an adequate dietary intake, it is recommended that enteral nutrition products should be used in order to improve nutritional status, even if these patients do not have a history of encephalopathy.

Bioinspiration From Nano to Micro Scales

CERN Document Server

2012-01-01

Methods in bioinspiration and biomimicking have been around for a long time. However, due to current advances in modern physical, biological sciences, and technologies, our understanding of the methods have evolved to a new level. This is due not only to the identification of mysterious and fascinating phenomena but also to the understandings of the correlation between the structural factors and the performance based on the latest theoretical, modeling, and experimental technologies. Bioinspiration: From Nano to Micro Scale provides readers with a broad view of the frontiers of research in the area of bioinspiration from the nano to macroscopic scales, particularly in the areas of biomineralization, antifreeze protein, and antifreeze effect. It also covers such methods as the lotus effect and superhydrophobicity, structural colors in animal kingdom and beyond, as well as behavior in ion channels. A number of international experts in related fields have contributed to this book, which offers a comprehensive an...

Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation.

Science.gov (United States)

Zhao, Kailou; Yang, Li; Wang, Xuejiao; Bai, Quan; Yang, Fan; Wang, Fei

2012-08-30

We have explored a novel dual-function stationary phase which combines both strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) characteristics. The novel dual-function stationary phase is based on porous and spherical silica gel functionalized with ligand containing sulfonic and benzyl groups capable of electrostatic and hydrophobic interaction functionalities, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed. As a result, it can be employed to separate proteins with SCX and HIC modes, respectively. The resolution and selectivity of the dual-function stationary phase were evaluated under both HIC and SCX modes with standard proteins and can be comparable to that of conventional IEC and HIC columns. More than 96% of mass and bioactivity recoveries of proteins can be achieved in both HIC and SCX modes, respectively. The results indicated that the novel dual-function column could replace two individual SCX and HIC columns for protein separation. Mixed retention mechanism of proteins on this dual-function column based on stoichiometric displacement theory (SDT) in LC was investigated to find the optimal balance of the magnitude of electrostatic and hydrophobic interactions between protein and the ligand on the silica surface in order to obtain high resolution and selectivity for protein separation. In addition, the effects of the hydrophobicity of the ligand of the dual-function packings and pH of the mobile phase used on protein separation were also investigated in detail. The results show that the ligand with suitable hydrophobicity to match the electrostatic interaction is very important to prepare the dual-function stationary phase, and a better resolution and selectivity can be obtained at pH 6.5 in SCX mode. Therefore, the dual-function column can replace two individual SCX and HIC columns for protein separation and be used to set up two-dimensional liquid

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...

[Prokaryotic expression of Nanog gene and preparation of anti-Nanog antibody].

Science.gov (United States)

Li, Jun; Wang, Xiao-min; Dou, Zhong-ying; Li, Yong

2012-07-01

To express Nanog fusion protein in Escherichia coli ( E.coli), and to prepare rabbit anti-mouse polyclonal antibodies to the Nanog fusion protein. Mouse Nanog gene was amplified from the pNA992 recombinant plasmid and inserted into pET-32a vector to construct a recombinant expression vector pET-32a-Nanog. The recombinant vector was transfected into E.coli BL21 and induced by IPTG to express in them. The acquired Nanog fusion protein was purified with HisTrap affinity column and injected as an antigen into rabbits for preparing polyclonal antibodies. At last, the titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA, Western blotting and immunocytochemical staining, respectively. The recombinant expression vector pET-32a-Nanog was successfully prepared, transfected and induced to obtain the high expression of the Nanog fusion protein in a form of inclusion bodies in E.coli. After purification, its purity was up to 97%. The titer of anti-Nanog antibodies was 1:32 000 in the immunized rabbit serum, and exhibited a high specificity to Nanog protein. The rabbit anti-mouse polyclonal antibodies have been prepared successfully with a high titer and specificity to the Nanog fusion protein.

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

International Nuclear Information System (INIS)

Nawaz, H.; Nadeem, U.; Solangi, B.; Hany, O.E.

2012-01-01

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

Preparation of functional lupine protein fractions by dry separation

NARCIS (Netherlands)

Pelgrom, P.J.M.; Berghout, J.A.M.; Goot, van der A.J.; Boom, R.M.; Schutyser, M.A.I.

2014-01-01

Lupine protein concentrate is a promising ingredient that can be obtained by a combination of milling and air classification, generally called dry fractionation. This is a more sustainable route than conventional wet extraction and delivers a protein concentrate with native functional properties.

[Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

Science.gov (United States)

Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

2011-08-01

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

NARCIS (Netherlands)

Pannekoek, H.; van Meijer, M.; Gaardsvoll, H.; van Zonneveld, A. J.

1995-01-01

Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the

Nanochemistry of protein-based delivery agents

Science.gov (United States)

Rajendran, Subin; Udenigwe, Chibuike; Yada, Rickey

2016-07-01

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

Dicty_cDB: Contig-U15057-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available ( U58944 |pid:none) Dissostichus mawsoni AFGP antifreeze g... 44 0.012 C81265( C81265 )probable lipoprotein ...U43149_1( U43149 |pid:none) Dissostichus mawsoni antifreeze glycop... 36 3.2 ( P24856 ) RecName: Full=Ice-st

Quercetin loaded biopolymeric colloidal particles prepared by simultaneous precipitation of quercetin with hydrophobic protein in aqueous medium.

Science.gov (United States)

Patel, Ashok R; Heussen, Patricia C M; Hazekamp, Johan; Drost, Ellen; Velikov, Krassimir P

2012-07-15

Quercetin loaded biopolymeric colloidal particles were prepared by precipitating quercetin (water insoluble polyphenol) and zein (hydrophobic protein), simultaneously, by adding their hydro-alcoholic solution to aqueous solution in presence of sodium caseinate as an electrosteric stabiliser. The presence of protein resulted in altering the shape of quercetin precipitates from needle-like to spherical shape at higher zein proportions, as confirmed by transmission electron microscopy. The average particle size of zein:quercetin composite particles was below 200 nm (130-161 nm) with negative surface charge (-30 to -41 mV), as confirmed by dynamic light scattering and electrophoretic mobility data. Solid state characterisation (X-ray diffraction) and spectroscopic measurements (UV-Vis and IR spectroscopy) confirmed characteristic changes in quercetin due to the entrapment in the biopolymeric matrix of colloidal particles. Results from anti-oxidant study demonstrated the advantage of entrapping quercetin in the colloidal particles in terms of the chemical stability in the alkaline pH and against photodegradation under UV-light irradiation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Preparation of protein- and cell-resistant surfaces by hyperthermal hydrogen induced cross-linking of poly(ethylene oxide).

Science.gov (United States)

Bonduelle, Colin V; Lau, Woon M; Gillies, Elizabeth R

2011-05-01

The functionalization of surfaces with poly(ethylene oxide) (PEO) is an effective means of imparting resistance to the adsorption of proteins and the attachment and growth of cells, properties that are critical for many biomedical applications. In this work, a new hyperthermal hydrogen induced cross-linking (HHIC) method was explored as a simple one-step approach for attaching PEO to surfaces through the selective cleavage of C-H bonds and subsequent cross-linking of the resulting carbon radicals. In order to study the effects of the process on the polymer, PEO-coated silicon wafers were prepared and the effects of different treatment times were investigated. Subsequently, using an optimized treatment time and a modified butyl polymer with increased affinity for PEO, the technique was applied to butyl rubber surfaces. All of the treated surfaces exhibited significantly reduced protein adsorption and cell growth relative to control surfaces and compared favorably with surfaces that were functionalized with PEO using conventional chemical methods. Thus HHIC is a simple and effective means of attaching PEO to non-functional polymer surfaces.

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

Nanochemistry of protein-based delivery agents

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Subin R.C.K. Rajendran

2016-07-01

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

Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

Science.gov (United States)

Okegawa, Yuki; Koshino, Masanori; Okushima, Teruya; Motohashi, Ken

2016-02-01

Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams). Copyright © 2015 Elsevier Inc. All rights reserved.

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

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

International Nuclear Information System (INIS)

Verma, Vipin; Verma, Poonam; Ray, Alok R; Ray, Pratima

2008-01-01

Human hair proteins were isolated and purified for the fabrication of tissue-engineering scaffolds. Their cellular compatibility was studied using NIH3T3 mice fibroblast cells. The proteins were characterized using FTIR spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis for molecular weights and two-dimensional polyacrylamide gel electrophoresis for their isoelectric points (pIs). The molecular weights of keratins were in the range of 40-60 kilo-Daltons (kDa) and of matrix proteins were in the range of 15-30 kDa. The pIs of keratins were found to be in the range of 4.5-5.3. Sponges of the proteins were formed by lyophilization. Scanning electron microscopy was performed to examine the surface. Swelling studies were carried out in phosphate buffer saline at physiological pH 7.4. The hydrophilic character of the protein surface was studied by determining an average contact angle, which came to be 37 0 . The wells of tissue culture plates were coated with these proteins for studying the attachment and morphology of the cells. The protein detachment study was done to ensure the adsorption of proteins on the wells until the completion of the experiments. The cellular growth on a protein-coated surface showed three-dimensional 'bulged' morphology due to cell-cell and cell-matrix contacts. The sponges of human hair proteins supported more cells for a longer period than control. The morphology and cell proliferation studies exhibited by NIH3T3 cells on these proteins have shown their potential to be used as tissue-engineering scaffolds with better cell-cell contacts and leucine-aspartic acid-valine (LDV)-mediated cell-matrix interactions

Preparation of the Secretory Recombinant ALV-J gp85 Protein Using Pichia pastoris and Its Immunoprotection as Vaccine Antigen Combining with CpG-ODN Adjuvant.

Science.gov (United States)

Jing, Weifang; Zhou, Jinrun; Wang, Chunyang; Qiu, Jianhua; Guo, Huijun; Li, Hongmei

2018-04-26

This study focuses on preparing the secretory recombinant J subgroup of avian leukosis virus (ALV-J) gp85 protein using Pichia pastoris and evaluating its immunoprotection as vaccine antigen combining with CpG-ODN adjuvant. The secretory recombinant plasmid pPIC9-gp85 containing ALV-J gp85 gene was designed and was transfected into the genome of P. pastoris (GS115) cells. The recombinant plasmid was expressed under the induction of methanol. The expressed products in the medium of the cells were purified and identified with endoglycosidase digestion assay and western blot mediated with monoclonal antibody (MAb) JE9. The purified product combining with CpG-ODN adjuvant was inoculated intramuscularly into 7-day-old chickens and three booster inoculations were performed on 21 days post first inoculation (dpfi), 42, and 56 dpfi. The antibody responses and cellular immune responses were detected, and the protective effects were analyzed after challenge with ALV-J. The results showed that the secretory pPIC9-gp85 plasmid was successfully constructed and could be stably expressed in GS115 cells. The expressed products were N-acetylglucosylated and could specifically combine with MAb (JE9). The secreted gp85 protein combining with CpG-ODN adjuvant could induce higher antibody response and spleen lymphocyte proliferation response and IFN-γ-inducing response, and could protect all the inoculated chickens against the viremia and the immunosuppressive lesions caused by ALV-J challenge. The results of neutralizing test in vitro suggested that the antisera with some ALV-J antibody titers could neutralize ALV-J strain and inhibit the growth of virus in vitro. The result of IFA showed that IgG antibody in the antisera could specifically combine with ALV-J strain in cells. It can be concluded that the secretory recombinant gp85 protein, as a new acetylglucosylated gp85 protein, was successfully prepared and combining with CpG-ODN adjuvant could protect the inoculated chickens

First report of suspected ethylene glycol poisoning in 2 dogs in South Africa : clinical communication

Directory of Open Access Journals (Sweden)

N. Keller

2005-06-01

Full Text Available Ethylene glycol (anti-freeze toxicity is a serious emergency in both veterinary and human medicine. Ethylene glycol (E/G is the active anti-freeze principle in radiator water additives. It is odourless, colourless and has a sweet taste. As little as 5 mℓ or 20 mℓ is sufficient to kill a cat or a dog, respectively. Ethylene glycol is rapidly absorbed and metabolised in the liver to oxalate, which is deposited as calcium oxalate in the kidneys causing irreversible damage. This report describes 2 dogs that were suspected to have ingested ethylene glycol. The report contains a description of the 3 stages of ethylene glycol toxicity as well as a short discussion of the treatment. Public awareness about the dangers of anti-freeze will help in limiting exposure of pets and humans to this potentially fatal toxin. Veterinarians need to be aware of anti-freeze toxicity as delayed recognition and treatment will lead to the death of the patient.

[Preparation and characterization of monoclonal antibodies against Micrococcus luteus Rpf domain].

Science.gov (United States)

Fan, Ai-lin; Shi, Chang-hong; Su, Ming-quan; Ma, Jing; Bai, Yin-lan; Cheng, Xiao-dong; Xu, Zhi-kai; Hao, Xiao-ke

2008-05-01

To express Micrococcus luteus Rpf domain in prokaryotic cells and prepare monoclonal antibodies against Rpf domain. The gene encoding Micrococcus luteus Rpf domain was amplified from genome of Micrococcus luteus by polymerase chain reaction(PCR), and inserted into cloning vector pUC-19. After sequenced, Micrococcus luteus Rpf domain gene was subcloned into the expression vector pPro-EXHT and transfected into E.coli DH5alpha. After induced by IPTG, the bacteria controlled by T7 promoter expressed the fused Micrococcus luteus Rpf domain protein with a hexahistidine tail at its N-terminal and the target protein was purified under denaturing conditions. Using this protein as antigen to immunize the BALB/c mice and prepare monoclonal antibodies against Micrococcus luteus Rpf domain. Then specifities and relative affinities of mAbs were identified by ELISA. The fusion protein was purified by metal chelate affinity chromatography under denaturing condition. Three cloned mAbs were prepared from the mice immunized by Rpf domain. All of them could recognize Rpf domain. specifically. The prepared mAbs against Rpf domain have strong specificity with high titers, which provides useful tools for further study of the function of Rpf domain in TB prevention.

Effect of perioperative application of L-asrginine combined with intacted protein compound preparations on postoperative antitumor immunity and tumor load in patients with gastric cancer

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Xiu-Lan Jiang

2016-10-01

Full Text Available Objective: To analyze the effect of perioperative application of L-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 of L-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.

A Proteomics Sample Preparation Method for Mature, Recalcitrant Leaves of Perennial Plants

Science.gov (United States)

Na, Zhang; Chengying, Lao; Bo, Wang; Dingxiang, Peng; Lijun, Liu

2014-01-01

Sample preparation is key to the success of proteomics studies. In the present study, two sample preparation methods were tested for their suitability on the mature, recalcitrant leaves of six representative perennial plants (grape, plum, pear, peach, orange, and ramie). An improved sample preparation method was obtained: Tris and Triton X-100 were added together instead of CHAPS to the lysis buffer, and a 20% TCA-water solution and 100% precooled acetone were added after the protein extraction for the further purification of protein. This method effectively eliminates nonprotein impurities and obtains a clear two-dimensional gel electrophoresis array. The method facilitates the separation of high-molecular-weight proteins and increases the resolution of low-abundance proteins. This method provides a widely applicable and economically feasible technology for the proteomic study of the mature, recalcitrant leaves of perennial plants. PMID:25028960

A proteomics sample preparation method for mature, recalcitrant leaves of perennial plants.

Directory of Open Access Journals (Sweden)

Deng Gang

Full Text Available Sample preparation is key to the success of proteomics studies. In the present study, two sample preparation methods were tested for their suitability on the mature, recalcitrant leaves of six representative perennial plants (grape, plum, pear, peach, orange, and ramie. An improved sample preparation method was obtained: Tris and Triton X-100 were added together instead of CHAPS to the lysis buffer, and a 20% TCA-water solution and 100% precooled acetone were added after the protein extraction for the further purification of protein. This method effectively eliminates nonprotein impurities and obtains a clear two-dimensional gel electrophoresis array. The method facilitates the separation of high-molecular-weight proteins and increases the resolution of low-abundance proteins. This method provides a widely applicable and economically feasible technology for the proteomic study of the mature, recalcitrant leaves of perennial plants.

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

Science.gov (United States)

Borsuk, Sibele; Newcombe, Jane; Mendum, Tom A; Dellagostin, Odir A; McFadden, Johnjoe

2009-11-01

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

International Nuclear Information System (INIS)

Ruan, Guihua; Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui; Du, Fuyou

2016-01-01

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N_α-benzoyl-L-arginine ethyl ester to N_α-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

Energy Technology Data Exchange (ETDEWEB)

Ruan, Guihua, E-mail: guihuaruan@hotmail.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China); Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Du, Fuyou, E-mail: dufu2005@126.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China)

2016-04-22

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N{sub α}-benzoyl-L-arginine ethyl ester to N{sub α}-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

Preparations of prokaryotic expression system, standard protein and antiserum, of human Y- box binding protein 1%人YB-1的高效原核表达及其标准蛋白与抗血清的制备

Institute of Scientific and Technical Information of China (English)

李朴; 史静; 郭变琴; 钟梁; 梁勤东; 涂植光

2011-01-01

Aim： To construct a GST- expression system of human Y - box binding protein 1 （YB - 1）, prepare the YB - 1 standard protein and its antiserum. Methods ： The code sequence of YB - 1 was subcloned to the expression vector pGEX - 6P - 1. The recombinant vector was transformed into E. coli BL21 to express fusion protein GST- YB1. SDS- PAGE was applied to analyze the expression level and form of fusion protein. Then, YB- 1 standard protein was obtained by the means of GST - affinity chromatography and column - protease - digestion. The rabbit was immunized with YB - 1 protein to prepare the anti - YB1 polyclonal antibody. Results： The recombinant expression vector was constructed. SDS - PAGE and Western blot results showed that the GST - fusion protein was high- level expressed with soluble - form, and YB - 1 standard protein and its antisermn were obtained successfully. Conclusions： An economical, rapid method to prepare YB - 1 standard protein is established, and further obtained the high titer and affinity YB - 1 polyclonal antibody, which lay foundations for preparation of YB - 1 monoclonal antibody and development of YB - 1 quantitative analysis methods.%目的：构建YB1-GST表达系统，建立经济高效YB-1蛋白制备方法并制备其多抗。方法：将YB-1编码序列亚克隆至表达载体pGEX-6P-1；转化表达菌并确定可溶性表达最佳条件；采用GST亲和层析与层析柱上PSP酶切融合蛋白获取无标签蛋白的YB-1，经超滤浓缩及Western blot鉴定后，进-步真空冷冻于燥，制备YB-1标准蛋白；采用大剂量YB-1长程免疫方案免疫家兔以制备其抗体。结果：成功构建了表达载体pGEX—YB1；SDS—PAGE结果显示，YB1-GST融合蛋白以可溶性表达为主；Western blot结果证实，表达产物经GST亲和层析、PSP酶切以及真空冷冻干燥后获得了纯度较高的YB-1�

The Proteome of Ulcerative Colitis in Colon Biopsies from Adults - Optimized Sample Preparation and Comparison with Healthy Controls.

Science.gov (United States)

Schniers, Armin; Anderssen, Endre; Fenton, Christopher Graham; Goll, Rasmus; Pasing, Yvonne; Paulssen, Ruth Hracky; Florholmen, Jon; Hansen, Terkel

2017-12-01

The purpose of the study was to optimize the sample preparation and to further use an improved sample preparation to identify proteome differences between inflamed ulcerative colitis tissue from untreated adults and healthy controls. To optimize the sample preparation, we studied the effect of adding different detergents to a urea containing lysis buffer for a Lys-C/trypsin tandem digestion. With the optimized method, we prepared clinical samples from six ulcerative colitis patients and six healthy controls and analysed them by LC-MS/MS. We examined the acquired data to identify differences between the states. We improved the protein extraction and protein identification number by utilizing a urea and sodium deoxycholate containing buffer. Comparing ulcerative colitis and healthy tissue, we found 168 of 2366 identified proteins differently abundant. Inflammatory proteins are higher abundant in ulcerative colitis, proteins related to anion-transport and mucus production are lower abundant. A high proportion of S100 proteins is differently abundant, notably with both up-regulated and down-regulated proteins. The optimized sample preparation method will improve future proteomic studies on colon mucosa. The observed protein abundance changes and their enrichment in various groups improve our understanding of ulcerative colitis on protein level. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation of denatured protein bone sterilized with gamma radiation

International Nuclear Information System (INIS)

Luna Z, D.

2005-01-01

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)

Evaluation of preparation methods for MS-based analysis of intestinal epithelial cell proteomes

DEFF Research Database (Denmark)

Hesselager, Marianne Overgaard; Codrea, Marius Cosmin; Bendixen, Emøke

2015-01-01

analyzed by LC and electrospray QTOF-MS. The methods were evaluated according to efficiency, purity, transmembrane protein recovery, as well as for suitability to large-scale preparations. Our data clearly demonstrate that mucosal shaving is by far the best-suited method for in-depth MS analysis in terms...... are low in abundance, and large amounts of sample is needed for their preparation and for undertaking MS-based analysis. The aim of this study was to evaluate three different methods for isolation and preparation of pig intestinal epithelial cells for MS-based analysis of the proteome. Samples were...... of ease and speed of sample preparation, as well as protein recovery. In comparison, more gentle methods where intestinal epithelial cells are harvested by shaking are more time consuming, result in lower protein yield, and are prone to increased technical variation due to multiple steps involved....

Scientific and technical guidance for the preparation and presentation of an application for authorisation of an infant and/or follow-on formula manufactured from protein hydrolysates

DEFF Research Database (Denmark)

Sjödin, Anders Mikael

2017-01-01

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to provide scientific and technical guidance for the preparation and presentation of applications for authorisation of infant and/or follow-on formula manufactured from...... protein hydrolysates. This guidance document addresses the information and data to be submitted to EFSA on infant and follow-on formulae manufactured from protein hydrolysates with respect to the safety and suitability of the specific formula and/or the formula’s efficacy in reducing the risk...

Systematic study on the preparation of a food grade soybean protein

Energy Technology Data Exchange (ETDEWEB)

Bazinet, L.; Labrecque, R.; Toupin, R. [Institut de Recherche d`Hydro-Quebec, Varennes, PQ (Canada); Boulet, M.; Ippersiel, D.; Lamarche, F. [Food Research and Development Centre, St. Hyacinthe, PQ (Canada)

1996-05-01

A new application of electrodialysis for the precipitation and separation of soy proteins, called electro-acidification, was evaluated. The compositional properties of electro-acidified proteins were compared to those of commercial standards. Since protein solutions have physico-chemical properties that make their behaviour difficult to predict, the limiting current, and the factors which influence them, were determined. These factors included KCl concentration, protein concentration, the recirculation rate in the stack, the concentration of added salt and the temperature of the protein solution. Three approaches to electro-acidification were developed, i. e., bipolar-membrane (most rapid with the lowest energy consumption), alternating anionic and cationic membranes (most time-consuming), and anodic proton generation (intermediate between the other two). The compositional quality of electro-acidified samples was shown to be superior or equal to that of commercial standards as confirmed by the solubility profile of the products. It was therefore concluded that using electrodialysis to produce protein isolates was technologically feasible and energy efficient. 76 refs., 4 tabs., 33 figs.

Radiation-induced preparation of core/shell gold/albumin nanoparticles

Science.gov (United States)

Flores, Constanza Y.; Achilli, Estefania; Grasselli, Mariano

2018-01-01

Nanoparticles (NPs) are one of the most promising nanomaterials to be used in the biomedical field. Gold NPs (Au-NPs) have been covered with monolayers of many different molecules and macromolecules to prepare different kinds of biosensors. However, these coatings based on physisorption methods are not stable enough to prepare functional nanomaterials to be used in complex mixtures or in vivo applications. The aim of this work was to prepare a protein coating of Au-NPs based on a protein multilayer covering, stabilized by a novel radiation-induced crosslinking process. Albumins from human and bovine source were added to Au-NPs suspension and followed by ethanol addition to induce protein aggregation. Samples were irradiated with a gamma source at 10 kGy to induce a protein crosslinking according to recent findings. Samples containing 30%v/v ethanol showed a plasmon peak at about 532 nm, demonstrating the presence of non-aggregated Au-NPs. Using higher ethanol concentrations, the absorbance of plasmon peak showed NP aggregation. By Dynamic Light Scattering measurements, a new particle population with an average diameter of about 60 nm was found. Moreover, TEM images showed that the NPs had spherical shape and the presence of a low-density halo around the metal core confirmed the presence of the protein shell. An irradiation dose of one kGy was enough to show changes in the plasmon peak characteristics. The increase in the chemical stability of protein shell was demonstrated by the reduction in the NP dissolution kinetics in presence of cyanate.

Investigation of in-situ poly(lactic acid)/soy protein concentrate composites: Composite preparation, properties and foam application development

Science.gov (United States)

Liu, Bo

2011-12-01

In this study, soy protein (SP), the residue of oil crushing, was used for preparation of value-added thermoplastics. Novel poly(lactic acid) (PLA)/soy protein concentrate (SPC) blends were investigated and foaming of the resulting blends was developed. PLA/SPC blends were prepared by twin-screw extrusion and test specimens by injection molding. Unlike the practice elsewhere SP was used as a filler in mixing with other polymers, SPC was processed as a plastic component in blending process in this work. Processing SPC as plastic component, water played an important role in terms of the deformability and the morphology of SP thus the properties of the blends. Plasticization of SP, compatibilization of the blends and structure-property relationship of the PLA/SPC blends were studied. In the literature water and glycerol were often used together in preparing SP plastics or plastic blends, but this study found that this traditional combination did not provide the best results in terms of morphology and mechanical properties. Water is only recommended in plasticizing SP in the blends. This study showed water as a plasticizer was a domain factor on control of morphology and properties of PLA/SPC blends. The due to the evaporation of water after extrusion, SP domain lost its deformability thus resulted in in-situ composites. Interconnected SPC phase structure was achieved by control water content in the pre-formulated SPC and SPC content in the blends. A novel dual compatibilization method was developed to improve the properties of PLA/SPC blends. Poly(2-ethyl-2-oxazoline) was used to improve the dispersion of SPC in the blending stage, and polymeric methylene diphenyl diisocyanate was used to improve the interfacial adhesion between SPC and PLA in the subsequent processing. The result showed excellent mechanical properties and improved thermal properties of PLA/SPC blends. Using processing aids is an effective way to decrease processing temperature and thermal degradation

Acid-Induced Cold Gelation of Globular Proteins: Effects of Protein Aggregate Characteristics and Disulfide Bonding on Rheological Properties

NARCIS (Netherlands)

Alting, A.C.; Weijers, M.; Hoog, E.H.A. de; Pijpekamp, A.M. van de; Cohen Stuart, M.A.; Hamer, R.J.; Kruif, C.G. de; Visschers, R.W.

2004-01-01

The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked

Adamantane-based amphiphiles (ADAs) for membrane protein study: importance of a detergent hydrophobic group in membrane protein solubilisation.

Science.gov (United States)

Chae, Pil Seok; Bae, Hyoung Eun; Das, Manabendra

2014-10-21

We prepared adamantane-containing amphiphiles and evaluated them using a large membrane protein complex in terms of protein solubilisation and stabilization efficacy. These agents were superior to conventional detergents, especially in terms of the membrane protein solubilisation efficiency, implying a new detergent structure-property relationship.

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

Science.gov (United States)

Deng, Shanggui; Huo, Jiancong; Xie, Chao

2008-08-01

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.

Electrophoresis in ice surface grooves for probing protein affinity to a specific plane of ice crystal.

Science.gov (United States)

Inagawa, Arinori; Okada, Yusuke; Okada, Tetsuo

2018-06-01

Channel-like grooves are formed on the surface of frozen aqueous sucrose. They are filled with a freeze concentrated solution (FCS) and act as an efficient size-tunable separation field for micro and nanoparticles. The width of the channel can be easily varied by changing the temperature. Because the channel width decreases with decreasing temperature, particles become immobilized due to physical interference from the ice wall when the temperature reaches a threshold point specific to the particle size. Surface modification of particles can add a factor of chemical interaction between the particles and ice walls. In this study, anti-freeze proteins (AFPs) are anchored on 1µm-polystyrene (PS) particles, and their behavior in the surface grooves on the ice is studied. The threshold temperature is an effective criterion for evaluating chemical interactions between particles and ice walls. The AFP binding on 1µm PS particles lowers the threshold temperature by 2.5°C, indicating interactions between AFPs on the PS particles and the ice wall. Because the AFPs studied here show selectivity towards the prism plane, it is critical that the prism plane of the ice crystal is in contact with the FCS in the surface grooves. Copyright © 2017 Elsevier B.V. All rights reserved.

Water-based preparation of spider silk films as drug delivery matrices.

Science.gov (United States)

Agostini, Elisa; Winter, Gerhard; Engert, Julia

2015-09-10

The main focus of this work was to obtain a drug delivery matrix characterized by biocompatibility, water insolubility and good mechanical properties. Moreover the preparation process has to be compatible with protein encapsulation and the obtained matrix should be able to sustain release a model protein. Spider silk proteins represent exceptional natural polymers due to their mechanical properties in combination with biocompatibility. As both hydrophobic and slowly biodegrading biopolymers, recombinant spider silk proteins fulfill the required properties for a drug delivery system. In this work, we present the preparation of eADF4(C16) films as drug delivery matrices without the use of any organic solvent. Water-based spider silk films were characterized in terms of protein secondary structure, thermal stability, zeta-potential, solubility, mechanical properties, and water absorption and desorption. Additionally, this study includes an evaluation of their application as a drug delivery system for both small molecular weight drugs and high molecular weight molecules such as proteins. Our investigation focused on possible improvements in the film's mechanical properties including plasticizers in the film matrix. Furthermore, different film designs were prepared, such as: monolayer, coated monolayer, multilayer (sandwich), and coated multilayer. The release of the model protein BSA from these new systems was studied. Results indicated that spider silk films are a promising protein drug delivery matrix, capable of releasing the model protein over 90 days with a release profile close to zero order kinetic. Such films could be used for several pharmaceutical and medical purposes, especially when mechanical strength of a drug eluting matrix is of high importance. Copyright © 2015 Elsevier B.V. All rights reserved.

Biosynthetic preparation of 35-S labelled methionine

International Nuclear Information System (INIS)

Freud, A.; Hirshfeld, N.; Teitelbaum, Z.; Heimer, Y.

1986-11-01

High specific activity methionine with sulfur-35 was prepared in our laboratory by growing Baker's yeast cells, in a medium containing 35 S-sulfate. L-S 35 methionine was prepared from the acid hydrolyzate of the proteins by chromatography on whatman paper. The specific activity was determined using o-phtaladehyde as a fluorophore to form a fluorescent complex. The specific activity was found to be usually greater than 800 Ci/mmol. (Author)

Preparation of polymethacrylic acid-grafted HEMA/PVP microspheres and preliminary study on basic protein adsorption.

Science.gov (United States)

Gao, Baojiao; Hu, Hongyan; Guo, Jianfeng; Li, Yanbin

2010-06-01

The crosslinked copolymeric microspheres (HEMA/NVP) of N-vinylpyrrolidone (NVP) and 2-hydroxyethyl methacrylate (HEMA) were prepared using inverse suspension polymerization method. Subsequently, the reaction of methacryloyl chloride with the hydroxyl groups on the surfaces of HEMA/NVP microspheres was performed, leading to the introduction of polymerisable double bonds onto the surfaces of microspheres HEMA/NVP. Afterward, methacrylic acid was allowed to be graft-polymerized on microspheres HEMA/NVP in the manner of "grafting from", resulting in the grafted microspheres PMAA-HEMA/NVP. The grafted microspheres PMAA-HEMA/NVP were fully characterized with several means. The graft-polymerization of MAA on microspheres HEMA/NVP was studied in detail, and the optimal reaction conditions were determined. Thereafter, the adsorption property of the grafted microspheres PMAA-HEMA/NVP for lysozyme as a basic protein model was preliminarily examined to explore the feasibility of removing deleterious basic protein such as density lipoprotein from blood. The experimental results indicate that the PMAA grafting degree on microspheres HEMA/NVP is limited because an enwinding polymer layer as a kinetic barrier on the surfaces of HEMA/NVP microspheres will be formed during the graft-polymerization, and block the graft-polymerization. In order to enhance PMAA grafting degree, reaction temperature, monomer concentration and the used amount of initiator should be effectively controlled. The experimental results also reveal that the grafted microspheres PMAA-HEMA/NVP possess very strong adsorption ability for lysozyme by right of strong electrostatic interaction. Copyright 2010 Elsevier B.V. All rights reserved.

Pressure cryocooling protein crystals

Science.gov (United States)

Kim, Chae Un [Ithaca, NY; Gruner, Sol M [Ithaca, NY

2011-10-04

Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

Preparation and characterization of a radioiodinated bacterial lipopolysaccharide

Energy Technology Data Exchange (ETDEWEB)

Ulevitch, R J [Scripps Clinic and Research Foundation, La Jolla, Calif. (USA)

1978-03-01

Radioiodinated lipopolysaccharide (LPS) from E.coli 0111:B4 has been prepared by reacting p-OH methylbenzimidate with 0111:B4 LPS at alkaline pH. The resulting LPS derivative has been radiolabeled with Na/sup 125/I. Specific activities of up to 5..mu..Ci/..mu..g LPS may be obtained by this technique and significantly the preparation of the radioiodinated LPS does not alter the biophysical, immunologic or biologic properties of 0111:B4 LPS. The methods described here are applicable to any 'protein free' LPS preparation containing primary amino groups.

Preparation and Characterization of Soluble Eggshell Membrane Protein/PLGA Electro spun Nano fibers for Guided Tissue Regeneration Membrane

International Nuclear Information System (INIS)

Jia, J.; Liu, G.; Duan, Y.; Guo, Z.; Yu, J.

2012-01-01

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) nano fibers using electro spinning method for GTR membrane application. SEP/PLGA nano fibers were successfully prepared with various blending ratios. The morphology, chemical composition, surface wettability, and mechanical properties of the nano fibers 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 nano fibers and investigate the interaction between cells and nano fibers. Results showed that the SEP/PLGA electro spun membrane was composed of uniform, bead-free nano fibers, 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 nano fibers could enhance cell attachment, spreading, and proliferation. The study indicated the potential of SEP/PLGA nano fibers for GTR application and provided a basis for future optimization

Microbial viability in preparations packaged for single use.

Science.gov (United States)

Obayashi, Akiko; Oie, Shigeharu; Kamiya, Akira

2003-05-01

We evaluated microbial viability in preparations packaged for single use only which mandate that residual solution be discarded such as albumin and globulin preparations as blood products, preparations containing albumin (such as urokinase and interferon), fat emulsions, and a preparation containing fat emulsions (propofol). In most preparations, Serratia marcescens and Burkholderia cepacia proliferated rapidly at 30 degrees C. However, in globulin preparations containing 1-2.25% glycine to prevent protein degradation (Gamma-Venin P, Venilon-I, Globulin Injection, and Ahlbulin), no growth of S. marcescens and B. cepacia was detected over 24 h at 30 degrees C. For globulin preparations containing 1-2.25% glycine, the injunction to "Discard residual solution after the package has been used" in the package inserts can be revised to "It is possible to use residual solution within 24 h after the package has been used with storage in a cool place."

Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane.

Science.gov (United States)

Yang, Yongxin; Anderson, Elizabeth; Zhang, Sheng

2018-04-12

Proteomic analysis of membrane proteins is challenged by the proteins solubility and detergent incompatibility with MS analysis. No single perfect protocol can be used to comprehensively characterize the proteome of membrane fraction. Here, we used cow milk fat globule membrane (MFGM) proteome analysis to assess six sample preparation procedures including one in-gel and five in-solution digestion approaches prior to LC-MS/MS analysis. The largest number of MFGM proteins were identified by suspension trapping (S-Trap) and filter-aided sample preparation (FASP) methods, followed by acetone precipitation without clean-up of tryptic peptides method. Protein identifications with highest average coverage was achieved by Chloroform/MeOH, in-gel and S-Trap methods. Most distinct proteins were identified by FASP method, followed by S-Trap. Analyses by Venn diagram, principal-component analysis, hierarchical clustering and the abundance ranking of quantitative proteins highlight differences in the MFGM fraction by the all sample preparation procedures. These results reveal the biased proteins/peptides loss occurred in each protocol. In this study, we found several novel proteins that were not observed previously by in-depth proteomics characterization of MFGM fraction in milk. Thus, a combination of multiple procedures with orthologous properties of sample preparation was demonstrated to improve the protein sequence coverage and expression level accuracy of membrane samples. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Expression, purification and antibody preparation of recombinat SARS-CoV X5 protein].

Science.gov (United States)

Wang, Li-Na; Kong, Jian-Qiang; Zhu, Ping; Du, Guan-Hua; Wang, Wei; Cheng, Ke-Di

2008-11-01

X5 protein is one of the putative unknown proteins of SARS-CoV. The recombinant protein has been successfully expressed in E. coli in the form of insoluble inclusion body. The inclusion body was dissolved in high concentration of urea. Affinity Chromatography was preformed to purify the denatured protein, and then the product was refolded in a series of gradient solutions of urea. The purified protein was obtained with the purity of > 95% and the yield of 93.3 mg x L(-1). Polyclonal antibody of this protein was obtained, and Western blotting assay indicated that the X5 protein has the strong property of antigen. Sixty-eight percent of the recombinant protein sequence was confirmed by LC-ESI-MS/MS analysis.

Serve Size and Estimated Energy and Protein Contents of Meals Prepared by 'Meals on Wheels' South Australia Inc.: Findings from a Meal Audit Study.

Science.gov (United States)

Arjuna, Tony; Miller, Michelle; Soenen, Stijn; Chapman, Ian; Visvanathan, Renuka; Luscombe-Marsh, Natalie D

2018-02-20

An audit of 'standard' (STD) and 'energy and protein fortified' (HEHP) meals from Meals on Wheels (MOW) South Australia's summer menu was conducted to evaluate the consistency, and serve size and nutrient contents, of their menu items. Twenty soups, 20 mains and 20 desserts from each of the STD and HEHP menus were prepared at the MOW South Australia's kitchen and delivered to three 'sham(dummy)-clients' over a 5-week period. Each meal component was weighed in triplicate, to the nearest gram, the variation within the serve weight was calculated, and the overall energy and protein content of each meal was determined using FoodWorks (Xyris Software, Highgate Hill, Queensland, Australia). On average, the variability for soups and mains was ≤6% and for desserts was ≤10% and although the measured serve sizes of the MOW meals were consistently smaller than prescribed serve size, the differences were minor. As a percentage of recommended daily intakes (RDIs) for adults aged over 60 years, we calculated that the STD meals contained 21-39% for energy and 42-63% for protein while the HEHP meals contained 29-55% for energy and 46-69% for protein. These findings demonstrate that MOW meals currently meet the voluntary meal guidelines for energy and protein.

Hypoxic-induced stress protein expression in rat cardiac myocytes

International Nuclear Information System (INIS)

Howard, G.; Geoghegan, T.E.

1986-01-01

Mammalian stress proteins can be induced in cells and tissues exposed to a variety of conditions including hyperthermia and diminished O 2 supply. The authors have previously shown that the expression of three stress proteins (71, 85, and 95 kDa) was induced in cardiac tissue from mice exposed to hypoxic conditions. The expression of mRNAs coding for the 85 and 95 kDa proteins increase with time of exposure to hypoxia, while the mRNA coding for the 71 kDa protein is transiently induced. The authors extended these studies to investigate the expression of stress proteins in isolated rat cardiac myocytes. Freshly prepared myocytes were exposed to control, hypoxic, anoxic, or heat-shock environments for up to 16 h. The proteins were then labeled for 6 hours with [ 35 S]methionine. Analysis of the solubilized proteins by SDS-PAGE and autoradiography showed that there was a 6-fold increase in synthesis of the 85 kDa protein upon exposure to hypoxia but not heat-shock conditions. The 71 kDa protein was present at high levels in both control and treated myocyte protein preparations, and presumably had been induced during the isolation procedure. Total RNA isolated from intact rat heart and isolated myocytes was compared by cell-free translation analysis and showed induction of RNAs coding for several stress proteins in the myocyte preparation. The induced proteins at 85 and 95 kDa have molecular weights similar to reported cell stress and/or glucose-regulated proteins

Antifreeze poisoning

Science.gov (United States)

... and breathing machine Chest x-ray CT scan (advanced brain imaging) ECG (electrocardiogram or heart tracing) Intravenous ... Nelson ME. Toxic alcohols. In: Walls RM, Hockberger RS, Gausche-Hill M, eds. Rosen's Emergency Medicine: Concepts ...

Effects of Preparation Conditions on Morphology of Polyacrylonitrile Micro/Ultrafiltration Membrane and Its Application in Protein and Fat Separation from Milk

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Seyed Ali Alavi

2014-04-01

Full Text Available Polyacrylonitrile (PAN micro/ultrafiltration membranes were prepared by phase inversion method. The effects of various preparation conditions including polymeric solution concentration, evaporation time, temperature, composition and residence time of the coagulation bath were investigated. Various important membrane characteristics such as pore size, bulk porosity, and mechanical and morphological properties were taken into the consideration. The characterizations were performed by measuring the bubble point, water flux, tensile strength and scanning electron microscopy (SEM analyses. The results showed that by increasing the polymeric solution concentration from 13 to 17 wt%, the porosity and water flux were decreased. Moreover, the membrane skin layer was considerably thickened with a very significant decrease in its pore sizes which was achieved in ultrafiltration region. By increasing the evaporation time at atmospheric pressure, membrane skin layer was thickened and the pore sizes were decreased. Low coagulation bath temperatures (below 30°C resulted in lower pore size, water flux, and an increase in membrane mechanical strength. Introduction of isopropanol (IPA into the water coagulation bath led to lower coagulation rate and consequently, the formation of smaller pores became possible by using pure isopropanol as coagulation bath. Furthermore, by increasing the residence time in coagulation bath, a more porous structure with more uniform pore sizes were formed that showed better mechanical properties. Finally, the so-called ultrafiltration membranes were applied in concentration process of protein and milk fat. A protein rejection more than 93% was attained while a complete removal of milk fat was achieved.

Multiple acquisition of magic angle spinning solid-state NMR experiments using one receiver: Application to microcrystalline and membrane protein preparations

Science.gov (United States)

Gopinath, T.; Veglia, Gianluigi

2015-04-01

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

Proof of concept of a "greener" protein purification/enrichment method based on carboxylate-terminated carbosilane dendrimer-protein interactions.

Science.gov (United States)

González-García, Estefanía; Maly, Marek; de la Mata, Francisco Javier; Gómez, Rafael; Marina, María Luisa; García, María Concepción

2016-11-01

Protein sample preparation is a critical and an unsustainable step since it involves the use of tedious methods that usually require high amount of solvents. The development of new materials offers additional opportunities in protein sample preparation. This work explores, for the first time, the potential application of carboxylate-terminated carbosilane dendrimers to the purification/enrichment of proteins. Studies on dendrimer binding to proteins, based on protein fluorescence intensity and emission wavelengths measurements, demonstrated the interaction between carboxylate-terminated carbosilane dendrimers and proteins at all tested pH levels. Interactions were greatly affected by the protein itself, pH, and dendrimer concentration and generation. Especially interesting was the interaction at acidic pH since it resulted in a significant protein precipitation. Dendrimer-protein interactions were modeled observing stable complexes for all proteins. Carboxylate-terminated carbosilane dendrimers at acidic pH were successfully used in the purification/enrichment of proteins extracted from a complex sample. Graphical Abstract Images showing the growing turbidity of solutions containing a mixture of proteins (lysozyme, myoglobin, and BSA) at different protein:dendrimer ratios (1:0, 1:1, 1:8, and 1:20) at acidic pH and SDS-PAGE profiles of the corresponsing supernatants. Comparison of SDS-PAGE profiles for the pellets obtained during the purification of proteins present in a complex sample using a conventional "no-clean" method based on acetone precipitation and the proposed "greener" method using carboxylate-terminated carbosilane dendrimer at a 1:20 protein:dendrimer ratio.

Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells.

Science.gov (United States)

Glatter, Timo; Ahrné, Erik; Schmidt, Alexander

2015-11-06

We evaluated different in-solution and FASP-based sample preparation strategies for absolute protein quantification. Label-free quantification (LFQ) was employed to compare different sample preparation strategies in the bacterium Pseudomonas aeruginosa and human embryonic kidney cells (HEK), and organismal-specific differences in general performance and enrichment of specific protein classes were noted. The original FASP protocol globally enriched for most proteins in the bacterial sample, whereas the sodium deoxycholate in-solution strategy was more efficient with HEK cells. Although detergents were found to be highly suited for global proteome analysis, higher intensities were obtained for high-abundant nucleic acid-associated protein complexes, like the ribosome and histone proteins, using guanidine hydrochloride. Importantly, we show for the first time that the observable total proteome mass of a sample strongly depends on the sample preparation protocol, with some protocols resulting in a significant underestimation of protein mass due to incomplete protein extraction of biased protein groups. Furthermore, we demonstrate that some of the observed abundance biases can be overcome by incorporating a nuclease treatment step or, alternatively, a correction factor for complementary sample preparation approaches.

Full replacement of menhaden fish meal protein by low-gossypol cottonseed flour protein in the diet of juvenile black sea bass Centropristis striata

Science.gov (United States)

Eight iso-nitrogeneous (46% crude protein) and iso-lipidic (14% crude lipid) diets were formulated and prepared to replace menhaden fish meal (FM) protein (59.5% CP) by low-gossypol glandless meal (GCSM) protein (50.4% CP), solvent-extracted cottonseed meal (SCSM) protein (53.8% protein) and high go...

Preparation and evaluation of microparticles from thiolated polymers via air jet milling.

Science.gov (United States)

Hoyer, Herbert; Schlocker, Wolfgang; Krum, Kafedjiiski; Bernkop-Schnürch, Andreas

2008-06-01

Microparticles were formulated by incorporation of the model protein horseradish peroxidase in (thiolated) chitosan and (thiolated) poly(acrylic acid) via co-precipitation. Dried protein/polymer complexes were ground with an air jet mill and resulting particles were evaluated regarding size distribution, shape, zeta potential, drug load, protein activity, release pattern, swelling behaviour and cytotoxicity. The mean particle size distribution was 0.5-12 microm. Non-porous microparticles with a smooth surface were prepared. Microparticles from (thiolated) chitosan had a positive charge whereas microparticles from (thiolated) poly(acrylic acid) were negatively charged. The maximum protein load for microparticles based on chitosan, chitosan-glutathione (Ch-GSH), poly(acrylic acid) (PAA) and for poly(acrylic acid)-glutathione (PAA-GSH) was 7+/-1%, 11+/-2%, 4+/-0.2% and 7+/-2%, respectively. The release profile of all microparticles followed a first order release kinetic. Chitosan (0.5mg), Ch-GSH, PAA and PAA-GSH particles showed a 31.4-, 13.8-, 54.2- and a 42.2-fold increase in weight, respectively. No significant cytotoxicity could be found. Thiolated microparticles prepared by jet milling technique were shown to be stable and to have controlled drug release characteristics. After further optimizations the preparation method described here might be a useful tool for the production of protein loaded drug delivery systems.

The two capsid proteins of maize rayado fino virus contain common peptide sequences.

Science.gov (United States)

Falk, B W; Tsai, J H

1986-01-01

Virions of maize rayado fino virus (MRFV) were purified and two major capsid proteins (ca. Mr 29,000 and 22,000) were resolved by SDS-PAGE. When the two major capsid proteins were isolated from gels and compared by one-dimensional peptide mapping after digestion with Staphylococcus aureus V-8 protease, indistinguishable peptide maps were obtained, suggesting that these two proteins contain common peptide sequences. Some preparations also showed minor protein components that were intermediate between the Mr 22,000 and Mr 29,000 capsid proteins. One of the minor proteins, ca. Mr 27,000, gave a peptide map indistinguishable from the major capsid proteins. In vitro ageing of partially purified preparations or virion treatment with proteolytic enzymes failed to show conversion of the Mr 29,000 protein to a Mr 22,000. Protease inhibitors added to the buffers used for virion purification did not affect the apparent 1:3 ratio of 29,000 to 22,000 proteins in the purified preparations.

Virosome and ISCOM vaccines against Newcastle disease: preparation, characterization and immunogenicity

NARCIS (Netherlands)

Homhuan, A.; Prakongpan, S.; Poomvises, P.; Maas, H.A.; Krommelin, D.; Kersten, G.; Jiskoot, W.

2004-01-01

The purpose of this study was to prepare and characterize virosomes and ISCOMs containing envelope proteins of Newcastle disease virus (NDV) and to evaluate their immunogenicity in target animals (chickens). Virosomes were prepared by solubilization of virus with either Triton X-100 or octyl

Expression of human protein S100A7 (psoriasin, preparation of antibody and application to human larynx squamous cell carcinoma

Directory of Open Access Journals (Sweden)

Barbieri Manuela R

2011-11-01

Full Text Available Abstract Background Up-regulation of S100A7 (Psoriasin, a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. Results The rS100A7 (His-tag protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag rabbit serum (polyclonal antibody anti-rS100A7. The molecular weight of rS100A7 (His-tag protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da. Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. Conclusions The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

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

Model to predict inhomogeneous protein-sugar distribution in powders prepared by spray drying

NARCIS (Netherlands)

Grasmeijer, Niels; Frijlink, Henderik W.; Hinrichs, Wouter L. J.

2016-01-01

A protein can be stabilized by spray drying an aqueous solution of the protein and a sugar, thereby incorporating the protein into a glassy sugar matrix. For optimal stability, the protein should be homogeneously distributed inside the sugar matrix. The aim of this study was to develop a model that

Modulating surface rheology by electrostatic protein/polysaccharide interactions

NARCIS (Netherlands)

Ganzevles, R.A.; Zinoviadou, K.; Vliet, van T.; Cohen Stuart, M.A.; Jongh, de H.H.J.

2006-01-01

There is a large interest in mixed protein/polysaccharide layers at air-water and oil-water interfaces because of their ability to stabilize foams and emulsions. Mixed protein/polysaccharide adsorbed layers at air-water interfaces can be prepared either by adsorption of soluble protein/

Cryo-electron microscopy of membrane proteins.

Science.gov (United States)

Goldie, Kenneth N; Abeyrathne, Priyanka; Kebbel, Fabian; Chami, Mohamed; Ringler, Philippe; Stahlberg, Henning

2014-01-01

Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.

Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

Directory of Open Access Journals (Sweden)

Zarei Saeed

2011-08-01

Full Text Available Abstract Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also

Novel Aflatoxin Derivatives and Protein Conjugates

Directory of Open Access Journals (Sweden)

Reinhard Niessner

2007-03-01

Full Text Available Aflatoxins, a group of structurally related mycotoxins, are well known for their toxic and carcinogenic effects in humans and animals. Aflatoxin derivatives and protein conjugates are needed for diverse analytical applications. This work describes a reliable and fast synthesis of novel aflatoxin derivatives, purification by preparative HPLC and characterisation by ESI-MS and one- and two-dimensional NMR. Novel aflatoxin bovine serum albumin conjugates were prepared and characterised by UV absorption and MALDI-MS. These aflatoxin protein conjugates are potentially interesting as immunogens for the generation of aflatoxin selective antibodies with novel specificities.

Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Micro-organisms.

Science.gov (United States)

Hasan, Muhammad; Fayter, Alice E R; Gibson, Matthew I

2018-06-22

All modern molecular biology and microbiology is underpinned not only by the tools to handle and manipulate microorganisms, but also those to store, bank and transport them. Glycerol is the current gold-standard cryoprotectant but it is intrinsically toxic to most micro-organisms: only a fraction of cells survive freezing and the presence of glycerol can impact down-stream applications and assays. Extremophile organisms survive repeated freeze/thaw cycles by producing antifreeze proteins which are potent ice recrystallization inhibitors. Here we introduce a new concept for the storage/transport of micro-organisms by using ice recrystallization inhibiting poly(vinyl alcohol) in tandem with poly(ethylene glycol). This cryopreserving formulation is shown to result in a 4-fold increase in E. coli yield post-thaw, compared to glycerol, utilizing lower concentrations, with successful cryopreservation at just 1.1 weight percent of additive. The mechanism of protection is demonstrated to be linked to inhibiting ice recrystallization (by comparison to a recombinant antifreeze protein) but also to the significantly lower toxicity of the polymers compared to glycerol. Optimized formulations are presented and shown to be broadly applicable to the cryopreservation of a panel of Gram negative, Gram positive and Mycobacteria strains. This represents a step-change in how micro-organisms will be stored by the design of new macromolecular ice growth inhibitors; it should enable a transition from traditional solvent-based to macromolecular microbiology storage methods.

Improvement of a sample preparation method assisted by sodium deoxycholate for mass-spectrometry-based shotgun membrane proteomics.

Science.gov (United States)

Lin, Yong; Lin, Haiyan; Liu, Zhonghua; Wang, Kunbo; Yan, Yujun

2014-11-01

In current shotgun-proteomics-based biological discovery, the identification of membrane proteins is a challenge. This is especially true for integral membrane proteins due to their highly hydrophobic nature and low abundance. Thus, much effort has been directed at sample preparation strategies such as use of detergents, chaotropes, and organic solvents. We previously described a sample preparation method for shotgun membrane proteomics, the sodium deoxycholate assisted method, which cleverly circumvents many of the challenges associated with traditional sample preparation methods. However, the method is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of sodium deoxycholate when it is used at relatively low concentrations such as 1%. Hence, we present an enhanced sodium deoxycholate sample preparation strategy that first uses a high concentration of sodium deoxycholate (5%) to lyse membranes and extract/solubilize hydrophobic membrane proteins, and then dilutes the detergent to 1% for a more efficient digestion. We then applied the improved method to shotgun analysis of proteins from rat liver membrane enriched fraction. Compared with other representative sample preparation strategies including our previous sodium deoxycholate assisted method, the enhanced sodium deoxycholate method exhibited superior sensitivity, coverage, and reliability for the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Collagen targeting using multivalent protein-functionalized dendrimers

NARCIS (Netherlands)

Breurken, M.; Lempens, E.H.M.; Temming, R.P.; Helms, B.A.; Meijer, E.W.; Merkx, M.

2011-01-01

Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic

VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis.

Science.gov (United States)

Xu, G; Wilson, W; Mecham, J; Murphy, K; Zhou, E M; Tabachnick, W

1997-07-01

The importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overalay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM-2 insect cells.

Adhesives from modified soy protein

Science.gov (United States)

Sun, Susan [Manhattan, KS; Wang, Donghai [Manhattan, KS; Zhong, Zhikai [Manhattan, KS; Yang, Guang [Shanghai, CN

2008-08-26

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

Validation of protein carbonyl measurement: A multi-centre study

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Edyta Augustyniak

2015-04-01

Full Text Available Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial kits. We have further explored the potential causes of variance in carbonyl analysis in a ring study. A soluble protein fraction was prepared from rat liver and exposed to 0, 5 and 15 min of UV irradiation. Lyophilised preparations were distributed to six different laboratories that routinely undertook protein carbonyl analysis across Europe. ELISA and Western blotting techniques detected an increase in protein carbonyl formation between 0 and 5 min of UV irradiation irrespective of method used. After irradiation for 15 min, less oxidation was detected by half of the laboratories than after 5 min irradiation. Three of the four ELISA carbonyl results fell within 95% confidence intervals. Likely errors in calculating absolute carbonyl values may be attributed to differences in standardisation. Out of up to 88 proteins identified as containing carbonyl groups after tryptic cleavage of irradiated and control liver proteins, only seven were common in all three liver preparations. Lysine and arginine residues modified by carbonyls are likely to be resistant to tryptic proteolysis. Use of a cocktail of proteases may increase the recovery of oxidised peptides. In conclusion, standardisation is critical for carbonyl analysis and heavily oxidised proteins may not be effectively analysed by any existing technique.

Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

International Nuclear Information System (INIS)

Senogles, S.E.; Caron, M.G.

1986-01-01

The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

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.

Protective effect of a coffee preparation (Nescafe pure) against carbon tetrachloride-induced liver fibrosis in rats.

Science.gov (United States)

Shi, Hongyang; Dong, Lei; Zhang, Yong; Bai, Yanhua; Zhao, Juhui; Zhang, Li

2010-06-01

We examined the effects of a coffee preparation on liver fibrosis induced by carbon tetrachloride (CCl(4)) and explored the possible mechanisms. Rats were divided randomly into four groups: control, CCl(4), and two coffee preparation groups. Except for the control group, liver fibrosis was induced in male Sprague-Dawley (SD) rats by subcutaneous injection with 40% CCl(4) twice a week for 8 weeks. At the same time, a coffee preparation (300 mg/kg and 150 mg/kg) was administered to the two coffee preparation groups intragastrically once daily. Upon pathological examination, a coffee preparation treatment significantly reduced liver damage and symptoms of liver fibrosis. The mRNA expression of collagen I, collagen III, bcl-2, vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGF-beta1) were markedly increased by CCl(4) treatment but suppressed by a coffee preparation treatment. Whereas compared with the CCl(4) group, the mRNA expression of Bax was increased in the coffee preparation group. The protein expression of Bax and bcl-2 were confirmed by western blot. Intragastric administration of a coffee preparation reduced the protein expression of alpha-smooth muscle actin (alpha-SMA) and the glucose-regulated proteins (GRP) 78 and 94 in rats increased by CCl(4). Our data indicate that a coffee preparation can efficiently inhibit CCl(4)-induced liver fibrosis in rats. The coffee preparation may therefore be a potential functional food for preventing liver fibrosis. Copyright 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Polymers for Protein Conjugation

Directory of Open Access Journals (Sweden)

Gianfranco Pasut

2014-01-01

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

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.

Large-scale preparation of plasmid DNA.

Science.gov (United States)

Heilig, J S; Elbing, K L; Brent, R

2001-05-01

Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

[Prokaryotic expression of recombinant prochymosin gene and its antiserum preparation].

Science.gov (United States)

Li, Xin-ping; Liu, Huan-huan; Pu, Yan; Zhang, Fu-chun; Li, Yi-jie

2012-07-01

To optimize the prochymosin (pCHY) gene codons and express the gene in Escherichia coli (E.coli), and to prepare its antiserum and detect chymosin protein specifically. According to codon usage bias of E.coli, prochymosin gene sequence was synthesized based on the conserved sequences of prochymosin gene from bovine, lamb and camel, and then cloned into the plasmid pET-30a and pcDNA3-AAT-COMP-C3d3 (pcD-ACC), respectively. pET-30a-pCHY was expressed, as the detected antigen, in E.coli BL21(DE3) after IPTG induction. RT-PCR was used to detect prochymosin mRNA expression in liver from the mice injected pcDNA3-AAT-COMP-pCHY-C3d3(pACCC) by hydrodynamics-based transfection method. To prepare the antiserum of prochymosin, pACCC and GST-pCHY proteins were used to immunize New Zealand rabbits in accordance with DNA prime-protein boost strategy. Antibody levels were tested by ELISA. Western blotting showed the molecular weight of His-pCHY protein was about 55 000, similar to the expected molecular size. ELISA demonstrated that the titer level of prochymosin antiserum was high. Based on the codon optimization, we have obtained high-titer prochymosin antiserum through DNA vaccine vector pcD-ACC combined with DNA prime-protein boost strategy, similar to that by protein vaccine.

In Vitro Digestibility of Rapeseed and Bovine Whey Protein Mixtures.

Science.gov (United States)

Joehnke, Marcel Skejovic; Rehder, Alina; Sørensen, Susanne; Bjergegaard, Charlotte; Sørensen, Jens Christian; Markedal, Keld Ejdrup

2018-01-24

Partial replacement of animal protein sources with plant proteins is highly relevant for the food industry, but potential effects on protein digestibility need to be established. In this study, the in vitro protein digestibility (IVPD) of four protein sources and their mixtures (50:50 w/w ratio) was investigated using a transient pepsin hydrolysis (1 h) followed by pancreatin (1 h). The protein sources consisted of napin-rich rapeseed (Brassica napus L.) protein concentrates (RPCs; RP1, RP2) prepared in pilot scale and major bovine whey proteins (WPs; α-LA, alpha-lactalbumin; β-LG, beta-lactoglobulin). IVPD of individual protein sources was higher for WPs compared to RPCs. The RP2/β-LG mixture resulted in an unexpected high IVPD equivalent to β-LG protein alone. Protein mixtures containing RP1 showed a new IVPD response type due to the negative influence of a high trypsin inhibitor activity (TIA) level. Improved IVPD of RP1 alone and in protein mixtures was obtained by lowering the TIA level using dithiothreitol (DTT). These results showed that napin-rich protein products prepared by appropriate processing can be combined with specific WPs in mixtures to improve the IVPD.

A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

Science.gov (United States)

Kostyleva, E V; Sereda, A S; Velikoretskaya, I A; Nefedova, L I; Sharikov, A Yu; Tsurikova, N V; Lobanov, N S; Semenova, M V; Sinitsyn, A P

2016-07-01

Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.

Serve Size and Estimated Energy and Protein Contents of Meals Prepared by ‘Meals on Wheels’ South Australia Inc.: Findings from a Meal Audit Study

Science.gov (United States)

Arjuna, Tony; Miller, Michelle; Soenen, Stijn; Chapman, Ian; Visvanathan, Renuka; Luscombe-Marsh, Natalie D

2018-01-01

An audit of ‘standard’ (STD) and ‘energy and protein fortified’ (HEHP) meals from Meals on Wheels (MOW) South Australia’s summer menu was conducted to evaluate the consistency, and serve size and nutrient contents, of their menu items. Twenty soups, 20 mains and 20 desserts from each of the STD and HEHP menus were prepared at the MOW South Australia’s kitchen and delivered to three ‘sham(dummy)-clients’ over a 5-week period. Each meal component was weighed in triplicate, to the nearest gram, the variation within the serve weight was calculated, and the overall energy and protein content of each meal was determined using FoodWorks (Xyris Software, Highgate Hill, Queensland, Australia). On average, the variability for soups and mains was ≤6% and for desserts was ≤10% and although the measured serve sizes of the MOW meals were consistently smaller than prescribed serve size, the differences were minor. As a percentage of recommended daily intakes (RDIs) for adults aged over 60 years, we calculated that the STD meals contained 21–39% for energy and 42–63% for protein while the HEHP meals contained 29–55% for energy and 46–69% for protein. These findings demonstrate that MOW meals currently meet the voluntary meal guidelines for energy and protein. PMID:29461476

Serve Size and Estimated Energy and Protein Contents of Meals Prepared by ‘Meals on Wheels’ South Australia Inc.: Findings from a Meal Audit Study

Directory of Open Access Journals (Sweden)

Tony Arjuna

2018-02-01

Full Text Available An audit of ‘standard’ (STD and ‘energy and protein fortified’ (HEHP meals from Meals on Wheels (MOW South Australia’s summer menu was conducted to evaluate the consistency, and serve size and nutrient contents, of their menu items. Twenty soups, 20 mains and 20 desserts from each of the STD and HEHP menus were prepared at the MOW South Australia’s kitchen and delivered to three ‘sham(dummy-clients’ over a 5-week period. Each meal component was weighed in triplicate, to the nearest gram, the variation within the serve weight was calculated, and the overall energy and protein content of each meal was determined using FoodWorks (Xyris Software, Highgate Hill, Queensland, Australia. On average, the variability for soups and mains was ≤6% and for desserts was ≤10% and although the measured serve sizes of the MOW meals were consistently smaller than prescribed serve size, the differences were minor. As a percentage of recommended daily intakes (RDIs for adults aged over 60 years, we calculated that the STD meals contained 21–39% for energy and 42–63% for protein while the HEHP meals contained 29–55% for energy and 46–69% for protein. These findings demonstrate that MOW meals currently meet the voluntary meal guidelines for energy and protein.

Microwave-Assisted Hydrothermal Rapid Synthesis of Calcium Phosphates: Structural Control and Application in Protein Adsorption.

Science.gov (United States)

Cai, Zhu-Yun; Peng, Fan; Zi, Yun-Peng; Chen, Feng; Qian, Qi-Rong

2015-07-31

Synthetic calcium phosphate (CaP)-based materials have attracted much attention in the biomedical field. In this study, we have investigated the effect of pH values on CaP nanostructures prepared using a microwave-assisted hydrothermal method. The hierarchical nanosheet-assembled hydroxyapatite (HAP) nanostructure was prepared under weak acidic conditions (pH 5), while the HAP nanorod was prepared under neutral (pH 7) and weak alkali (pH 9) condition. However, when the pH value increases to 11, a mixed product of HAP nanorod and tri-calcium phosphate nanoparticle was obtained. The results indicated that the pH value of the initial reaction solution played an important role in the phase and structure of the CaP. Furthermore, the protein adsorption and release performance of the as-prepared CaP nanostructures were investigated by using hemoglobin (Hb) as a model protein. The sample that was prepared at pH = 11 and consisted of mixed morphologies of nanorods and nanoprisms showed a higher Hb protein adsorption capacity than the sample prepared at pH 5, which could be explained by its smaller size and dispersed structure. The results revealed the relatively high protein adsorption capacity of the as-prepared CaP nanostructures, which show promise for applications in various biomedical fields such as drug delivery and protein adsorption.

Microwave-Assisted Hydrothermal Rapid Synthesis of Calcium Phosphates: Structural Control and Application in Protein Adsorption

Directory of Open Access Journals (Sweden)

Zhu-Yun Cai

2015-07-01

Full Text Available Synthetic calcium phosphate (CaP-based materials have attracted much attention in the biomedical field. In this study, we have investigated the effect of pH values on CaP nanostructures prepared using a microwave-assisted hydrothermal method. The hierarchical nanosheet-assembled hydroxyapatite (HAP nanostructure was prepared under weak acidic conditions (pH 5, while the HAP nanorod was prepared under neutral (pH 7 and weak alkali (pH 9 condition. However, when the pH value increases to 11, a mixed product of HAP nanorod and tri-calcium phosphate nanoparticle was obtained. The results indicated that the pH value of the initial reaction solution played an important role in the phase and structure of the CaP. Furthermore, the protein adsorption and release performance of the as-prepared CaP nanostructures were investigated by using hemoglobin (Hb as a model protein. The sample that was prepared at pH = 11 and consisted of mixed morphologies of nanorods and nanoprisms showed a higher Hb protein adsorption capacity than the sample prepared at pH 5, which could be explained by its smaller size and dispersed structure. The results revealed the relatively high protein adsorption capacity of the as-prepared CaP nanostructures, which show promise for applications in various biomedical fields such as drug delivery and protein adsorption.

Fibril formation from pea protein and subsequent gel formation.

Science.gov (United States)

Munialo, Claire Darizu; Martin, Anneke H; van der Linden, Erik; de Jongh, Harmen H J

2014-03-19

The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20 h at pH 2.0. Following heating of pea proteins, it was observed that all of the proteins were hydrolyzed into peptides and that 50% of these peptides were assembled into fibrils. Changes on a structural level in pea proteins were studied using circular dichroism, transmission electron microscopy, and particle size analysis. During the fibril assembly process, an increase in aggregate size was observed, which coincided with an increase in thioflavin T binding, indicating the presence of β-sheet aggregates. Fibrils made using pea proteins were more branched and curly. Gel formation of preformed fibrils was induced by slow acidification from pH 7.0 to a final pH of around pH 5.0. The ability of pea protein-based fibrillar gels to fracture during an amplitude sweep was comparable to those of soy protein and whey protein-based fibrillar gels, although gels prepared from fibrils made using pea protein and soy protein were weaker than those of whey protein. The findings show that fibrils can be prepared from pea protein, which can be incorporated into protein-based fibrillar gels.

Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples

Directory of Open Access Journals (Sweden)

Peter Feist

2015-02-01

Full Text Available Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.

Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples

Science.gov (United States)

Feist, Peter; Hummon, Amanda B.

2015-01-01

Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed. PMID:25664860

Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples.

Science.gov (United States)

Feist, Peter; Hummon, Amanda B

2015-02-05

Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.

Supported silver clusters as nanoplasmonic transducers for protein sensing

DEFF Research Database (Denmark)

Fojan, Peter; Hanif, Muhammad; Bartling, Stephen

2015-01-01

Transducers for optical sensing of proteins are prepared using cluster beam deposition on quartz substrates. Surface plasmon resonance phenomenon of the supported silver clusters is used for the detection. It is shown that surface immobilisation procedure providing adhesion of the silver clusters...... stages and protein immobilisation scheme the sensing of protein of interest can be assured using a relatively simple optical spectroscopy method....... an enhancement of the plasmon absorption band used for the detection. Atomic force microscopy study allows to suggest that immobilisation of antibodies on silver clusters has been achieved, thus giving a possibility to incubate and detect an antigen of interest. Hence, by applying the developed preparation...

Interaction of a non-histone chromatin protein (high-mobility group protein 2) with DNA

International Nuclear Information System (INIS)

Goodwin, G.H.; Shooter, K.V.; Johns, E.W.

1975-01-01

The interaction with DNA of the calf thymus chromatin non-histone protein termed the high-mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125 I-labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263-270] for the interaction of high-mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high-mobility group protein 2 differs from high-mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form. The molecular weight of high-mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26,000 was obtained. A low level of nuclease activity detected in one preparation of high-mobility group protein 2 has been investigated. (orig.) [de

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.

Current strategies for protein production and purification enabling membrane protein structural biology.

Science.gov (United States)

Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

2016-12-01

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

The Young's Modulus, Fracture Stress, and Fracture Strain of Gellan Hydrogels Filled with Whey Protein Microparticles.

Science.gov (United States)

Lam, Cherry Wing Yu; Ikeda, Shinya

2017-05-01

Texture modifying abilities of whey protein microparticles are expected to be dependent on pH during heat-induced aggregation of whey protein in the microparticulation process. Therefore, whey protein microparticles were prepared at either pH 5.5 or 6.8 and their effects on small and large deformation properties of gellan gels containing whey protein microparticles as fillers were investigated. The majority of whey protein microparticles had diameters around 2 μm. Atomic force microscopy images showed that whey protein microparticles prepared at pH 6.8 partially collapsed and flatted by air-drying, while those prepared at pH 5.5 did not. The Young's modulus of filled gels adjusted to pH 5.5 decreased by the addition of whey protein microparticles, while those of filled gels adjusted to pH 6.8 increased with increasing volume fraction of filler particles. These results suggest that filler particles were weakly bonded to gel matrices at pH 5.5 but strongly at pH 6.8. Whey protein microparticles prepared at pH 5.5 showed more enhanced increases in the Young's modulus than those prepared at pH 6.8 at volume fractions between 0.2 and 0.4, indicating that microparticles prepared at pH 5.5 were mechanically stronger. The fracture stress of filled gels showed trends somewhat similar to those of the Young's modulus, while their fracture strains decreased by the addition of whey protein microparticles in all examined conditions, indicating that the primary effect of these filler particles was to enhance the brittleness of filled gels. © 2017 Institute of Food Technologists®.

CHEMICAL COMPOSITION AND FUNCTIONAL PROPERTIES OF RICE PROTEIN CONCENTRATES

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V. V. Kolpakova

2015-01-01

Full Text Available Traditionally rice and products of its processing are used to cook porridge, pilaf, lettuce, confectionery, fish, dairy and meat products. At the same time new ways of its processing with releasing of protein products for more effective using, including the use of a glutenfree diet, are developing. The task of this study was a comparative research of nutrition and biological value and functional properties of protein and protein-calcium concentrates produced from rice flour milled from white and brown rice. The traditional and special methods were used. Concentrates were isolated with enzyme preparations of xylanase and amylolytic activity with the next dissolution of protein in diluted hydrochloric acid. Concentrates differed in the content of mineral substances (calcium, zinc, iron and other elements, amino acids and functional properties. The values of the functional properties and indicators of the nutritional value of concentrates from white rice show the advisability of their using in food products, including gluten-free products prepared on the basis of the emulsion and foam systems, and concentrates from brown rice in food products prepared on the basis of using of the emulsion systems. Protein concentrates of brown rice have a low foaming capacity and there is no foam stability at all.

Preparation of shrimp waste as aqua feedstuff: a study of physicochemical properties and in vitro digestibility

Directory of Open Access Journals (Sweden)

Karun Thongprajukeaw

2014-12-01

Full Text Available The effects of different preparation methods (boiling, microwave irradiation, oven-drying, soaking, and sun-drying for improving the protein quality of shrimp waste (SW when used as aqua feedstuff were observed. The findings from this study indicate that microwave irradiation maintained both the chemical composition and total carotenoid concentration of the SW. This method controlled the appropriate physicochemical characteristics thus promoting proteolytic digestion, as indicated by measurement of changes in pH, water solubility, microstructures, and thermal transition. The protein digestibility based on the use of digestive enzymes from Nile tilapia (Oreochromis niloticus also increased after preparation with microwave irradiation. Based on this study, improvement in the protein quality in SW may be achieved by microwave irradiation and this method may be used as an alternative method for preparing aqua feedstuff.

Comparison and functionalization study of microemulsion-prepared magnetic iron oxide nanoparticles.

Science.gov (United States)

Okoli, Chuka; Sanchez-Dominguez, Margarita; Boutonnet, Magali; Järås, Sven; Civera, Concepción; Solans, Conxita; Kuttuva, Gunaratna Rajarao

2012-06-05

Magnetic iron oxide nanoparticles (MION) for protein binding and separation were obtained from water-in-oil (w/o) and oil-in-water (o/w) microemulsions. Characterization of the prepared nanoparticles have been performed by TEM, XRD, SQUID magnetometry, and BET. Microemulsion-prepared magnetic iron oxide nanoparticles (ME-MION) with sizes ranging from 2 to 10 nm were obtained. Study on the magnetic properties at 300 K shows a large increase of the magnetization ~35 emu/g for w/o-ME-MION with superparamagnetic behavior and nanoscale dimensions in comparison with o/w-ME-MION (10 emu/g) due to larger particle size and anisotropic property. Moringa oleifera coagulation protein (MOCP) bound w/o- and o/w-ME-MION showed an enhanced performance in terms of coagulation activity. A significant interaction between the magnetic nanoparticles and the protein can be described by changes in fluorescence emission spectra. Adsorbed protein from MOCP is still retaining its functionality even after binding to the nanoparticles, thus implying the extension of this technique for various applications.

Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls

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Canut Hervé

2006-05-01

Full Text Available Abstract Background The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Reaching such a goal closely depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific difficulties: (i the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP during the isolation procedure, (ii polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50% of predicted intracellular proteins. Since isolated cell walls should hold secreted proteins, one can imagine alternative procedures to prepare cell walls containing a lower proportion of contaminant proteins. Results The rationales of several published procedures to isolate cell walls for proteomics were analyzed, with regard to the bioinformatic-predicted subcellular localization of the identified proteins. Critical steps were revealed: (i homogenization in low ionic strength acid buffer to retain CWP, (ii purification through increasing density cushions, (iii extensive washes with a low ionic strength acid buffer to retain CWP while removing as many cytosolic proteins as possible, and (iv absence of detergents. A new procedure was developed to prepare cell walls from etiolated hypocotyls of Arabidopsis thaliana. After salt extraction, a high proportion of proteins predicted to be secreted was released (73%, belonging to the same functional classes as proteins identified using previously described protocols. Finally, removal of intracellular proteins was obtained using detergents, but their amount represented less than 3% in mass of the total protein extract, based on protein quantification. Conclusion The

21 CFR 184.1498 - Microparticulated protein product.

Science.gov (United States)

2010-04-01

... ingredient statement on both bulk and packaged food must include the source of the protein (e.g., “microparticulated egg white protein”), followed by a parenthetical listing of each of the ingredients in the... preparation of the microparticulated protein product must be used in compliance with the limitations of the...

Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation

DEFF Research Database (Denmark)

Malmström, Johan; Larsen, Kristoffer; Hansson, Lennart

2002-01-01

-dimensional electrophoresis was interfaced to miniaturized sample preparation techniques using microcapillary extraction. Four protein groups were identified; cytoskeletal, adhesion, scavenger and metabolic proteins. These patient's proteomes showed a high degree of heterogeneity between patients but larger homogeneity...

Biological evaluation of a nutritional supplement prepared with QPM Maize cultivar BR 473 and other traditional food items

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Paula Heberth de

2004-01-01

Full Text Available Quality Protein Maize (QPM cultivar BR 473 was employed, together with soybean flour, brown sugar, banana meal and oat meal, for the preparation of a nutritional supplement.. 21-day old male Fisher rats were fed diets containing the supplement as a protein source, both with and without soybean flour; casein diets with 10 or 7% protein served as respective controls. Protein Efficiency Ratio (PER, Net Protein Utilization (NPU, Net Protein Retention (NPR and Digestibility were determined. Blood biochemical parameters (glucose, cholesterol, urea, hemoglobin, albumin and total protein were also measured in the animals and showed that all were in good health condition at the end of the experiment. The obtained results for PER, NPU and NPR indicated that the supplement prepared with QPM maize cultivar BR 473 was a good protein source, especially when soybean flour was added.

Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (¤Pisum sativum¤) symbiosomes

DEFF Research Database (Denmark)

Saalbach, G.; Erik, P.; Wienkoop, S.

2002-01-01

PBM preparations from pea symbiosomes always contain abundant amounts of bacteroid proteins as a contaminate. Interestingly, in addition to a few PS/PBM proteins a number of endomembrane proteins (less likely representing a contaminate), including V-ATPase, BIP, and an integral membrane protein known...

Immunostimulatory mouse granuloma protein.

Science.gov (United States)

Fontan, E; Fauve, R M; Hevin, B; Jusforgues, H

1983-10-01

Earlier studies have shown that from subcutaneous talc-induced granuloma in mice, a fraction could be extracted that fully protected mice against Listeria monocytogenes. Using standard biochemical procedures--i.e., ammonium sulfate fractionation, preparative electrophoresis, gel filtration chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis--we have now purified an active factor to homogeneity. A single band was obtained in NaDodSO4/polyacrylamide gel with an apparent Mr of 55,000. It migrated with alpha 1-globulins and the isoelectric point was 5 +/- 0.1. The biological activity was destroyed with Pronase but not with trypsin and a monospecific polyclonal rabbit antiserum was obtained. The intravenous injection of 5 micrograms of this "mouse granuloma protein" fully protects mice against a lethal inoculum of L. monocytogenes. Moreover, after their incubation with 10 nM mouse granuloma protein, mouse peritoneal cells became cytostatic against Lewis carcinoma cells.

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.

Water-in-Oil Microemulsions for Protein Delivery: Loading Optimization and Stability.

Science.gov (United States)

Perinelli, Diego R; Cespi, Marco; Pucciarelli, Stefania; Vincenzetti, Silvia; Casettari, Luca; Lam, Jenny K W; Logrippo, Serena; Canala, Elisa; Soliman, Mahmoud E; Bonacucina, Giulia; Palmieri, Giovanni F

2017-01-01

Microemulsions are attractive delivery systems for therapeutic proteins and peptides due to their ability to enhance bioavailability. Although different proteins and peptides have been successfully delivered through such ternary systems, no information can be found about protein loading and the formulation stability when such microemulsions are prepared with pharmaceuticallyapproved oils and surfactants. The aim of this work was to optimise a ternary system consisting of water/ ethyl oleate/Span® 80-Tween® 80 and to determine its protein loading capacity and stability, using bovine serum albumin (BSA) as a model of biomolecule. The optimization was carried out using a Central Composite Design and all the prepared formulations were characterised through dynamic light scattering, rheology, optical and polarized microscopy. Subsequently, the maximum loading capacity was determined and the stability of the final microemulsion with the highest content of protein was followed over six months. To investigate the structural features of the protein, BSA was recovered from the microemulsion and analysed through fluorescence spectroscopy. After incorporation of the protein in the microemulsion, a decrease of its aqueous solubility was observed. However, the formulation remained stable over six months and the native-like state of the recovered protein was demonstrated by fluorescence spectroscopy Conclusion: This study demonstrated the feasibility of preparing microemulsions with the highest content of protein and their long-term stability. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Engineered mammalian cells for production of recombinant proteins

DEFF Research Database (Denmark)

2017-01-01

The present invention relates to mammalian cells modified to provide for improved expression of a recombinant protein of interest. In particular, the invention relates to CHO cells and other host cells in which the expression of one or more endogenous secreted proteins has been disrupted, as well...... as to the preparation, identification and use of such cells in the production of recombinant proteins....

Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

Science.gov (United States)

Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

1999-01-01

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

International Nuclear Information System (INIS)

Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

1987-01-01

The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

Immunoelectrophoretic studies on pig intestinal brush border proteins

DEFF Research Database (Denmark)

Danielsen, Erik Michael; Sjöström, H; Norén, O

1977-01-01

Brush borders were prepared from pig intestinal mucosa and the membrane proteins solubilized with either Triton X-100 or papain. Proteins, thus released, were used as antigens to raise antisera in rabbits. The immunoglobulin G fractions were isolated and shown by the double layer immunofluorescence...

Preparation, Characterization and In vitro Evaluation of Theophylline ...

African Journals Online (AJOL)

Methods: SPI-based nanoparticles were prepared with soy protein-dextran conjugates obtained by titanium dioxide (TiO2) photocatalysis using a simple ionic gelation method. Formation of the conjugates was monitored spectrophotometrically for free amino group content (A340nm) and by Fourier transform infrared ...

LC-MS analysis of the plasma metabolome–a novel sample preparation strategy

DEFF Research Database (Denmark)

Skov, Kasper; Hadrup, Niels; Smedsgaard, Jørn

2015-01-01

Blood plasma is a well-known body fluid often analyzed in studies on the effects of toxic compounds as physiological or chemical induced changes in the mammalian body are reflected in the plasma metabolome. Sample preparation prior to LC-MS based analysis of the plasma metabolome is a challenge...... as plasma contains compounds with very different properties. Besides, proteins, which usually are precipitated with organic solvent, phospholipids, are known to cause ion suppression in electrospray mass spectrometry. We have compared two different sample preparation techniques prior to LC-qTOF analysis...... of plasma samples: The first is protein precipitation; the second is protein precipitation followed by solid phase extraction with sub-fractionation into three sub-samples; a phospholipid, a lipid and a polar sub-fraction. Molecular feature extraction of the data files from LC-qTOF analysis of the samples...

Physicochemical Pro~rti~ of Curd Prepared from Melon Seeds

African Journals Online (AJOL)

Key~~;ds~ Mel6n curd, coagulati'an, protein, calcium, sulphate, acceptabilit:" . ' .' ~ I[. ,.} ..... from where they were prepared, Fat contents,~ar ied among ... Table 1: Chemical compositio'o ~f' Melon Seed Milk (IWSM) Raw Melon Curd RMC' .' -'.

Optical and structural properties of protein/gold hybrid bio-nanofilms prepared by layer-by-layer method.

Science.gov (United States)

Pál, Edit; Hornok, Viktória; Sebok, Dániel; Majzik, Andrea; Dékány, Imre

2010-08-01

Lysozyme/gold thin layers were prepared by layer-by-layer (LbL) self-assembly method. The build-up of the films was followed by UV-vis-absorbance spectra, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) techniques. The structural property of films was examined by X-ray diffraction (XRD) measurements, while their morphology was studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM). It was found that gold nanoparticles (NPs) had cubic crystalline structure, the primary particles form aggregates in the thin layer due to the presence of lysozyme molecules. The UV-vis measurements prove change in particle size while the colour of the film changes from wine-red to blue. The layer thickness of films was determined using the above methods and the loose, porous structure of the films explains the difference in the results. The vapour adsorption property of hybrid layers was also studied by QCM using different saturated vapours and ammonia gas. The lysozyme/Au films were most sensitive for ammonia gas among the tested gases/vapours due to the strongest interaction between the functional groups of the protein. Copyright 2010 Elsevier B.V. All rights reserved.

Competitive protein binding assay

International Nuclear Information System (INIS)

Kaneko, Toshio; Oka, Hiroshi

1975-01-01

The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

Photodynamic tissue adhesion with chlorin(e6) protein conjugates.

Science.gov (United States)

Khadem, J; Veloso, A A; Tolentino, F; Hasan, T; Hamblin, M R

1999-12-01

To test the hypothesis that a photodynamic laser-activated tissue solder would perform better in sealing scleral incisions when the photosensitizer was covalently linked to the protein than when it was noncovalently mixed. Conjugates and mixtures were prepared between the photosensitizer chlorin(e6) and various proteins (albumin, fibrinogen, and gelatin) in different ratios and used to weld penetrating scleral incisions made in human cadaveric eyes. A blue-green (488-514 nm) argon laser activated the adhesive, and the strength of the closure was measured by increasing the intraocular pressure until the wound showed leakage. Both covalent conjugates and noncovalent mixtures showed a light dose-dependent increase in leaking pressure. A preparation of albumin chlorin(e6) conjugate with additional albumin added (2.5 protein to chlorin(e6) molar ratio) showed significantly higher weld strength than other protein conjugates and mixtures. This is the first report of dye-protein conjugates as tissue solders. These conjugates may have applications in ophthalmology.

Radioimmunoassay of measles virus hemagglutinin protein G

International Nuclear Information System (INIS)

Lund, G.A.; Salmi, A.A.

1982-01-01

Guinea pig and rabbit antisera from animals immunized with purified measles virus hemagglutinin (G) protein were used to establish a solid-phase four-layer radioimmunoassay for quantitative measurement of the G protein. The sensitivity of the assay was 2 ng of purified G protein, and 200 μg of protein from uninfected Vero cells neither decreased the sensitivity nor reacted non-specifically in the assay. Radioimmunoassay standard dose-response curves were established and unknown values interpolated from these using the logit program of a desktop computer. Using this procedure, a measles virus growth curve in infected Vero cells was determined by measurement of G protein production. Under these same conditions, hemagglutination was not sensitive enough to detect early hemagglutinin production. Viral antigens in canine distemper virus, Newcastle disease virus, parainfluenza viruses 1-4, simian virus 5, and respiratory syncytial virus-infected cell lysates did not cross-react in the radioimmunoassay. A small degree of cross-reactivity was detected with mumps viral antigens, both with Vero cell-derived (wild-type strain) and egg-derived (Enders strain) purified virus preparations and with a cell lysate antigen prepared from wild-type mumps virus-infected Vero cells. (Auth.)

Radioimmunoassay of measles virus hemagglutinin protein G

Energy Technology Data Exchange (ETDEWEB)

Lund, G A; Salmi, A A [Turku Univ. (Finland)

1982-08-01

Guinea pig and rabbit antisera from animals immunized with purified measles virus hemagglutinin (G) protein were used to establish a solid-phase four-layer radioimmunoassay for quantitative measurement of the G protein. The sensitivity of the assay was 2 ng of purified G protein, and 200 ..mu..g of protein from uninfected Vero cells neither decreased the sensitivity nor reacted non-specifically in the assay. Radioimmunoassay standard dose-response curves were established and unknown values interpolated from these using the logit program of a desktop computer. Using this procedure, a measles virus growth curve in infected Vero cells was determined by measurement of G protein production. Under these same conditions, hemagglutination was not sensitive enough to detect early hemagglutinin production. Viral antigens in canine distemper virus, Newcastle disease virus, parainfluenza viruses 1-4, simian virus 5, and respiratory syncytial virus-infected cell lysates did not cross-react in the radioimmunoassay. A small degree of cross-reactivity was detected with mumps viral antigens, both with Vero cell-derived (wild-type strain) and egg-derived (Enders strain) purified virus preparations and with a cell lysate antigen prepared from wild-type mumps virus-infected Vero cells.

Radioimmunoassay of protein hormones

International Nuclear Information System (INIS)

Talas, M.; Fingerova, H.

1976-01-01

A survey is presented of the history of RIA methods for FSH, LH, HCG, HPL and prolactin determinations with special regard to the double antibody method in a kinetic system. Problems are shown in 125 I-labelling protein hormones in preparing own antisera. (L.O.)

The Influence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores.

Science.gov (United States)

Abhyankar, Wishwas R; Kamphorst, Kiki; Swarge, Bhagyashree N; van Veen, Henk; van der Wel, Nicole N; Brul, Stanley; de Koster, Chris G; de Koning, Leo J

2016-01-01

Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14 N spores prepared on solid Schaeffer's-glucose (SG) agar plates and 15 N metabolically labeled spores prepared in shake flasks containing 3-( N -morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14 N: 15 N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the

The Influence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores

Science.gov (United States)

Abhyankar, Wishwas R.; Kamphorst, Kiki; Swarge, Bhagyashree N.; van Veen, Henk; van der Wel, Nicole N.; Brul, Stanley; de Koster, Chris G.; de Koning, Leo J.

2016-01-01

Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14N spores prepared on solid Schaeffer’s-glucose (SG) agar plates and 15N metabolically labeled spores prepared in shake flasks containing 3-(N-morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14N:15N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the

The influence of sporulation conditions on the spore coat protein composition of Bacillus subtilis spores.

Directory of Open Access Journals (Sweden)

Wishwas R. Abhyankar

2016-10-01

Full Text Available Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14N spores prepared on solid SG agar plates and 15N metabolically labelled spores prepared in shake flasks containing MOPS buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14N: 15N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and

Benchmarking sample preparation/digestion protocols reveals tube-gel being a fast and repeatable method for quantitative proteomics.

Science.gov (United States)

Muller, Leslie; Fornecker, Luc; Van Dorsselaer, Alain; Cianférani, Sarah; Carapito, Christine

2016-12-01

Sample preparation, typically by in-solution or in-gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in-gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS-PAGE is a time-consuming approach. Tube-gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label-free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label-free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG-prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein Adsorption and Antibacterial Behavior for Hydroxyapatite Nanocrystals Prepared by Hydrothermal Method

OpenAIRE

笠原, 英充; 小形, 信男; 荻原, 隆

2005-01-01

Homogeneous hydroxyapatite nanocrystals which have aspect ratio with more than four were synthesized by hydrothermal method. X-ray fluorescence analysis revealed that the Ca/P ratio of hydroxyapatite nanocrystals was maintaining start composition. The protein adsorption properties and bacteria-resistant of hydroxyapatite nanocrystals were investigated. The protein adsorption properties of hydroxyapatite nanocrystals were improvement after the hydrothermal treatment. Bacteria-resistant behavio...

A recent case of Antarctic bioprospecting from Japan

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Akiho Shibata

2010-03-01

Full Text Available Antarctic bioprospecting, namely the search for valuable genetic or chemical compounds in Antarctic nature, has been the subject of intense discussion within Antarctic Treaty Consultative Meetings. In this discussion, based on the so-called "end-users view point," utilizing the patent database to see how much Antarctic biological material has been used in patents, Antarctic bioprospecting has been depicted as a lucrative commercial activity operated by big multinational companies. This paper, instead, proposes an "access view point" for Antarctic bioprospecting, by examining a recent Japanese case in which scientists participating in the Japanese Antarctic Research Expedition in 2007 collected some sediment from Antarctic lakes near Syowa Station, isolated and cultured a particular fungus, and found the first evidence of the presence of antifreezing activity in oomycetes. In 2009, the scientists' affiliate institutions, including the National Institute of Advanced Industrial Science and Technology, applied for a patent on Antarctomyces psychrotrophicus Syw-1 and the antifreeze protein obtained from it. A detailed examination of this case demonstrates that the dichotomy of Antarctic bioprospecting into "commercial" and "scientific" does not reflect the reality of bioprospecting activities and, therefore, does not provide an appropriate ground for legal and policy discussion on Antarctic bioprospecting.

Efficient preparation and analysis of membrane and membrane protein systems

Czech Academy of Sciences Publication Activity Database

Javanainen, M.; Martinez-Seara, Hector

2016-01-01

Roč. 1858, č. 10 (2016), s. 2468-2482 ISSN 0005-2736 Institutional support: RVO:61388963 Keywords : tools and software * membrane building * protein insertion * molecular dynamics * lipid bilayer Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.498, year: 2016

Protein adsorption at polymer-grafted surfaces: Comparison between a mixture of saliva proteins and some well-defined model proteins

NARCIS (Netherlands)

Kawasaki, K.; Kambara, M.; Matsumura, H.; Norde, W.

2003-01-01

Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10-15 nm2, 5.5 nm2 and 4 nm2 per polymer chain, respectively. The

Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina.

Science.gov (United States)

Azad, Md Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2006-11-30

The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.

[Preparation and characterization of mouse polyclonal antibody against conserved region of human FOXO3].

Science.gov (United States)

Li, Lei; Lyu, Dan

2017-06-01

Objective To purify the recombinant protein specific to conserved region of forkhead box O3 (FOXO3) and prepare mouse anti-human FOXO3 polyclonal antibody. Methods The DNA fragment (aa290-472) encoding conserved domain of FOXO3 was amplified by PCR, and subsequently cloned into pET28a vector. Following transformation into E.coli BL21, the soluble fusion protein His-FOXO3 was induced by IPTG and purified by Ni-NTA affinity chromatography. The purified protein was used to immunize BALB/c mice to generate polyclonal antibody. The characteristics of the polyclonal antibody were assessed by ELISA, Western blotting and immunoprecipitation assays. Results We successfully prepared the expression vector pET28a-FOXO3 (aa290-472) and expressed the purified fusion protein in a soluble form. By immunizing mice with the fusion protein, we obtained anti-human FOXO3 polyclonal antibody. ELISA and Western blotting showed that the mouse antibody could recognize specifically the endogenous FOXO3 protein. Conclusion The polyclonal antibody against conserved domain of FOXO3 can identify the endogenous FOXO3 protein. It can be used to analyze the endogenous FOXO3 expression level.

Single-cell and population NF-κB dynamic responses depend on lipopolysaccharide preparation.

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Miriam V Gutschow

Full Text Available Lipopolysaccharide (LPS, found in the outer membrane of gram-negative bacteria, elicits a strong response from the transcription factor family Nuclear factor (NF-κB via Toll-like receptor (TLR 4. The cellular response to lipopolysaccharide varies depending on the source and preparation of the ligand, however. Our goal was to compare single-cell NF-κB dynamics across multiple sources and concentrations of LPS.Using live-cell fluorescence microscopy, we determined the NF-κB activation dynamics of hundreds of single cells expressing a p65-dsRed fusion protein. We used computational image analysis to measure the nuclear localization of the fusion protein in the cells over time. The concentration range spanned up to nine orders of magnitude for three E. coli LPS preparations. We find that the LPS preparations induce markedly different responses, even accounting for potency differences. We also find that the ability of soluble TNF receptor to affect NF-κB dynamics varies strikingly across the three preparations.Our work strongly suggests that the cellular response to LPS is highly sensitive to the source and preparation of the ligand. We therefore caution that conclusions drawn from experiments using one preparation may not be applicable to LPS in general.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. II - Enzymatic and chemical sample preparation.

Science.gov (United States)

Bobaly, Balazs; D'Atri, Valentina; Goyon, Alexandre; Colas, Olivier; Beck, Alain; Fekete, Szabolcs; Guillarme, Davy

2017-08-15

The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g. N-glycans) and/or by decreasing the molecular size of the tested protein by enzymatic or chemical fragmentation. These approaches make the sample more suitable for chromatographic and mass spectrometric analysis. Structural elucidation and quality control (QC) analysis of biopharmaceutics are usually performed at intact, subunit and peptide levels. In this paper, general sample preparation approaches used to attain peptide, subunit and glycan level analysis are overviewed. Protocols are described to perform tryptic proteolysis, IdeS and papain digestion, reduction as well as deglycosylation by PNGase F and EndoS2 enzymes. Both historical and modern sample preparation methods were compared and evaluated using rituximab and trastuzumab, two reference therapeutic mAb products approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA). The described protocols may help analysts to develop sample preparation methods in the field of therapeutic protein analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein Nanoparticles as Drug Delivery Carriers for Cancer Therapy

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Warangkana Lohcharoenkal

2014-01-01

Full Text Available Nanoparticles have increasingly been used for a variety of applications, most notably for the delivery of therapeutic and diagnostic agents. A large number of nanoparticle drug delivery systems have been developed for cancer treatment and various materials have been explored as drug delivery agents to improve the therapeutic efficacy and safety of anticancer drugs. Natural biomolecules such as proteins are an attractive alternative to synthetic polymers which are commonly used in drug formulations because of their safety. In general, protein nanoparticles offer a number of advantages including biocompatibility and biodegradability. They can be prepared under mild conditions without the use of toxic chemicals or organic solvents. Moreover, due to their defined primary structure, protein-based nanoparticles offer various possibilities for surface modifications including covalent attachment of drugs and targeting ligands. In this paper, we review the most significant advancements in protein nanoparticle technology and their use in drug delivery arena. We then examine the various sources of protein materials that have been used successfully for the construction of protein nanoparticles as well as their methods of preparation. Finally, we discuss the applications of protein nanoparticles in cancer therapy.

Protein nanoparticles as drug delivery carriers for cancer therapy.

Science.gov (United States)

Lohcharoenkal, Warangkana; Wang, Liying; Chen, Yi Charlie; Rojanasakul, Yon

2014-01-01

Nanoparticles have increasingly been used for a variety of applications, most notably for the delivery of therapeutic and diagnostic agents. A large number of nanoparticle drug delivery systems have been developed for cancer treatment and various materials have been explored as drug delivery agents to improve the therapeutic efficacy and safety of anticancer drugs. Natural biomolecules such as proteins are an attractive alternative to synthetic polymers which are commonly used in drug formulations because of their safety. In general, protein nanoparticles offer a number of advantages including biocompatibility and biodegradability. They can be prepared under mild conditions without the use of toxic chemicals or organic solvents. Moreover, due to their defined primary structure, protein-based nanoparticles offer various possibilities for surface modifications including covalent attachment of drugs and targeting ligands. In this paper, we review the most significant advancements in protein nanoparticle technology and their use in drug delivery arena. We then examine the various sources of protein materials that have been used successfully for the construction of protein nanoparticles as well as their methods of preparation. Finally, we discuss the applications of protein nanoparticles in cancer therapy.

Determination and Quantification of Molecular Interactions in Protein Films: A Review

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Felicia Hammann

2014-12-01

Full Text Available Protein based films are nowadays also prepared with the aim of replacing expensive, crude oil-based polymers as environmentally friendly and renewable alternatives. The protein structure determines the ability of protein chains to form intra- and intermolecular bonds, whereas the degree of cross-linking depends on the amino acid composition and molecular weight of the protein, besides the conditions used in film preparation and processing. The functionality varies significantly depending on the type of protein and affects the resulting film quality and properties. This paper reviews the methods used in examination of molecular interactions in protein films and discusses how these intermolecular interactions can be quantified. The qualitative determination methods can be distinguished by structural analysis of solutions (electrophoretic analysis, size exclusion chromatography and analysis of solid films (spectroscopy techniques, X-ray scattering methods. To quantify molecular interactions involved, two methods were found to be the most suitable: protein film swelling and solubility. The importance of non-covalent and covalent interactions in protein films can be investigated using different solvents. The research was focused on whey protein, whereas soy protein and wheat gluten were included as further examples of proteins.

Determination and Quantification of Molecular Interactions in Protein Films: A Review.

Science.gov (United States)

Hammann, Felicia; Schmid, Markus

2014-12-10

Protein based films are nowadays also prepared with the aim of replacing expensive, crude oil-based polymers as environmentally friendly and renewable alternatives. The protein structure determines the ability of protein chains to form intra- and intermolecular bonds, whereas the degree of cross-linking depends on the amino acid composition and molecular weight of the protein, besides the conditions used in film preparation and processing. The functionality varies significantly depending on the type of protein and affects the resulting film quality and properties. This paper reviews the methods used in examination of molecular interactions in protein films and discusses how these intermolecular interactions can be quantified. The qualitative determination methods can be distinguished by structural analysis of solutions (electrophoretic analysis, size exclusion chromatography) and analysis of solid films (spectroscopy techniques, X-ray scattering methods). To quantify molecular interactions involved, two methods were found to be the most suitable: protein film swelling and solubility. The importance of non-covalent and covalent interactions in protein films can be investigated using different solvents. The research was focused on whey protein, whereas soy protein and wheat gluten were included as further examples of proteins.

Preparation of patient-related allergens for hyposensitization. Qualitative aspects

DEFF Research Database (Denmark)

Poulsen, L K; Søndergaard, I; Weeke, B

1988-01-01

An affinity chromatography method for preparation of patient-related antigens from commercially available allergen extracts has been investigated. IgG1,2,4 from a patient previously hyposensitized with dog hair and dandruff allergen was bound to protein A-sepharose. Secondly, commercial allergen ...

Preparation of recombinant proteins in milk to improve human and animal health

OpenAIRE

Soler , Eric; Thépot , Dominique; Rival-Gervier , Sylvie; JOLIVET , Geneviève; Houdebine , Louis-Marie

2006-01-01

International audience; Milk is a very abundant source of proteins for animal and human consumption. Milk composition can be modified using transgenesis, including exogenous gene addition and endogenous gene inactivation. The study of milk protein genes has provided researchers with regulatory regions capable of efficiently and specifically driving the expression of foreign genes in milk. The projects underway are aimed at modifying milk composition, improving its nutritional value, reducing ...

A comparative evaluation of the ultrastructure and protein yields of ...

African Journals Online (AJOL)

... efficient as the detergent solubilized technique. Additionally some axial filaments were also released. The protein yield of L. hardjo from the two techniques of leptospire protein preparation was significantly lower than the other test serovars (P<0.01). Overall, the protein yield of antigens produced by the SDS solubilization ...

Soluble protein isolated from low cost fish and fish wastes

OpenAIRE

Lekshmy Nair, A.; Gopakumar, K.

1982-01-01

The method of preparation, composition, amino acid content, protein efficiency ratio and areas of possible application of water soluble protein isolates from low cost fish and fish wastes are discussed in detail in this communication.

Optimization Of Freeze-Dried Starter For Yogurt By Full Factorial Experimental Design

Directory of Open Access Journals (Sweden)

Chen He

2015-12-01

Full Text Available With the rapidly development of fermented milk product, it is significant for enhancing the performance of starter culture. This paper not only investigated the influence of anti-freeze factors and freeze-drying protective agents on viable count, freeze-drying survival rate and yield of Lactobacillus bulgaricus (LB and Streptococcus thermophilus (ST, but also optimized the bacteria proportion of freeze-dried starter culture for yogurt by full factorial experimental design. The results showed as following: the freeze-drying protective agents or anti-freeze factors could enhanced survival rate of LB and ST; the freeze-dried LB and ST powders containing both of anti-freeze factors and freeze-drying protective agents had higher viable count and freeze-drying survival rate that were 84.7% and 79.7% respectively; In terms of fermentation performance, the best group of freeze-dried starter for yogurt was the compound of LB3 and ST2.

Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

Science.gov (United States)

Gajdosik, Martina Srajer; Clifton, James; Josic, Djuro

2012-01-01

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. PMID:22520159

Purification of thyrotropin from human hypophysis: preliminary preparation

International Nuclear Information System (INIS)

Borghi, V.C.; Lin, L.H.; Bartolini, P.

1988-07-01

The adequacy of stored crude preparations for isolation of human tyrotropin (TSH) was evaluated according to Ross et al from a side fraction obtained during the purification of growth hormone from frozen pituitaries (SOMATORMON). Six crude TSH preparations were stored at - 20 0 C during several years for further purification. One of these preparations was purified by sucessive chromatographies on Sephadex G-100, hydroxylapatite and SP-Sephadex C50. The TSH content present in the chromatographic fractions and in the pools was assayed by specific radioimmunoassay developed at our laboratory. The protein determination of the fractions and pools was performed by absorbance at 280 nm and by the method of Lowry at al, respectively. The TSH activity increased eight times during the purification and the TSH purified had a radioimmunological potency around half that de scribed by Roos at al. The results suggest the fitness of long time stored preparations in the attainment of pure TSH. (author) [pt

Effects of Diatomite and SBS on Freeze-Thaw Resistance of Crumb Rubber Modified Asphalt Mixture

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Haibin Wei

2017-01-01

Full Text Available Asphalt mixture is susceptible to moisture damage under the effect of freeze-thaw (F-T cycles. In this paper, crumb rubber (CR was used to modify stone mastic asphalt (SMA and the effects of diatomite and styrene butadiene styrene (SBS on antifreezing performances of crumb rubber modified SMA (CRSMA were investigated. Regression analysis and modified grey model (MGM were used to construct the prediction models for properties of modified mixtures. CRSMA, CR and diatomite modified SMA (CRDSMA, and CR and SBS modified SMA (CRSSMA were prepared in laboratory, respectively. Process of F-T cycles was designed. Air void, indirect tensile strength (ITS, and indirect tensile stiffness modulus (ITSM were measured to evaluate the antifreezing performances of CRSMA, CRDSMA, and CRSSMA. Results indicate that air voids increase with the increasing of F-T cycles. ITS and ITSM all decrease with the increasing of F-T cycles. The addition of diatomite and SBS can reduce the air void and improve the ITS and ITSM of CRSMA. CRSSMA presents the lowest air void, highest tensile strength, and largest stiffness modulus, which reveals that CRSSMA has the best F-T resistance among three different kinds of mixtures. Moreover, MGM (1, 2 models present more favorable accuracy in prediction of air void and ITS compared with regression ones.

Rational and efficient preparation of a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide fused to human growth hormone in Escherichia coli.

Science.gov (United States)

Wang, Song; Shen, Mingqiang; Xu, Yang; Chen, Fang; Chen, Mo; Chen, Shilei; Wang, Aiping; Zhang, Zhou; Ran, Xinze; Cheng, Tianmin; Su, Yongping; Wang, Junping

2013-04-01

The 14-mer thrombopoietin mimetic peptide (TMP), especially in the form of dimer, displayed potent megakaryocytopoiesis activity in vitro. However, it is difficult to prepare such short peptide with high bioactivity through gene-engineering approaches. In this study, a chimeric protein containing a tandem dimer of TMP (dTMP) fused to human growth hormone (hGH), a kind of hematopoietic growth factor that activates the same signal pathways as thrombopoietin, was produced in Escherichia coli by soluble expression. By rational utilization of the XmnI and EcoRV restriction sites, a PCR fragment encoding dTMP-GH was inserted into the plasmid vector pMAL-p2X at the position right after Xa factor cleavage site, in frame with maltose-binding protein (MBP) gene. Under optimized conditions, a high-level expression of soluble MBP-dTMP-GH fusion protein was obtained. By application of amylose resin chromatography, Xa factor digestion, hydrophobic chromatography followed by gel filtration, the dTMP-GH fusion protein was separated. Finally, a relatively high yield of dTMP-GH fusion protein with high purity (>98%) and without redundant amino acid was achieved, as identified by high-performance liquid chromatography, mass spectrometry, and amino acid sequencing. The functional assays showed that dTMP-GH could promote the proliferation of megakaryoblast cells and maturation of murine megakaryocytes derived from bone marrow, in a dose-dependent manner. Moreover, an enhanced effect of dTMP-GH on megakaryocytopoiesis was found as compared with equimolar concentration of dTMP and rhGH. This work provides a new avenue to generate thrombopoietic agents based on TMP.

Towards a map of the Populus biomass protein-protein interaction network

Energy Technology Data Exchange (ETDEWEB)

Beers, Eric [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Brunner, Amy [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Helm, Richard [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Dickerman, Allan [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)

2015-07-31

-depth characterizations. Characterizations involved both in vivo and in vitro independent methods to confirm protein-protein interactions and the evaluation of novel phenotypes resulting from creation of transgenic poplar and Arabidopsis plants engineered for increased or decreased expression of the selected genes. Transgenic poplar trees were studied in growth chamber, greenhouse, and two separate replicated field trials involving over 25 distinct wood-associated proteins. In-depth characterizations yielding positive results include the following. First, a NAC domain transcription factor (NAC154) that is a promoter of stress response and dormancy in trees was discovered. Increasing expression of NAC154 caused stunted growth and premature senescence, while decreasing expression led to both delayed bud and leaf expansion in spring and delayed leaf drop (i.e., prolonged leaf retention) in fall. Second, we discovered and characterized a new connection between a negative regulator of wood formation, the NAC domain transcription factor XND1, and an important regulator of cell division and cell differentiation, RBR. Third, we identified a new network of interacting wood-associated transcription factors belonging to the MYB and HD families. One of the HD family proteins, WOX13, was used to prepare transgenic poplar for high-level expression, resulting in significantly increased lateral branch growth. Finally, we modeled and performed in vitro analyses of the insect protein rubber resilin and we prepared transgenic Arabidopsis plants for expression of resilin to test the feasibility of using resilin to modify lignin cross-linking in wood and reduce recalcitrance and improve yield of fermentable sugars for biofuels production. Analysis of these and additional transgenics created with this support is continuing.

In vitro degradation of the 32kDa PS II reaction centre protein

International Nuclear Information System (INIS)

Eckenswiller, L.C.; Greenberg, B.M.

1989-01-01

The 32kDa thylakoid membrane protein is an integral component of the PS II reaction centre. The protein, although stable in the dark, undergoes light dependent turnover. Light from the UV, visible and far-red spectral regions induce 32kDa protein degradation. To better understand 32kDa protein metabolism, an in vitro degradation system is being developed. It consists of isolated thylakoid membranes than contain radiolabelled protein. The 32kDa protein is actively and specifically degraded when the thylakoid preparation is exposed to UV or visible radiation. The protein is stable in the dark. The herbicides (atrazine and DCMU) inhibit degradation in the in vitro system as they do in vivo. Additionally, several methods of isolating thylakoids are being compared to optimize the 32kDa protein degradation reaction. The preparations will be evaluated based on their ability to permit light dependent degradation of the 32kDa protein without affecting the other membrane components

Expression of human mag-1 gene in E. coli and preparation of its antibody

International Nuclear Information System (INIS)

Lin Huiyun; Xu Yuanji; Wang Yan; Chen Huihua; Du Zhiyan; Tan Xiaogang; Lu Yinglin

2006-01-01

Objective: To further investigate the new metastasis associated gene, mag-1 expressed in E. coli and its anti-body was prepared in rabbit. Methods: mag-1 was amplified by PCR from pcDNA3-mag-1 and directly cloned into pET-28a vector. The fusion protein was expressed in BL21 and identified by Western blot using anti-His monoclonal antibody. Rabbit was immunized with partially purified fusion protein subcutaneously. Results: Sequence analysis revealed identity of the sequence obtained to the previous report. The recombinant His-mag-1 could be expressed in E. coli as a fusion protein of 18 x 10 3 . The recombinant protein was mostly expressed in the inclusion bodies on the induction by 0.1 mmol/L IPTG at 37 degree C for 6 hours. Western blot analysis showed that the recombinant protein could be recognized by His monoclonal anti-body. The titer of polyclonal antibody against mag-1 was 1:160000. Conclusion: The mag-1 gene is expressed in E. coli highly and its antibody is prepared successfully. (authors)

Comparative studies of human and chicken retinol-binding proteins and prealbumins.

Science.gov (United States)

Kopelman, M; Mokady, S; Cogan, U

1976-08-09

Microheterogeneity of retinol-binding proteins of human plasma and urine, and of chicken plasma was studied by polyacrylamide gel electrophoresis. All three protein systems were found microheterogenous. Incorporation of retinol into the protein preparations on the one hand, and depletion of these proteins from retinol on the other hand, enabled us to clarify the extent to which the presence or absence of the ligand affects the apparent heterogeneity. Upon electrophoresis, each of the native proteins displayed two pairs of protein zones. It appeared that within each pair the fast moving band corresponded to aporetinol-binding protein which upon binding of retinol was converted to a holoprotein with a slightly lower mobility. However, it did not seem that proteins of one pair were converted to proteins of the second pair upon binding of retinol, substantiating ghe microheterogenous character of this protein system. A rapid, two step procedure for isolation of prealbumins from plasma is described. The method which consists of DEAE-cellulose chromatography follwed by preparative electrophoresis was utilized to separate human and chicken prealbumins. Routine dodecyl sulphate electrophoresis resulted in partial dissociation of human prealbumin but in no dissociation of the chicken protein. More drastic treatments prior to electrophoresis were needed to effect complete disruption of both proteins into subunits.

Preparation and quality characterization of soy milk based non-dairy ice cream

Directory of Open Access Journals (Sweden)

Samreen Ahsan

2015-07-01

Full Text Available Soy milk made from soybean has prospective to be used as a substitute of milk due to its health benefits. It is a rich source of iso-flavones, omega-3-fatty acid, dietary fiber, vitamin C, carotenoids, protein and oligosaccharides. The current study was designed to examine the effects of galacto-manan on ice cream by using commercially available (silk and locally prepared soy milk. Galacto-mannan (guar gum was used in different concentration (0.3, 0.4, 0.5 and 0.6% for the preparation of ice cream. Ice cream was analyzed for physico-chemical and sensory characteristics at 0, 30 and 60 days of storage interval. Overrun, meltdown, viscosity, total solids, pH and acidity were affected significantly by ice cream samples as well as storage. While non-significant effects of stabilizer and storage were found on fat, protein, and ash contents of ice cream. On organoleptic evaluation, the highest scores were awarded to the ice cream sample prepared with 0.5% of guar gum. Ice cream manufactured with locally prepared soy milk and guar gum revealed comparable quality with lower cost.

Preparation and quality characterization of soy milk based non-dairy ice cream

Directory of Open Access Journals (Sweden)

Samreen Ahsan

2015-08-01

Full Text Available Soy milk made from soybean has prospective to be used as a substitute of milk due to its health benefits. It is a rich source of iso-flavones, omega-3-fatty acid, dietary fiber, vitamin C, carotenoids, protein and oligosaccharides. The current study was designed to examine the effects of galacto-manan on ice cream by using commercially available (silk and locally prepared soy milk. Galacto-mannan (guar gum was used in different concentration (0.3, 0.4, 0.5 and 0.6% for the preparation of ice cream. Ice cream was analyzed for physico-chemical and sensory characteristics at 0, 30 and 60 days of storage interval. Overrun, meltdown, viscosity, total solids, pH and acidity were affected significantly by ice cream samples as well as storage. While non-significant effects of stabilizer and storage were found on fat, protein, and ash contents of ice cream. On organoleptic evaluation, the highest scores were awarded to the ice cream sample prepared with 0.5% of guar gum. Ice cream manufactured with locally prepared soy milk and guar gum revealed comparable quality with lower cost.

Preparation of 131I-asialo-α1-acid glycoprotein

International Nuclear Information System (INIS)

Rijk, P.P. van

1975-01-01

α 1 -Acid glycoprotein (orosomucoid) was prepared from a byproduct of the ethanol plasma fractionation by means of ion-exchange procedures. Immunoelectrophoresis suggested a high degree of purity; the purified protein contained 13.5% sialic acid and 17.8% hexose. The α 1 -acid glycoprotein was modified by removal of sialic acid with neurominidase (E.C. 3.2.1.18) followed by iodination with 131 I. The purpose of the preparation, its potential use as a pharmacon for liver-function studies in nuclear medicine, is the subject of further study

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

Recovery of high purity proteins from polyacrylamide gels using ultraviolet scanning densitometry

International Nuclear Information System (INIS)

Bartolini, P.; Arkaten, R.; Ribela, M.T.C.P.

1988-07-01

We present here a technique for the purification of proteins carried out by a quantitative analytical method used in conjunction with a preparative gel electrophoresis. Both methods employ densitometric ultraviolet scanning of unstained protein bands, a procedure wich is particulary suitable for the purification and recovery of biologically active polypeptides. In short, the purified extracted protein, isolated in a segment cut out from a preparative gel, is recovered by a second (reversed) electrophoresis. We performed the extractions and recoveries of different amounts of two standard proteins (BSA and STI) and a polypeptide hormone (hGH). Our main interest, especially for the hormone is the complete protein recovery with retention of bio and immunoactivity and high purity. For the proteins tested, the mean recovery was of 93 + - 5% obtaining a mean purity of 95 + - 7%. We conclude that the proposed method should have interesting applications, particularly in the obtention of very pure hormones, as are needed for radioligand assays, for radiolabelling and specific antibody raising. We emphasize the simplicity and rapidity of the method (the entire preparative process: first electrophoresis, UV scanning and reversed electrophoresis can be performed in approximately six hours) and its efficiency in recovering pure proteins even on a milligram scale. We thank the support from the IAEA (4299/RB) and FINEP (43.86.0351.00) and CENE (Brazil). (author) [pt

Regulation of protein synthesis during sea urchin early development

International Nuclear Information System (INIS)

Kelso, L.C.

1989-01-01

Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. [ 32 P] labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development

Differences in nutrient and energy contents of commonly consumed dishes prepared in restaurants v. at home in Hunan Province, China.

Science.gov (United States)

Jia, Xiaofang; Liu, Jiawu; Chen, Bo; Jin, Donghui; Fu, Zhongxi; Liu, Huilin; Du, Shufa; Popkin, Barry M; Mendez, Michelle A

2018-05-01

Eating away from home is associated with poor diet quality, in part due to less healthy food choices and larger portions. However, few studies account for the potential additional contribution of differences in food composition between restaurant- and home-prepared dishes. The present study aimed to investigate differences in nutrients of dishes prepared in restaurants v. at home. Eight commonly consumed dishes were collected in twenty of each of the following types of locations: small and large restaurants, and urban and rural households. In addition, two fast-food items were collected from ten KFC, McDonald's and food stalls. Five samples per dish were randomly pooled from every location. Nutrients were analysed and energy was calculated in composite samples. Differences in nutrients of dishes by preparation location were determined. Hunan Province, China. Na, K, protein, total fat, fatty acids, carbohydrate and energy in dishes. On average, both the absolute and relative fat contents, SFA and Na:K ratio were higher in dishes prepared in restaurants than households (P < 0·05). Protein was 15 % higher in animal food-based dishes prepared in households than restaurants (P<0·05). Quantile regression models found that, at the 90th quantile, restaurant preparation was consistently negatively associated with protein and positively associated with the percentage of energy from fat in all dishes. Moreover, restaurant preparation also positively influenced the SFA content in dishes, except at the highest quantiles. These findings suggest that compared with home preparation, dishes prepared in restaurants in China may differ in concentrations of total fat, SFA, protein and Na:K ratio, which may further contribute, beyond food choices, to less healthy nutrient intakes linked to eating away from home.

Multi-scale carbon micro/nanofibers-based adsorbents for protein immobilization

Energy Technology Data Exchange (ETDEWEB)

Singh, Shiv; Singh, Abhinav [Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Bais, Vaibhav Sushil Singh; Prakash, Balaji [Department of Biological Science and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Verma, Nishith, E-mail: nishith@iitk.ac.in [Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Center for Environmental Science and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India)

2014-05-01

In the present study, different proteins, namely, bovine serum albumin (BSA), glucose oxidase (GOx) and the laboratory purified YqeH were immobilized in the phenolic resin precursor-based multi-scale web of activated carbon microfibers (ACFs) and carbon nanofibers (CNFs). These biomolecules are characteristically different from each other, having different structure, number of parent amino acid molecules and isoelectric point. CNF was grown on ACF substrate by chemical vapor deposition, using Ni nanoparticles (Nps) as the catalyst. The ultra-sonication of the CNFs was carried out in acidic medium to remove Ni Nps from the tip of the CNFs to provide additional active sites for adsorption. The prepared material was directly used as an adsorbent for proteins, without requiring any additional treatment. Several analytical techniques were used to characterize the prepared materials, including scanning electron microscopy, Fourier transform infrared spectroscopy, BET surface area, pore-size distribution, and UV–vis spectroscopy. The adsorption capacities of prepared ACFs/CNFs in this study were determined to be approximately 191, 39 and 70 mg/g for BSA, GOx and YqeH, respectively, revealing that the carbon micro-nanofibers forming synthesized multi-scale web are efficient materials for the immobilization of protein molecules. - Highlights: • Ni metal Np-dispersed carbon micro-nanofibers (ACFs/CNFs) are prepared. • ACFs/CNFs are mesoporous. • Significant adsorption of BSA, GOx and YqeH is observed on ACFs/CNFs. • Multi-scale web of ACFs/CNFs is effective for protein immobilization.

Preparation and characterization of the polyclonal antibody against ...

African Journals Online (AJOL)

To prepare the polyclonal antibody against GAR domain, cDNA encoding 466 amino acids protein of GAR domain was amplified from MG-63 cell by RT-PCR. The amplified cDNA, that exhibited 99% identity to the published sequence, was cloned into prokaryotic expression vector pQE-80L for the expression of GAR ...

Peptide/protein-polymer conjugates: synthetic strategies and design concepts.

Science.gov (United States)

Gauthier, Marc A; Klok, Harm-Anton

2008-06-21

This feature article provides a compilation of tools available for preparing well-defined peptide/protein-polymer conjugates, which are defined as hybrid constructs combining (i) a defined number of peptide/protein segments with uniform chain lengths and defined monomer sequences (primary structure) with (ii) a defined number of synthetic polymer chains. The first section describes methods for post-translational, or direct, introduction of chemoselective handles onto natural or synthetic peptides/proteins. Addressed topics include the residue- and/or site-specific modification of peptides/proteins at Arg, Asp, Cys, Gln, Glu, Gly, His, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val residues and methods for producing peptides/proteins containing non-canonical amino acids by peptide synthesis and protein engineering. In the second section, methods for introducing chemoselective groups onto the side-chain or chain-end of synthetic polymers produced by radical, anionic, cationic, metathesis and ring-opening polymerization are described. The final section discusses convergent and divergent strategies for covalently assembling polymers and peptides/proteins. An overview of the use of chemoselective reactions such as Heck, Sonogashira and Suzuki coupling, Diels-Alder cycloaddition, Click chemistry, Staudinger ligation, Michael's addition, reductive alkylation and oxime/hydrazone chemistry for the convergent synthesis of peptide/protein-polymer conjugates is given. Divergent approaches for preparing peptide/protein-polymer conjugates which are discussed include peptide synthesis from synthetic polymer supports, polymerization from peptide/protein macroinitiators or chain transfer agents and the polymerization of peptide side-chain monomers.

Automated multi-dimensional purification of tagged proteins.

Science.gov (United States)

Sigrell, Jill A; Eklund, Pär; Galin, Markus; Hedkvist, Lotta; Liljedahl, Pia; Johansson, Christine Markeland; Pless, Thomas; Torstenson, Karin

2003-01-01

The capacity for high throughput purification (HTP) is essential in fields such as structural genomics where large numbers of protein samples are routinely characterized in, for example, studies of structural determination, functionality and drug development. Proteins required for such analysis must be pure and homogenous and available in relatively large amounts. AKTA 3D system is a powerful automated protein purification system, which minimizes preparation, run-time and repetitive manual tasks. It has the capacity to purify up to 6 different His6- or GST-tagged proteins per day and can produce 1-50 mg protein per run at >90% purity. The success of automated protein purification increases with careful experimental planning. Protocol, columns and buffers need to be chosen with the final application area for the purified protein in mind.

QUALITY AND SHELF LIFE EVALUATION OF NUGGETS PREPARED FROM SPENT DUCK AND SPENT HEN MEAT

Directory of Open Access Journals (Sweden)

Rajesh Kumar

2015-12-01

Full Text Available A study was conducted to compare the quality of nuggets prepared from spent hen and duck meat. The cooked nuggets were analyzed for pH, thiobarbituric acid (TBA, tyrosine value (TV, moisture, fat, protein, total plate count (TPC and sensory evaluations. Nuggets prepared from spent hen meat showed significantly higher (p<0.05 moisture content however pH, fat and protein content were significantly higher (p<0.05 in duck nuggets. TBA values, TVs and (TPC were highest in duck nuggets but were within the acceptable level up to 7th day of refrigerated storage (4±1°C in both types of nuggets. Both nuggets maintain their sensory quality up to 7th day of refrigeration storage but spent hen nuggets were preferred by consumers compared to nuggets prepared from spent duck meat. Result of the study indicated that, despite the comparative differences among these nuggets, spent duck and hen meat could be used for preparation of nutritionally rich and acceptable nuggets.

Ubiquitination of specific mitochondrial matrix proteins

International Nuclear Information System (INIS)

Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G.; Ciechanover, Aaron

2016-01-01

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

Ubiquitination of specific mitochondrial matrix proteins

Energy Technology Data Exchange (ETDEWEB)

Lehmann, Gilad [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ziv, Tamar [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Braten, Ori [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Admon, Arie [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Udasin, Ronald G. [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ciechanover, Aaron, E-mail: aaroncie@tx.technion.ac.il [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel)

2016-06-17

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

Ultrasonic treatment of Viscozyme Cassava C preparation for improving cellulase activity

Science.gov (United States)

Tra, Tran Thi Thu; Vu, Huynh Minh; Man, Le Van Viet

2017-09-01

In this study, the effects of ultrasonic treatment on the cellulolytic activity of Viscozyme Cassava C preparation were investigated. The biocatalyst was treated with ultrasound at different enzyme concentrations (from 0.02 to 19.50 mg protein/mL), ultrasonic powers (from 0 to 12 W/mL) and times (from 0 to 120 seconds). The highest cellulase activity was achieved when the enzyme preparation was ultrasonicated at 7.3 W/mL for 40 sec, under which the cellulase activity increased by 18.1% over the control. The optimal pH and temperature of the sonicated and unsonicated biocatalysts were statistically similar. However, the half-life value of the sonicated preparation at 4 °C was 24.5% higher than that of the unsonicated preparation. This result indicated that ultrasonic treatment of the enzyme preparation could reduce its amount used in biocatalysis.

Preparation and characterization of amphiphilic copolymer PVDF-g-PMABS and its application in improving hydrophilicity and protein fouling resistance of PVDF membrane

Science.gov (United States)

Chen, Fengtao; Shi, Xingxing; Chen, Xiaobing; Chen, Wenxing

2018-01-01

A facile strategy to improve the hydrophilicity and the antifouling properties of poly(vinylidene fluoride) (PVDF) membranes, a functional monomer of 4-methacrylamidobenzenesulfonic acid (MABS), was designed and synthesized through the amidation reaction between 2-methylacryloyl chloride and sulfanilic acid. Utilizing PVDF and the obtained MABS as reaction monomers, a novel amphiphilic copolymer was firstly prepared by radical polymerization method. The resulting PVDF-g-PMABS was used as a hydrophilic additive in the fabrication of PVDF porous membranes via immersion precipitation process. The surface chemical compositions and structure morphologies of as-prepared blend membranes (PVDF-g-PMABS/PVDF) were characterized by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), respectively. Contact angle measurement and cross-flow permeation test were employed to evaluate the hydrophilicity and antifouling properties of the membranes. It was found that the blend membrane with 4 wt.% PVDF-g-PMABS exhibited a noticeable pure water flux (136.34 L m-2 h-1) and a remarkable flux recovery ratio (FRR) of 98.60% in comparison with the pristine PVDF membrane (63.37 L m-2 h-1 and 38.67%, respectively). The enhanced performance was attributed to the synergetic effects of the strong hydrogen bonding force and the electrostatic repulsion of sulfonic groups against the protein foulants.

Canola/rapeseed protein-functionality and nutrition

Directory of Open Access Journals (Sweden)

Wanasundara Janitha P.D.

2016-07-01

Full Text Available Protein rich meal is a valuable co-product of canola/rapeseed oil extraction. Seed storage proteins that include cruciferin (11S and napin (2S dominate the protein complement of canola while oleosins, lipid transfer proteins and other minor proteins of non-storage nature are also found. Although oil-free canola meal contains 36–40% protein on a dry weight basis, non-protein components including fibre, polymeric phenolics, phytates and sinapine, etc. of the seed coat and cellular components make protein less suitable for food use. Separation of canola protein from non-protein components is a technical challenge but necessary to obtain full nutritional and functional potential of protein. Process conditions of raw material and protein preparation are critical of nutritional and functional value of the final protein product. The storage proteins of canola can satisfy many nutritional and functional requirements for food applications. Protein macromolecules of canola also provide functionalities required in applications beyond edible uses; there exists substantial potential as a source of plant protein and a renewable biopolymer. Available information at present is mostly based on the protein products that can be obtained as mixtures of storage protein types and other chemical constituents of the seed; therefore, full potential of canola storage proteins is yet to be revealed.

Release of proteins via ion exchange from albumin-heparin microspheres

NARCIS (Netherlands)

Kwon, Glen S.; Bae, You Han; Cremers, H.F.M.; Cremers, Harry; Feijen, Jan; Kim, Sung Wan

1992-01-01

Albumin-heparin and albumin microspheres were prepared as ion exchange gels for the controlled release of positively charged polypeptides and proteins. The adsorption isotherms of chicken egg and human lysozyme, as model proteins, on microspheres were obtained. An adsorption isotherm of chicken egg

Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

International Nuclear Information System (INIS)

Hayashi, Kokoro; Kojima, Chojiro

2010-01-01

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in 1 H- 15 N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

PLGA/alginate composite microspheres for hydrophilic protein delivery

International Nuclear Information System (INIS)

Zhai, Peng; Chen, X.B.; Schreyer, David J.

2015-01-01

Poly(lactic-co-glycolic acid) (PLGA) microspheres and PLGA/alginate composite microspheres were prepared by a novel double emulsion and solvent evaporation technique and loaded with bovine serum albumin (BSA) or rabbit anti-laminin antibody protein. The addition of alginate and the use of a surfactant during microsphere preparation increased the encapsulation efficiency and reduced the initial burst release of hydrophilic BSA. Confocal laser scanning microcopy (CLSM) of BSA-loaded PLGA/alginate composite microspheres showed that PLGA, alginate, and BSA were distributed throughout the depths of microspheres; no core/shell structure was observed. Scanning electron microscopy revealed that PLGA microspheres erode and degrade more quickly than PLGA/alginate composite microspheres. When loaded with anti-laminin antibody, the function of released antibody was well preserved in both PLGA and PLGA/alginate composite microspheres. The biocompatibility of PLGA and PLGA/alginate microspheres were examined using four types of cultured cell lines, representing different tissue types. Cell survival was variably affected by the inclusion of alginate in composite microspheres, possibly due to the sensitivity of different cell types to excess calcium that may be released from the calcium cross-linked alginate. - Highlights: • A double emulsion technique is used to prepare protein-loaded PLGA or PLGA/alginate microspheres. • PLGA, alginate and protein are distributed evenly within microsphere structure. • Addition of alginate improves loading efficiency and slows degradation and protein release. • PLGA/alginate microspheres have favorable biocompatibility

PLGA/alginate composite microspheres for hydrophilic protein delivery

Energy Technology Data Exchange (ETDEWEB)

Zhai, Peng [Department of Anatomy and Cell Biology, University of Saskatchewan, S7N5E5 (Canada); Division of Biomedical Engineering, University of Saskatchewan, S7N5A9 (Canada); Chen, X.B. [Department of Mechanical Engineering, University of Saskatchewan, S7N5A9 (Canada); Division of Biomedical Engineering, University of Saskatchewan, S7N5A9 (Canada); Schreyer, David J., E-mail: david.schreyer@usask.ca [Department of Anatomy and Cell Biology, University of Saskatchewan, S7N5E5 (Canada); Division of Biomedical Engineering, University of Saskatchewan, S7N5A9 (Canada)

2015-11-01

Poly(lactic-co-glycolic acid) (PLGA) microspheres and PLGA/alginate composite microspheres were prepared by a novel double emulsion and solvent evaporation technique and loaded with bovine serum albumin (BSA) or rabbit anti-laminin antibody protein. The addition of alginate and the use of a surfactant during microsphere preparation increased the encapsulation efficiency and reduced the initial burst release of hydrophilic BSA. Confocal laser scanning microcopy (CLSM) of BSA-loaded PLGA/alginate composite microspheres showed that PLGA, alginate, and BSA were distributed throughout the depths of microspheres; no core/shell structure was observed. Scanning electron microscopy revealed that PLGA microspheres erode and degrade more quickly than PLGA/alginate composite microspheres. When loaded with anti-laminin antibody, the function of released antibody was well preserved in both PLGA and PLGA/alginate composite microspheres. The biocompatibility of PLGA and PLGA/alginate microspheres were examined using four types of cultured cell lines, representing different tissue types. Cell survival was variably affected by the inclusion of alginate in composite microspheres, possibly due to the sensitivity of different cell types to excess calcium that may be released from the calcium cross-linked alginate. - Highlights: • A double emulsion technique is used to prepare protein-loaded PLGA or PLGA/alginate microspheres. • PLGA, alginate and protein are distributed evenly within microsphere structure. • Addition of alginate improves loading efficiency and slows degradation and protein release. • PLGA/alginate microspheres have favorable biocompatibility.

Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

International Nuclear Information System (INIS)

Witt, P.L.; Bownds, M.D.

1987-01-01

Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger

Preparation of steroid radioligands for ultrafiltration assays by a solid-phase transport globulin method using concanavalin A-Sepharose

International Nuclear Information System (INIS)

Hiramatsu, R.; Nisula, B.C.

1985-01-01

Concanavalin A-Sepharose (Con A-Sepharose) was applied to separate non-protein-bound and albumin-bound radioactive impurities from steroid radioligands. Con A-Sepharose gel, plasma, and steroid radioligand were mixed, incubated, and then washed with buffer. This method was compared with an affinity diafiltration method which separates non-protein-bound radioactive impurities with a filter membrane. 3 H-Water and 3 H-estrone sulfate, chosen to serve as molecules representative of non-protein-bound and albumin-bound impurities, were removed quite effectively by the Con A-Sepharose method, while 85% of 3 H-estrone sulfate could not be removed by the diafiltration method. Plasma unbound cortisol (F) and testosterone (T) values determined by ultrafiltration using 3 H-F and 3 H-T prepared by the Con A-Sepharose method were significantly lower than those determined using the radioligands unprocessed or prepared by the diafiltration method. The whole procedure of the Con A-Sepharose method takes only 3-4 hours. This method is a simple, rapid, and effective technique for preparation of steroid radioligands for plasma protein binding studies

Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry.

Science.gov (United States)

Fairburn, B; Muthana, M; Hopkinson, K; Slack, L K; Mirza, S; Georgiou, A S; Espigares, E; Wong, C; Pockley, A G

2006-09-01

The stress protein gp96 exhibits a number of immunological activities, the majority of studies into which have used gp96 purified from a variety of tissues. On the basis of 1-D gel electrophoresis, the purity of these preparations has been reported to range between 70% and 99%. This study analyzed gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry (MS-MS). The procedure for purifying gp96 was reproducible, as similar protein profiles were observed in replicate gels of gp96 preparations. The purity of the preparations was typically around 70%, with minor co-purified proteins of varying molecular weights and mobilities being present. Dominant bands at 95-100 kDa in preparations from Wistar rats and C57BL/6 mice were identified as gp96 by ECL Western blotting. Multiple bands having similar, yet distinct molecular weights and differing pI mobility on ECL Western blots were confirmed as being gp96 in preparations from Wistar rats using MS-MS. The most striking feature of the 2-D gel analysis was the presence of additional dominant bands at 55 kDa in preparations from Wistar rats, and at 75-90 kDa in preparations from C57BL/6 mice. These were identified as gp96 by ECL Western blotting and, in the case of preparations from Wistar rats, by MS-MS. Although the lower molecular weight, gp96-related molecules might be partially degraded gp96, their reproducible presence, definition and characteristics suggest that they are alternative, species-specific isoforms of the molecule. A 55 kDa protein which exhibited a lower pI value than gp96 was present in all preparations and this was identified as calreticulin, another putative immunoregulatory molecule. This study confirms the reproducibility of the gp96 purification protocol and reveals the presence of multiple gp96 isoforms, some of which likely result from post-translational modifications such as differential glycosylation and

Protein immobilization on Ni(II) ion patterns prepared by microcontact printing and dip-pen nanolithography

NARCIS (Netherlands)

Wu, Chien-Ching; Reinhoudt, David N; Otto, Cees; Velders, Aldrik H; Subramaniam, Vinod

2010-01-01

An indirect method of protein patterning by using Ni(II) ion templates for immobilization via a specific metal-protein interaction is described. A nitrilotriacetic acid (NTA)-terminated self-assembled monolayer (SAM) allows oriented binding of histidine-tagged proteins via complexation with late

Optimizing human synovial fluid preparation for two-dimensional gel electrophoresis.

Science.gov (United States)

Chen, Carl Pc; Hsu, Chih-Chin; Yeh, Wen-Lin; Lin, Hsiu-Chu; Hsieh, Sen-Yung; Lin, Shih-Cherng; Chen, Tai-Tzung; Chen, Max Jl; Tang, Simon Ft

2011-10-11

Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly. The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

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Chen Max JL

2011-10-01

Full Text Available Abstract Background Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly. Results The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit

Encapsulation of Protein-Polysaccharide HIP Complex in Polymeric Nanoparticles

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Ripal Gaudana

2011-01-01

Full Text Available The objective of the present study is to formulate and characterize a nanoparticulate-based formulation of a macromolecule in a hydrophobic ion pairing (HIP complex form. So far, HIP complexation approach has been studied only for proteins with molecular weight of 10–20 kDa. Hence, we have selected bovine serum albumin (BSA having higher molecular weight (66.3 kDa as a model protein and dextran sulphate (DS as a complexing polymer to generate HIP complex. We have prepared and optimized the HIP complex formation process of BSA with DS. Ionic interactions between basic amino acids of BSA with sulphate groups of DS were confirmed by FTIR analysis. Further, nanoparticles were prepared and characterized with respect to size and surface morphology. We observed significant entrapment of BSA in nanoparticles prepared with minimal amounts of PLGA polymer. Finally, results of circular dichroism and intrinsic fluorescence assay have clearly indicated that HIP complexation and method of nanoparticle preparation did not alter the secondary and tertiary structures of BSA.

Plant protein-based hydrophobic fine and ultrafine carrier particles in drug delivery systems.

Science.gov (United States)

Malekzad, Hedieh; Mirshekari, Hamed; Sahandi Zangabad, Parham; Moosavi Basri, S M; Baniasadi, Fazel; Sharifi Aghdam, Maryam; Karimi, Mahdi; Hamblin, Michael R

2018-02-01

For thousands of years, plants and their products have been used as the mainstay of medicinal therapy. In recent years, besides attempts to isolate the active ingredients of medicinal plants, other new applications of plant products, such as their use to prepare drug delivery vehicles, have been discovered. Nanobiotechnology is a branch of pharmacology that can provide new approaches for drug delivery by the preparation of biocompatible carrier nanoparticles (NPs). In this article, we review recent studies with four important plant proteins that have been used as carriers for targeted delivery of drugs and genes. Zein is a water-insoluble protein from maize; Gliadin is a 70% alcohol-soluble protein from wheat and corn; legumin is a casein-like protein from leguminous seeds such as peas; lectins are glycoproteins naturally occurring in many plants that recognize specific carbohydrate residues. NPs formed from these proteins show good biocompatibility, possess the ability to enhance solubility, and provide sustained release of drugs and reduce their toxicity and side effects. The effects of preparation methods on the size and loading capacity of these NPs are also described in this review.

Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

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Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2004-05-01

A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

Optimization of protein buffer cocktails using Thermofluor.

Science.gov (United States)

Reinhard, Linda; Mayerhofer, Hubert; Geerlof, Arie; Mueller-Dieckmann, Jochen; Weiss, Manfred S

2013-02-01

The stability and homogeneity of a protein sample is strongly influenced by the composition of the buffer that the protein is in. A quick and easy approach to identify a buffer composition which increases the stability and possibly the conformational homogeneity of a protein sample is the fluorescence-based thermal-shift assay (Thermofluor). Here, a novel 96-condition screen for Thermofluor experiments is presented which consists of buffer and additive parts. The buffer screen comprises 23 different buffers and the additive screen includes small-molecule additives such as salts and nucleotide analogues. The utilization of small-molecule components which increase the thermal stability of a protein sample frequently results in a protein preparation of higher quality and quantity and ultimately also increases the chances of the protein crystallizing.

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

International Nuclear Information System (INIS)

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

2007-01-01

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 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°

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°.

Electrophoretic transfer protein zymography.

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Pan, Daniel; Hill, Adam P; Kashou, Anthony; Wilson, Karl A; Tan-Wilson, Anna

2011-04-15

Zymography detects and characterizes proteolytic enzymes by electrophoresis of protease-containing samples into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing a copolymerized protein substrate. The usefulness of zymography for molecular weight determination and proteomic analysis is hampered by the fact that some proteases exhibit slower migration through a gel that contains substrate protein. This article introduces electrophoretic transfer protein zymography as one solution to this problem. In this technique, samples containing proteolytic enzymes are first resolved in nonreducing SDS-PAGE on a gel without protein substrate. The proteins in the resolving gel are then electrophoretically transferred to a receiving gel previously prepared with a copolymerized protein substrate. The receiving gel is then developed as a zymogram to visualize clear or lightly stained bands in a dark background. Band intensities are linearly related to the amount of protease, extending the usefulness of the technique so long as conditions for transfer and development of the zymogram are kept constant. Conditions of transfer, such as the pore sizes of resolving and receiving gels and the transfer time relative to the molecular weight of the protease, are explored. Copyright © 2011 Elsevier Inc. All rights reserved.

Design, preparation and preliminary use of a novel biomimetic agent rFN/CAD

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Hong KUANG

2012-12-01

Full Text Available Objective 　To prepare a novel biomimetic agent with adhesive and osteoinduction capabilities, and provide a new approach for improving seeding cell adhesion and osteoblastic differentiation on a scaffold surface. Methods 　A novel bifunctional recombinant protein was engineered and prepared by bioinformatics, standard molecular cloning, protein expression and purification techniques. A centrifugal cell adhesive assay and an osteoinduction assay were employed to determine the biofunction ex vivo. Results 　A 1954bp long DNA chimera of FN Ⅲ7-10/CAD11 EC1-2 and recombinant FN Ⅲ7-10/CAD11 EC1-2 of 641 amino acids were obtained. Polystyrene surface coated with this protein significantly increased the adhesive property and osteodifferentiation of human bone marrow mesenchymal stem cells. Conclusion 　rFN/CAD possesses dual biofunctions of osteoblastic adhesion and differentiation, which can be further applied as an ideal biomimetic molecule for surface modification.

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...

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

DEFF Research Database (Denmark)

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

2013-01-01

Free flow electrophoresis is used for rapid and high-recovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e...

Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia.

Science.gov (United States)

Hernandez-Valladares, Maria; Aasebø, Elise; Selheim, Frode; Berven, Frode S; Bruserud, Øystein

2016-08-22

Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP) as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility.

The Effect of Gel Microstructure on Simulated Gastric Digestion of Protein Gels

NARCIS (Netherlands)

Opazo-Navarrete, Mauricio; Altenburg, Marte D.; Boom, Remko M.; Janssen, Anja E.M.

2018-01-01

The objective of this study was to analyse the impact of the gel structure obtained by different heat-induced temperatures on the in vitro gastric digestibility at pH 2. To achieve this, gels were prepared from soy protein, pea protein, albumin from chicken egg white and whey protein isolate at

Short communication. Impact of the amino acid proline on the cold hardiness of honey bee, Apis mellifera L.

Directory of Open Access Journals (Sweden)

M. Mollaei

2013-07-01

Full Text Available Like many insects, honey bee can increase its cold tolerance through freeze avoidance, using antifreeze proteins (AFPs to lower its supercooling point (SCP. Proline is the most dominant amino acid in honey bee hemolymph, which can be obtained by the insect through feeding. In the current study the antifreeze activity of this amino acid was evaluated on worker honey bees, immediately before the start of cold season. The experiment was established on four treatments including three different concentrations of proline (1%, 3% and 4.35% diluted in 1:1 water sucrose syrup, and the syrup without proline (control. Newly emerged worker honey bees were fed on the mentioned diets for 2 weeks, under cage condition, and then 20 bees from each treatment (cage were selected randomly for determination of cold hardiness inside a cooling bath. Using a CHY data logger, equipped with a K100 sensor attached to the bee’s gaster, the SCP, the amount of released heat and the rate of this release as measures of insect cold hardiness were recorded. Proline significantly reduced honey bees’ SCP. The lowest point, -7.67±0.2646°C, was observed in the concentration of 1% proline. The amount of released heat and the rate of this release were not significantly different across the treatments.

The genetic characteristics in cytology and plant physiology of two wheat (Triticum aestivum) near isogenic lines with different freezing tolerances.

Science.gov (United States)

Wang, Wenqiang; Hao, Qunqun; Wang, Wenlong; Li, Qinxue; Wang, Wei

2017-11-01

Freezing tolerance in taft plants relied more upon an ABA-independent- than an ABA-dependent antifreeze signaling pathway. Two wheat (Triticum aestivum) near isogenic lines (NIL) named tafs (freezing sensitivity) and taft (freezing tolerance) were isolated in the laboratory and their various cytological and physiological characteristics under freezing conditions were studied. Proplastid, cell membrane, and mitochondrial ultrastructure were less damaged by freezing treatment in taft than tafs plants. Chlorophyll, ATP, and thylakoid membrane protein contents were significantly higher, but malondialdehyde content was significantly lower in taft than tafs plants under freezing condition. Antioxidant capacity, as indicated by reactive oxygen species accumulation and antioxidant enzyme activity, and the relative gene expression were significantly greater in taft than tafs plants. Soluble sugars and abscisic acid (ABA) contents were significantly higher in taft plants than in tafs plants under both normal and freezing conditions. The upregulated expression levels of certain freezing tolerance-related genes were greater in taft than tafs plants under freezing treatment. The addition of sodium tungstate, an ABA synthesis inhibitor, led to only partial freezing tolerance inhibition in taft plants and the down-regulated expression of some ABA-dependent genes. Thus, both ABA-dependent and ABA-independent signaling pathways are involved in the freezing tolerance of taft plants. At the same time, freezing tolerance in taft plants relied more upon an ABA-independent- than an ABA-dependent antifreeze signaling pathway.

Analytical sample preparation strategies for the determination of antimalarial drugs in human whole blood, plasma and urine

DEFF Research Database (Denmark)

Casas, Monica Escolà; Hansen, Martin; Krogh, Kristine A

2014-01-01

the available sample preparation strategies combined with liquid chromatographic (LC) analysis to determine antimalarials in whole blood, plasma and urine published over the last decade. Sample preparation can be done by protein precipitation, solid-phase extraction, liquid-liquid extraction or dilution. After...

Characterization of microparticles prepared by emulsion method from pectin and protein

Science.gov (United States)

In this study, pectin was extracted from apple peel and formulated into microparticles in combination with zein, an edible food protein. The physical, chemical, and structural properties of the resultant pectin structures were evaluated. The resultant microparticles were also examined in vitro for c...

Inhibition of Ice Growth and Recrystallization by Zirconium Acetate and Zirconium Acetate Hydroxide

Science.gov (United States)

Mizrahy, Ortal; Bar-Dolev, Maya; Guy, Shlomit; Braslavsky, Ido

2013-01-01

The control over ice crystal growth, melting, and shaping is important in a variety of fields, including cell and food preservation and ice templating for the production of composite materials. Control over ice growth remains a challenge in industry, and the demand for new cryoprotectants is high. Naturally occurring cryoprotectants, such as antifreeze proteins (AFPs), present one solution for modulating ice crystal growth; however, the production of AFPs is expensive and inefficient. These obstacles can be overcome by identifying synthetic substitutes with similar AFP properties. Zirconium acetate (ZRA) was recently found to induce the formation of hexagonal cavities in materials prepared by ice templating. Here, we continue this line of study and examine the effects of ZRA and a related compound, zirconium acetate hydroxide (ZRAH), on ice growth, shaping, and recrystallization. We found that the growth rate of ice crystals was significantly reduced in the presence of ZRA and ZRAH, and that solutions containing these compounds display a small degree of thermal hysteresis, depending on the solution pH. The compounds were found to inhibit recrystallization in a manner similar to that observed in the presence of AFPs. The favorable properties of ZRA and ZRAH suggest tremendous potential utility in industrial applications. PMID:23555701

Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

Energy Technology Data Exchange (ETDEWEB)

Hayashi, Kokoro [Fujifilm Corporation, Analysis Technology Center (Japan); Kojima, Chojiro, E-mail: kojima@protein.osaka-u.ac.j [Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences (Japan)

2010-11-15

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in {sup 1}H-{sup 15}N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

Use of poultry protein isolate as a food ingredient: sensory and color characteristics of low-fat Turkey bologna.

Science.gov (United States)

Omana, Dileep A; Pietrasik, Zeb; Betti, Mirko

2012-07-01

The potential of using poultry protein isolate (PPI) as a food ingredient to substitute either soy protein isolate (SPI) or meat protein in turkey bologna was investigated. PPI was prepared from mechanically separated turkey meat using pH-shift technology and the prepared PPI was added to turkey bologna at 2 different concentrations (1.5% and 2% dry weight basis). Product characteristics were compared with those prepared with the addition of 2% SPI, 11% meat protein (control-1), or 13% meat protein (control-2). All the 5 treatments were subjected to sensory analysis to evaluate aroma, appearance, color, flavor, saltiness, juiciness, firmness, and overall acceptability of the turkey bologna samples using 9-point hedonic scales. A turkey bologna control sample with 11% meat protein appeared to be softer compared to other treatments as revealed by texture profile analysis while purge loss during storage in a retail display case was significantly (P Sensory analysis concluded that 1.5% PPI and 2% PPI could be used as substitute of SPI or lean meat and the treatments could be improved by increasing saltiness and decreasing firmness. The study revealed that with slight modifications in saltiness, turkey bologna can be prepared with the addition of poultry protein isolates as an acceptable substitute for soy protein isolate or meat protein. This will help to avoid usage of nonmeat ingredients (as SPI substitute) and to reduce the cost of production (as meat protein substitute) of low-fat turkey bologna. © 2012 Institute of Food Technologists®

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels.

Science.gov (United States)

Brzica, Hrvoje; Abdullahi, Wazir; Reilly, Bianca G; Ronaldson, Patrick T

2018-05-07

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Individual whey protein components influence lipid oxidation dependent on pH

DEFF Research Database (Denmark)

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

In emulsions, lipid oxidation is expected to be initiated at the oil-water interface. The properties of the emulsifier used and the composition at the interface is therefore expected to be of great importance for the resulting oxidation. Previous studies have shown that individual whey protein...... by affecting the preferential adsorption of whey protein components at the interface. The aim of the study was to compare lipid oxidation in 10% fish oil-in-water emulsions prepared with 1% whey protein having either a high concentration of α-lactalbumin, a high concentration of β-lactoglobulin or equal...... amounts of the two. Emulsions were prepared at pH4 and pH7. Emulsions were characterized by their droplet sizes, viscosities, and contents of proteins in the water phase. Lipid oxidation was assessed by PV and secondary volatile oxidation products. Results showed that pH greatly influenced the oxidative...

Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

Science.gov (United States)

Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

2010-10-13

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

Morphology-Variable Aggregates Prepared from Cholesterol-Containing Amphiphilic Glycopolymers: Their Protein Recognition/Adsorption and Drug Delivery Applications

Directory of Open Access Journals (Sweden)

Zhao Wang

2018-02-01

Full Text Available In this study, a series of diblock glycopolymers, poly(6-O-methacryloyl-d-galactopyranose-b-poly(6-cholesteryloxyhexyl methacrylate (PMAgala-b-PMAChols, with cholesterol/galactose grafts were prepared through a sequential reversible addition-fragmentation chain transfer (RAFT polymerization and deprotection process. The glycopolymers could self-assemble into aggregates with various morphologies depending on cholesterol/galactose-containing block weight ratios, as determined by transmission electronic microscopy (TEM and dynamic laser light scattering (DLS. In addition, the lectin (Ricinus communis agglutinin II, RCA120 recognition and bovine serum albumin (BSA adsorption of the PMAgala-b-PMAChol aggregates were evaluated. The SK-Hep-1 tumor cell inhibition properties of the PMAgala-b-PMAChol/doxorubicin (DOX complex aggregates were further examined in vitro. Results indicate that the PMAgala-b-PMAChol aggregates with various morphologies showed different interaction/recognition features with RCA120 and BSA. Spherical aggregates (d ≈ 92 nm possessed the highest RCA120 recognition ability and lowest BSA protein adsorption. In addition, the DOX-loaded spherical complex aggregates exhibited a better tumor cell inhibition property than those of nanofibrous complex aggregates. The morphology-variable aggregates derived from the amphiphilic glycopolymers may serve as multifunctional biomaterials with biomolecular recognition and drug delivery features.

Preparation and physicochemical properties of protein concentrate and isolate produced from Acacia tortilis (Forssk.) Hayne ssp. raddiana.

Science.gov (United States)

Embaby, Hassan E; Swailam, Hesham M; Rayan, Ahmed M

2018-02-01

The composition and physicochemical properties of defatted acacia flour (DFAF), acacia protein concentrate (APC) and acacia protein isolate (API) were evaluated. The results indicated that API had lower, ash and fat content, than DFAF and APC. Also, significant difference in protein content was noticed among DFAF, APC and API (37.5, 63.7 and 91.8%, respectively). Acacia protein concentrate and isolates were good sources of essential amino acids except cystine and methionine. The physicochemical and functional properties of acacia protein improved with the processing of acacia into protein concentrate and protein isolate. The results of scanning electron micrographs showed that DFAF had a compact structure; protein concentrate were, flaky, and porous type, and protein isolate had intact flakes morphology.

Protein instability and immunogenicity: roadblocks to clinical application of injectable protein delivery systems for sustained release.

Science.gov (United States)

Jiskoot, Wim; Randolph, Theodore W; Volkin, David B; Middaugh, C Russell; Schöneich, Christian; Winter, Gerhard; Friess, Wolfgang; Crommelin, Daan J A; Carpenter, John F

2012-03-01

Protein instability and immunogenicity are two main roadblocks to the clinical success of novel protein drug delivery systems. In this commentary, we discuss the need for more extensive analytical characterization in relation to concerns about protein instability in injectable drug delivery systems for sustained release. We then will briefly address immunogenicity concerns and outline current best practices for using state-of-the-art analytical assays to monitor protein stability for both conventional and novel therapeutic protein dosage forms. Next, we provide a summary of the stresses on proteins arising during preparation of drug delivery systems and subsequent in vivo release. We note the challenges and difficulties in achieving the absolute requirement of quantitatively assessing the degradation of protein molecules in a drug delivery system. We describe the potential roles for academic research in further improving protein stability and developing new analytical technologies to detect protein degradation byproducts in novel drug delivery systems. Finally, we provide recommendations for the appropriate approaches to formulation design and assay development to ensure that stable, minimally immunogenic formulations of therapeutic proteins are created. These approaches should help to increase the probability that novel drug delivery systems for sustained protein release will become more readily available as effective therapeutic agents to treat and benefit patients. Copyright © 2011 Wiley Periodicals, Inc.

Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis

International Nuclear Information System (INIS)

Li, Dan; Chan, Chiomui; Liang, Yu-He; Zheng, Xiaofeng; Li, Lanfen; Su, Xiao-Dong

2005-01-01

B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°

Processing of commercial peanut cake into food-grade meal and its utilization in preparation of cookies.

Science.gov (United States)

Tate, P V; Chavan, J K; Patil, P B; Kadam, S S

1990-04-01

The commercial cake produced during expeller pressing of peanuts was extracted with n-hexane, and 80% ethanol followed by sieving through 80 mesh, to remove residual oil, pigments, bitter taste and fibrous material. The processed meal exhibited comparable composition with defatted peanut flour prepared in the laboratory by solvent extraction of full-fat peanut meal. However, the processed cake meal exhibited low methionine content and in vitro protein digestibility as compared with defatted peanut flour. The processed cake meal can be blended with wheat flour to the extent of 10% (w/w) to prepare acceptable cookies with improved protein and mineral contents.

Intracellular Protein Delivery for Treating Breast Cancer

Science.gov (United States)

2014-08-01

les prepare likely here the numbe of relative action, we lick reactio itive protein nce spectra o ieties onto ancer target lp-His- Trp - ressed...While protein transduction domain (PTD)-fused apoptin has been delivered to cells(Sun et al., 2009; Tavassoli et al., 2004), this approach suffers from...forms the central spoke of the wheel- like structure (Figure 1b), with the larger MBP portion distributes around the apoptin. The planar arrangement

Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.