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Sample records for tag fused proteins

  1. Promoting Tag Removal of a MBP-Fused Integral Membrane Protein by TEV Protease.

    Science.gov (United States)

    Chen, Yanke; Li, Qichang; Yang, Jun; Xie, Hao

    2017-03-01

    Tag removal is a prerequisite issue for structural and functional analysis of affinity-purified membrane proteins. The present study took a MBP-fused membrane protein, MrpF, as a model to investigate the tag removal by TEV protease. Influences of the linking sequence between TEV cleavage site and MrpF on protein expression and predicted secondary structure were investigated. The steric accessibility of TEV protease to cleavage site of MBP-fused MrpF was explored. It was found that reducing the size of hydrophilic group of detergents and/or extending the linking sequence between cleavage site and target protein can significantly improve the accessibility of the cleavage site and promote tag removal by TEV protease.

  2. Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent

    Energy Technology Data Exchange (ETDEWEB)

    Amer, Brendan R.; Macdonald, Ramsay; Jacobitz, Alex W.; Liauw, Brandon; Clubb, Robert T., E-mail: rclubb@mbi.ucla.edu [University of California, Los Angeles, Department of Chemistry and Biochemistry (United States)

    2016-03-15

    Many proteins can’t be studied using solution NMR methods because they have limited solubility. To overcome this problem, recalcitrant proteins can be fused to a more soluble protein that functions as a solubility tag. However, signals arising from the solubility tag hinder data analysis because they increase spectral complexity. We report a new method to rapidly and efficiently add a non-isotopically labeled Small Ubiquitin-like Modifier protein (SUMO) solubility tag to an isotopically labeled protein. The method makes use of a newly developed SUMO-Sortase tagging reagent in which SUMO and the Sortase A (SrtA) enzyme are present within the same polypeptide. The SUMO-Sortase reagent rapidly attaches SUMO to any protein that contains the sequence LPXTG at its C-terminus. It modifies proteins at least 15-times faster than previously described approaches, and does not require active dialysis or centrifugation during the reaction to increase product yields. In addition, silently tagged proteins are readily purified using the well-established SUMO expression and purification system. The utility of the SUMO-Sortase tagging reagent is demonstrated using PhoP and green fluorescent proteins, which are ~90 % modified with SUMO at room temperature within four hours. SrtA is widely used as a tool to construct bioconjugates. Significant rate enhancements in these procedures may also be achieved by fusing the sortase enzyme to its nucleophile substrate.

  3. Specifically and wash-free labeling of SNAP-tag fused proteins with a hybrid sensor to monitor local micro-viscosity.

    Science.gov (United States)

    Wang, Chao; Song, Xinbo; Chen, Lingcheng; Xiao, Yi

    2017-05-15

    Viscosity, as one of the major factors of intracellular microenvironment, influences the function of proteins. To detect local micro-viscosity of a protein, it is a precondition to apply a viscosity sensor for specifically target to proteins. However, all the reported small-molecule probes are just suitable for sensing/imaging of macro-viscosity in biological fluids of entire cells or organelles. To this end, we developed a hybrid sensor BDP-V BG by connecting a viscosity-sensitive boron-dipyrromethene (BODIPY) molecular rotor (BDP-V) to O 6 -benzylguanine (BG) for specific detection of local micro-viscosity of SNAP-tag fused proteins. We measured and calculated the reaction efficiency between the sensor and SNAP-tag protein in vitro to confirm the high labeling specificity. We also found that the labeling reaction results in a 53-fold fluorescence enhancement for the rotor, which qualifies it as a wash-free sensor with ignorable background fluorescence. The high sensitivity of protein labeled sensor (BDP-V-SNAP) to the changes of local viscosity was evaluated by detecting the enhancement of fluorescence lifetimes. Further, with the sensor BDP-V BG, we achieved high specific labeling of cells expressing two SNAP-tag fused proteins (nuclear histone H2B and mitochondrial COX8A). Two-photon excited fluorescence lifetime imaging revealed that, the micro-viscosities nearby the SNAP-tag fused two proteins are distinct. The different changes of local micro-viscosity of SNAP-tag fused histone protein in apoptosis induced by three nucleus-targeted drugs were also characterized for the first time. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Enzyme-linked immunosorbent assays for insulin-like growth factor-I using six-histidine tag fused proteins

    International Nuclear Information System (INIS)

    Huang Yong; Shi Ruina; Zhong Xuefei; Wang Dan; Zhao Meiping; Li Yuanzong

    2007-01-01

    The fusion proteins of insulin-like growth factor-I (IGF-I) and six-histidine tag (IGF-I-6H, 6H-IGF-I-6H) were cloned, expressed, purified and renatured, with their immunoreaction properties and biological activities intact. The binding kinetics between these fusion proteins and anti-IGF-I antibody or anti-6H antibody were studied using surface plasmon resonance (SPR). Two enzyme-linked immunosorbent assay (ELISA) modes, which proved feasible in the measurement of human serum samples, were used to detect IGF-I with the help of the six-histidine tagged proteins. Furthermore, combining the production technique of the six-histidine tagged fusion protein with the competitive sandwich ELISA mode, using an enzyme labeled anti-6H antibody as a tracer, can be a universal immunochemical method to quantitate other polypeptides or proteins

  5. A Phosphorylation Tag for Uranyl Mediated Protein Purification and Photo Assisted Tag Removal

    DEFF Research Database (Denmark)

    Zhang, Qiang; Jørgensen, Thomas. J. D.; Nielsen, Peter E

    2014-01-01

    of enzymes available for this purpose. In the present study, we demonstrate the utility of the divalent uranyl ion in a new procedure for protein purification and tag removal. By employment of a GFP (green florescence protein) recombinant protein we show that uranyl binding to a phosphorylated C-terminal tag......Most protein purification procedures include an affinity tag fused to either the N or C-terminal end of the protein of interest as well as a procedure for tag removal. Tag removal is not straightforward and especially tag removal from the C-terminal end is a challenge due to the characteristics...... enables target protein purification from an E. coli extract by immobilized uranyl affinity chromatography. Subsequently, the tag can be efficiently removed by UV-irradiation assisted uranyl photocleavage. We therefore suggest that the divalent uranyl ion (UO22+) may provide a dual function in protein...

  6. Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag.

    Directory of Open Access Journals (Sweden)

    Aimee Shen

    2009-12-01

    Full Text Available We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD, an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP(6, a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His(6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP(6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.

  7. Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

    Directory of Open Access Journals (Sweden)

    Takuya Kobayashi

    Full Text Available BACKGROUND: Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel multifunctional green fluorescent protein (mfGFP tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis, streptavidin-binding peptide (SBP, and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM. These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. CONCLUSIONS AND SIGNIFICANCE: The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.

  8. A novel two-zone protein uptake model for affinity chromatography and its application to the description of elution band profiles of proteins fused to a family 9 cellulose binding module affinity tag.

    Science.gov (United States)

    Kavoosi, Mojgan; Sanaie, Nooshafarin; Dismer, Florian; Hubbuch, Jürgen; Kilburn, Douglas G; Haynes, Charles A

    2007-08-10

    A novel two-zone model (TZM) is presented to describe the rate of solute uptake by the stationary phase of a sorption-type chromatography column. The TZM divides the porous stationary-phase particle into an inner protein-free core and an outer protein-containing zone where intraparticle transport is limited by pore diffusion and binding follows Langmuir theory. The TZM and the classic pore-diffusion model (PDM) of chromatography are applied to the prediction of stationary-phase uptake and elution bands within a cellulose-based affinity chromatography column designed to selectively purify proteins genetically labelled with a CBM9 (family 9 cellulose binding module) affinity tag. Under both linear and nonlinear loading conditions, the TZM closely matches rates of protein uptake within the stationary phase particles as measured by confocal laser scanning microscopy, while the PDM deviates from experiment in the linear-binding region. As a result, the TZM is shown to provide improved predictions of product breakthrough, including elution behavior from a bacterial lysate feed.

  9. A method for labeling proteins with tags at the native genomic loci in budding yeast.

    Science.gov (United States)

    Wang, Qian; Xue, Huijun; Li, Siqi; Chen, Ying; Tian, Xuelei; Xu, Xin; Xiao, Wei; Fu, Yu Vincent

    2017-01-01

    Fluorescent proteins and epitope tags are often used as protein fusion tags to study target proteins. One prevailing technique in the budding yeast Saccharomyces cerevisiae is to fuse these tags to a target gene at the precise chromosomal location via homologous recombination. However, several limitations hamper the application of this technique, such as the selectable markers not being reusable, tagging of only the C-terminal being possible, and a "scar" sequence being left in the genome. Here, we describe a strategy to solve these problems by tagging target genes based on a pop-in/pop-out and counter-selection system. Three fluorescent protein tag (mCherry, sfGFP, and mKikGR) and two epitope tag (HA and 3×FLAG) constructs were developed and utilized to tag HHT1, UBC13 or RAD5 at the chromosomal locus as proof-of-concept.

  10. A method for labeling proteins with tags at the native genomic loci in budding yeast.

    Directory of Open Access Journals (Sweden)

    Qian Wang

    Full Text Available Fluorescent proteins and epitope tags are often used as protein fusion tags to study target proteins. One prevailing technique in the budding yeast Saccharomyces cerevisiae is to fuse these tags to a target gene at the precise chromosomal location via homologous recombination. However, several limitations hamper the application of this technique, such as the selectable markers not being reusable, tagging of only the C-terminal being possible, and a "scar" sequence being left in the genome. Here, we describe a strategy to solve these problems by tagging target genes based on a pop-in/pop-out and counter-selection system. Three fluorescent protein tag (mCherry, sfGFP, and mKikGR and two epitope tag (HA and 3×FLAG constructs were developed and utilized to tag HHT1, UBC13 or RAD5 at the chromosomal locus as proof-of-concept.

  11. A General Method for Intracellular Protein Delivery through 'E-tag' Protein Engineering and Arginine Functionalized Gold Nanoparticles.

    Science.gov (United States)

    Mout, Rubul; Rotello, Vincent M

    2017-12-20

    In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and nanoparticles were mixed, they formed nanoassemblies, which got fused to cell membrane upon incubation and directly released the E-tagged protein into cell cytosol. We used this method to deliver a wide variety of proteins with different sizes, charges, and functions in various cell lines (Mout et al ., 2017). To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant E-tagged proteins). Laboratory-synthesis of gold nanoparticles has been previously described (Yang et al ., 2011). Desired E-tagged proteins can be cloned from the corresponding genes, and expressed and purified using standard laboratory procedures. We will use E-tagged green fluorescent protein (GFP) as a reference protein here. Users can simply insert an E-tag into their protein of interest, at either terminus. To achieve maximum delivery efficiency, we suggest users testing different length of E-tags. For example, we inserted E = 0 to 20 (E0 means no E-tag insertion, and E20 means 20 glutamic acids insertion in a row) to most of the proteins we tested, and screened for optimal E-tagged length for highest delivery efficiency. E10-tagged proteins gave us the highest delivery efficiency for most of the proteins (except for Cas9, where E20 tag showed highest delivery efficiency). Once these materials are ready, it takes about ~10 min to make the E-tagged protein and nanoparticle nanoassemblies, which are immediately used for delivery. Complete delivery (~100% for GFP-E10) is achieved in less than 3 h.

  12. Recombinant tagging system using ribosomal frameshifting to monitor protein expression.

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    Han, Se Jong; Cho, Sayeon; Lowehhaupt, Ky; Park, So-Young; Sim, Sang Jun; Kim, Yang-Gyun

    2013-03-01

    For rapid and accurate quantitation of recombinant proteins during expression and after purification, we introduce a new tagging strategy that expresses both target proteins and limitedly tagged target proteins together in a single cell at a constant ratio by utilizing cis-elements of programmed -1 ribosomal frameshifting (-1RFS) as an embedded device. -1RFS is an alternative reading mechanism that effectively controls protein expression by many viruses. When a target gene is fused to the enhanced green fluorescent protein (EGFP) gene with a -1RFS element implanted between them, the unfused target and the target-GFP fusion proteins are expressed at a fixed ratio. The expression ratio between these two protein products is adjustable simply by changing -1RFS signals. This limited-tagging system would be valuable for the real-time monitoring of protein expression when optimizing expression condition for a new protein, and in monitoring large-scale bioprocesses without a large metabolic burden on host cells. Furthermore, this strategy allows for the direct measurement of the quantity of a protein on a chip surface and easy application to proteomewide study of gene products. Copyright © 2012 Wiley Periodicals, Inc.

  13. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  14. Overview of the recombinant proteins purification by affinity tags and ...

    African Journals Online (AJOL)

    From protein within isolation process which the same matter increases labor costs further and prevents application of these tags in industrial scale. Therefore proper replacement is emphasized for enzymatic removal of purification tags. Keywords: protein purification; recombinant proteins; self-cleavable tags; Intein tags; ...

  15. Production of recombinant proteins GST L1, E6 and E7 tag HPV 16 ...

    African Journals Online (AJOL)

    In the present work recombinant proteins were produced for used in LUMINEX in order to undergo serological study of Tunisian female population. HPV types 16 L1, E6 and E7 sequences fused to their 3'-end to a sequence encoding the terminal undecapeptide of the SV40 large T-antigen (tag) were isolated from plasmids ...

  16. In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.

    Science.gov (United States)

    Visone, Valeria; Han, Wenyuan; Perugino, Giuseppe; Del Monaco, Giovanni; She, Qunxin; Rossi, Mosè; Valenti, Anna; Ciaramella, Maria

    2017-01-01

    Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.

  17. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase.

    Science.gov (United States)

    Yin, Jun; Straight, Paul D; McLoughlin, Shaun M; Zhou, Zhe; Lin, Alison J; Golan, David E; Kelleher, Neil L; Kolter, Roberto; Walsh, Christopher T

    2005-11-01

    An 11-residue peptide with the sequence DSLEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyl transferase-catalyzed protein labeling by small molecule-CoA conjugates. We name this peptide the "ybbR tag," because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the underlined Ser residue, and the ybbR tag was found to have a strong tendency for adopting an alpha-helical conformation in solution. Here we demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-molecule probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.

  18. Inntags: small self-structured epitopes for innocuous protein tagging.

    Science.gov (United States)

    Georgieva, Maya V; Yahya, Galal; Codó, Laia; Ortiz, Raúl; Teixidó, Laura; Claros, José; Jara, Ricardo; Jara, Mònica; Iborra, Antoni; Gelpí, Josep Lluís; Gallego, Carme; Orozco, Modesto; Aldea, Martí

    2015-10-01

    Protein tagging is widely used in approaches ranging from affinity purification to fluorescence-based detection in live cells. However, an intrinsic limitation of tagging is that the native function of the protein may be compromised or even abolished by the presence of the tag. Here we describe and characterize a set of small, innocuous protein tags (inntags) that we anticipate will find application in a variety of biological techniques.

  19. Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

    Science.gov (United States)

    Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

    2016-11-11

    A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Endogenous gene tagging with fluorescent proteins.

    Science.gov (United States)

    Fetter, John; Samsonov, Andrey; Zenser, Nathan; Zhang, Fan; Zhang, Hongyi; Malkov, Dmitry

    2015-01-01

    Human genome manipulation has become a powerful tool for understanding the mechanisms of numerous diseases including cancer. Inserting reporter sequences in the desired locations in the genome of a cell can allow monitoring of endogenous activities of disease related genes. Native gene expression and regulation is preserved in these knock-in cells in contrast to cell lines with target overexpression under an exogenous promoter as in the case of transient transfection or stable cell lines with random integration. The fusion proteins created using the modern genome editing tools are expressed at their physiological level and thus are more likely to retain the characteristic expression profile of the endogenous proteins in the cell. Unlike biochemical assays or immunostaining, using a tagged protein under endogenous regulation avoids fixation artifacts and allows detection of the target's activity in live cells. Multiple gene targets could be tagged in a single cell line allowing for the creation of effective cell-based assays for compound screening to discover novel drugs.

  1. Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteins

    Directory of Open Access Journals (Sweden)

    Siepert Eva-Maria

    2012-09-01

    Full Text Available Abstract Background Conventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP. Results As an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv Ki-4(scFv and the anti-MucI single-chain fragment variable M12(scFv. During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal

  2. In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

    DEFF Research Database (Denmark)

    Visone, Valeria; Han, Wenyuan; Perugino, Giuseppe

    2017-01-01

    , and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell...... to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity......, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms....

  3. High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

    Science.gov (United States)

    Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

    2018-02-01

    Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility

    International Nuclear Information System (INIS)

    Kobayashi, Hiroshi; Swapna, G. V. T.; Wu, Kuen-Phon; Afinogenova, Yuliya; Conover, Kenith; Mao, Binchen; Montelione, Gaetano T.; Inouye, Masayori

    2012-01-01

    A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS 2 ) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS 2 -tag is replaced with non-isotope labeled PrS 2 -tag, silencing the NMR signals from PrS 2 -tag in isotope-filtered 1 H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS 2 -tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS 2 (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS 2 -tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS 2 -tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone 1 H, 15 N and 13 C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear 1 H– 15 N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.

  5. Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

    Science.gov (United States)

    Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

    2017-11-01

    Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  6. Highly efficient one-step scarless protein tagging by type IIS restriction endonuclease-mediated precision cloning.

    Science.gov (United States)

    Xu, Zhen; Rui, Yan-Ning; Balzeau, Julien; Menezes, Miriam R; Niu, Airu; Hagan, John P; Kim, Dong H

    2017-08-12

    Protein tagging with a wide variety of epitopes and/or fusion partners is used routinely to dissect protein function molecularly. Frequently, the required DNA subcloning is inefficient, especially in cases where multiple constructs are desired for a given protein with unique tags. Additionally, the generated clones have unwanted junction sequences introduced. To add versatile tags into the extracellular domain of the transmembrane protein THSD1, we developed a protein tagging technique that utilizes non-classical type IIS restriction enzymes that recognize non-palindromic DNA sequences and cleave outside of their recognition sites. Our results demonstrate that this method is highly efficient and can precisely fuse any tag into any position of a protein in a scarless manner. Moreover, this method is cost-efficient and adaptable because it uses commercially available type IIS restriction enzymes and is compatible with the traditional cloning system used by many labs. Therefore, precision tagging technology will benefit a number of researchers by providing an alternate method to integrate an array of tags into protein expression constructs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Efficient refolding and immobilization of PMMA-tag-fused single-chain Fv antibodies for sensitive immunological detection on a PMMA plate.

    Science.gov (United States)

    Kumada, Yoichi; Ishikawa, Yasuyuki; Fujiwara, Yusuke; Takeda, Rui; Miyamoto, Ryosuke; Niwa, Daisuke; Momose, Shun; Kang, Bongmun; Kishimoto, Michimasa

    2014-09-01

    In this study, we investigated the efficient refolding and site-specific immobilization of single-chain variable fragments (scFvs) genetically fused with a poly(methylmethacrylate)-binding peptide (PMMA-tag). According to the results of an aggregation test of a scFv-PM in the presence of 0.5 M urea, aggregation was hardly detectable at a weak-alkaline pH (8.5) with lower concentrations of NaCl. Consequently, more than 93% recovery of the anti-RNase scFv-PM model was attained, when it was refolded by dialysis against 50 mM TAPS (pH8.5). These results suggested that the apparent isoelectric point (pI) of a target scFv was decreased to a great extent by the genetic fusion of a PMMA-tag containing 5 acidic amino acids, and, thus, the solubility of the scFv-PM in its semi-denatured form was considerably improved. We also designed alternative peptide-tags composed of plural aspartic acid residues (D5, D10 and D15-tags) to decrease the apparent pI value of the fusion protein. As a consequence, scFv-D5, scFv-D10 and scFv-D15 were also efficiently refolded with yields of more than 95%. It is noteworthy that even scFv-PS-D15, which had both a positively charged polystyrene-binding peptide (PS-tag) and a negatively charged D15-tag, was serially connected at the C-terminal region of scFvs, and also refolded with a yield of 96.1%. These results clearly indicate that controlling the apparent pI value of scFvs by the fusion of oligo-peptides composed of acidic amino acids at the C-terminus resulted in a high degree of recovery via dialysis refolding. According to the results of a sandwich ELISA using scFv-PMs, scFv-D15 and scFv-PS-D15 as ligands, high antigen-binding signals were detected from both the PMMA and phi-PS plates immobilized with scFv-PMs. Furthermore, the high antigen-binding activity of scFv-PMs was maintained in an adsorption state when it was immobilized on the surface of not only PMMA, but also hydrophilic PS (phi-PS) and polycarbonate (PC). These results

  8. LocFuse: human protein-protein interaction prediction via classifier fusion using protein localization information.

    Science.gov (United States)

    Zahiri, Javad; Mohammad-Noori, Morteza; Ebrahimpour, Reza; Saadat, Samaneh; Bozorgmehr, Joseph H; Goldberg, Tatyana; Masoudi-Nejad, Ali

    2014-12-01

    Protein-protein interaction (PPI) detection is one of the central goals of functional genomics and systems biology. Knowledge about the nature of PPIs can help fill the widening gap between sequence information and functional annotations. Although experimental methods have produced valuable PPI data, they also suffer from significant limitations. Computational PPI prediction methods have attracted tremendous attentions. Despite considerable efforts, PPI prediction is still in its infancy in complex multicellular organisms such as humans. Here, we propose a novel ensemble learning method, LocFuse, which is useful in human PPI prediction. This method uses eight different genomic and proteomic features along with four types of different classifiers. The prediction performance of this classifier selection method was found to be considerably better than methods employed hitherto. This confirms the complex nature of the PPI prediction problem and also the necessity of using biological information for classifier fusion. The LocFuse is available at: http://lbb.ut.ac.ir/Download/LBBsoft/LocFuse. The results revealed that if we divide proteome space according to the cellular localization of proteins, then the utility of some classifiers in PPI prediction can be improved. Therefore, to predict the interaction for any given protein pair, we can select the most accurate classifier with regard to the cellular localization information. Based on the results, we can say that the importance of different features for PPI prediction varies between differently localized proteins; however in general, our novel features, which were extracted from position-specific scoring matrices (PSSMs), are the most important ones and the Random Forest (RF) classifier performs best in most cases. LocFuse was developed with a user-friendly graphic interface and it is freely available for Linux, Mac OSX and MS Windows operating systems. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Directed Supramolecular Surface Assembly of SNAP-tag Fusion Proteins

    NARCIS (Netherlands)

    Uhlenheuer, D.A.; Wasserberg, D.; Haase, C.; Nguyen, Hoang D.; Schenkel, J.H.; Huskens, Jurriaan; Ravoo, B.J.; Jonkheijm, Pascal; Brunsveld, Luc

    2012-01-01

    Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized

  10. Expression and Purification of Recombinant Proteins in Escherichia coli with a His6or Dual His6-MBP Tag.

    Science.gov (United States)

    Raran-Kurussi, Sreejith; Waugh, David S

    2017-01-01

    Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His 6 - or a dual His 6 -MBP tagged fusion protein by Gateway ® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His 6 tag or a His 6 -MBP tag can be made on the basis of this solubility test.

  11. Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein

    NARCIS (Netherlands)

    Hink, M.A.; Griep, R.A.; Borst, J.W.; Hoek, van A.; Eppink, M.H.M.; Schots, A.; Visser, A.J.W.G.

    2000-01-01

    Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a

  12. Plasticized protein for 3D printing by fused deposition modeling

    Science.gov (United States)

    Chaunier, Laurent; Leroy, Eric; Della Valle, Guy; Lourdin, Denis

    2016-10-01

    The developments of Additive Manufacturing (AM) by Fused Deposition Modeling (FDM) now target new 3D printable materials, leading to novel properties like those given by biopolymers such as proteins: degradability, biocompatibility and edibility. Plasticized materials from zein, a storage protein issued from corn, present interesting thermomechanical and rheological properties, possibly matching with AM-FDM specifications. Thus commercial zein plasticized with 20% glycerol has a glass transition temperature (Tg) at about 42°C, after storage at intermediate relative humidity (RH=59%). Its principal mechanical relaxation at Tα ≈ 50°C leads to a drop of the elastic modulus from about 1.1 GPa, at ambient temperature, to 0.6 MPa at Tα+100°C. These values are in the same range as values obtained in the case of standard polymers for AM-FDM processing, as PLA and ABS, although relaxation mechanisms are likely different in these materials. Such results lead to the setting up of zein-based compositions printable by AM-FDM and allow processing bioresorbable printed parts, with designed 3D geometry and structure.

  13. A novel strategy for the purification of a recombinant protein using ceramic fluorapatite-binding peptides as affinity tags.

    Science.gov (United States)

    Islam, Tuhidul; Aguilar-Yañez, José Manuel; Simental-Martínez, Jesús; Ortiz-Alcaraz, Cesar Ivan; Rito-Palomares, Marco; Fernandez-Lahore, Marcelo

    2014-04-25

    In recent years, affinity fusion-tag systems have become a popular technique for the purification of recombinant proteins from crude extracts. However, several drawbacks including the high expense and low stability of ligands, their leakage during operation, and difficulties in immobilization, make it important to further develop the method. The present work is concerned with the utilization of a ceramic fluorapatite (CFT)-based chromatographic matrix to overcome these drawbacks. A heptapeptide library exhibiting a range of properties have been synthesized and subjected to ceramic fluorapatite (CFT) chromatography to characterize their retention behavior as a function of pH and composition of the binding buffer. The specific binding and elution behavior demonstrates the possible application of CFT-binding peptides as tags for enhancing the selective recovery of proteins by CFT chromatography. To materialize this strategy, a phage-derived CFT-specific sequence KPRSVSG (Tag1) with/without a consecutive hexalysine sequence, KKKKKKKPRSVSG (Tag2), were fused at the C-terminus of an enhanced green fluorescent protein (eGFP). The resulting gene constructs H-eGFP, H-eGFP-Tag1 and H-eGFP-Tag2 were expressed in Escherichia coli strain BL-21, and the clarified cell lysate was applied to the CFT column equilibrated with binding buffer (20-50mM sodium phosphate, pH 6-8.4). Sodium phosphate (500mM) or 1M NaCl in the respective binding buffer was used to elute the fused proteins, and the chromatographic fractions were analyzed by gel electrophoresis. Both the yield and purity were over 90%, demonstrating the potential application of the present strategy. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli.

    Science.gov (United States)

    Yu, Xuelian; Sun, Jiaqi; Wang, Weiyu; Jiang, Li; Cheng, Beijiu; Fan, Jun

    2017-06-01

    In this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (TEV) protease (TEVp) variant in Escherichia coli strains BL21 (DE3) and Rosetta™ (DE3) are investigated. Combination of the augmenting rare transfer RNAs (tRNAs) and the fused expressivity tag (N-terminal seven amino acid residues of E. coli translation initiation factor II) promotes the soluble TEVp partner expressed at relatively high level. Attachment of the maltose-binding protein (MBP) tag increases soluble expression of the protease released from the fusion protein in E. coli cells, but the incorporated TEVp recognition sequence slightly decreases expressivity of the fusion construct. Except for the green fluorescent protein, the attached expressivity tag shows less efficiency than the MBP tag in enhancing expression levels of the selected five target proteins in the Rosetta™ (DE3) cells under different induction conditions. Our results identified that high-level production of the functional target protein as the fusion partner in E. coli is combined with the intrinsic property of fusion tag, fusion protein stability, inherent folding of target protein, rare tRNA abundance, and the incorporated linker. Purified TEVp fusion constructs with the N-terminal expressivity tag, as well as the MBP partner, are the ideal alternatives for removing fusion tag.

  15. RFP tags for labeling secretory pathway proteins

    Energy Technology Data Exchange (ETDEWEB)

    Han, Liyang; Zhao, Yanhua [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Zhang, Xi; Peng, Jianxin [College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Xu, Pingyong, E-mail: pyxu@ibp.ac.cn [Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Huan, Shuangyan, E-mail: shuangyanhuan@163.com [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Zhang, Mingshu, E-mail: mingshu1984@gmail.com [Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-05-09

    Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.

  16. Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography.

    Science.gov (United States)

    Cabanne, Charlotte; Pezzini, Jérôme; Joucla, Gilles; Hocquellet, Agnès; Barbot, Caroline; Garbay, Bertrand; Santarelli, Xavier

    2009-05-15

    Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50-60% range for affinity resins, and in the 80-85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.

  17. Rheological characterization of plasticized corn proteins for fused deposition modeling

    Science.gov (United States)

    Chaunier, Laurent; Dalgalarrondo, Michèle; Della Valle, Guy; Lourdin, Denis; Marion, Didier; Leroy, Eric

    2017-10-01

    Additive Manufacturing (AM) of tailored natural biopolymer-based objects by Fused Deposition Modeling (FDM) opens new perspectives for applications such as biomedical temporary devices, or pharmaceutical tablets. This exploits the biocompatibility, resorbability and edibility properties of biopolymers. When adequately plasticized, zeins, storage proteins from endosperm of maize kernels, displayed thermomechanical properties possibly matching FDM processing requirements at a convenient temperature Tprinting=130°C. Indeed, with 20% glycerol added (Tg=42°C), plasticized zeins present a high modulus, E'>1GPa, at ambient conditions, which drops below 0.6 MPa at the processing temperature T=130°C, before flowing in the molten state. The rheological characterization shows that the processing window is limited by a progressive increase of viscosity linked to proteins aggregation and crosslinking by S-S bonding between cysteine amino acid residues, which can lead to gelation. However, for short residence time typical of FDM, the viscosity of plasticized zeins is comparable to the one of standard polymers, like ABS or PLA in their FDM processing conditions: indeed, in presence of glycerol, the molten zeins show a shear-thinning behavior with |η*|≈3kPa.s at 1s-1, decreasing to |η*|≈0.3kPa.s at 100s-1, at 130°C. Moreover, zeins presenting both hydrophilic and hydrophobic domains, amphiphilic plasticizers can be used supplementary to tune their rheological behavior. With 20% oleic acid added to the previous composition, the viscosity is divided down to a ratio about 1/2 at 100s-1 at 130°C, below the value of a standard polymer as PLA at its printing temperature. These results show the possible enhancement of the printability of zein-based materials in the molten state, by combining polar and amphiphilic plasticizers.

  18. Protein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro-tag.

    Science.gov (United States)

    Lewandowski, Angela T; Yi, Hyunmin; Luo, Xiaolong; Payne, Gregory F; Ghodssi, Reza; Rubloff, Gary W; Bentley, William E

    2008-02-15

    We report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface-assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices. (c) 2007 Wiley Periodicals, Inc.

  19. 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.

  20. Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases.

    Science.gov (United States)

    Zhou, Zhe; Cironi, Pablo; Lin, Alison J; Xu, Yangqing; Hrvatin, Sinisa; Golan, David E; Silver, Pamela A; Walsh, Christopher T; Yin, Jun

    2007-05-22

    Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small molecules of diverse structures to the target proteins with high efficiency and specificity.

  1. Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit

    Directory of Open Access Journals (Sweden)

    Floridi Aristide

    2008-10-01

    Full Text Available Abstract Background Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β2-adrenergic receptor (β2AR and Gαs indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gαs-fused β2AR. Results The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization. Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β2AR returned rapidly to the plasma membrane, β2AR-Gαs did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis. Conclusion The covalent linkage between β2AR and Gαs does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes

  2. Subcellular localization and functional expression of the glycerol uptake protein 1 (GUP1) of Saccharomyces cerevisiae tagged with green fluorescent protein

    OpenAIRE

    Bleve, Gianluca; Zacheo, Giuseppe; Cappello, Maria Stella; Dellaglio, Franco; Grieco, Francesco

    2005-01-01

    GFP (green fluorescent protein) from Aequorea victoria was used as an in vivo reporter protein when fused to the N- and C-termini of the glycerol uptake protein 1 (Gup1p) of Saccharomyces cerevisiae. The subcellular localization and functional expression of biologically active Gup1–GFP chimaeras was monitored by confocal laser scanning and electron microscopy, thus supplying the first study of GUP1 dynamics in live yeast cells. The Gup1p tagged with GFP is a functional glycerol transporter lo...

  3. Stable isotope, site-specific mass tagging for protein identification

    Science.gov (United States)

    Chen, Xian

    2006-10-24

    Proteolytic peptide mass mapping as measured by mass spectrometry provides an important method for the identification of proteins, which are usually identified by matching the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. The present invention describes a method for increasing the specificity, accuracy and efficiency of the assignments of particular proteolytic peptides and consequent protein identification, by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence without the need for ultrahigh instrumental accuracy. Selected amino acid(s) are labeled with .sup.13C/.sup.15N/.sup.2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tag(s) can then be readily distinguished from other peptides in mass spectra. The present method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency and accuracy for protein identifications.

  4. Extending the scope of site-specific cysteine bioconjugation by appending a prelabeled cysteine tag to proteins using protein trans-splicing.

    Science.gov (United States)

    Dhar, Tulika; Kurpiers, Thomas; Mootz, Henning D

    2011-01-01

    Incorporating synthetic probes site-specifically into proteins is of central interest in several areas of biotechnology and protein chemistry. Bioconjugation techniques provide a simple and effective means of chemically modifying a protein. In particular, covalent chemical modifications of cysteine residues belong to one of the most important reactions due to the unique reactivity of its thiol moiety and the relatively low abundance of this amino acid in proteins. However, such types of modifications cannot be performed in a regioselective fashion when one or more additional cysteines are present. To address this limitation, we have developed an approach where a short cysteine-containing tag (Cys-Tag) fused to one part of a split intein and modified at its sulfhydryl group can be used to label proteins by trans-splicing with a protein of interest (POI) fused to the other half of the split intein. In this way, it is possible to selectively label a protein containing multiple cysteines. The artificially split Mycobacterium xenopi GyrA intein and the Synechocystis sp. DnaB intein were highly suitable for this purpose and were successfully used for the labeling of several proteins. This approach enables a simple route for labeling proteins by site-specific cysteine bioconjugation with any one of several commercially available cysteine-modifying probes.

  5. Structural Insight into the Photochemistry of Split Green Fluorescent Proteins: A Unique Role for a His-Tag.

    Science.gov (United States)

    Deng, Alan; Boxer, Steven G

    2018-01-10

    Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e., split GFP with a β-strand removed, that were found to behave differently in the presence of light. In these structures, the N-terminal His-tag and several neighboring residues play a highly unusual structural and functional role in stabilizing the truncated GFP by substituting as a surrogate β-strand in the groove vacated by the native strand. This finding provides an explanation for the seemingly very different peptide binding and photodissociation properties of split proteins involving β-strands 10 and 11. We show that these truncated GFPs can bind other non-native sequences, and this promiscuity invites the possibility for rational design of sequences optimized for strand binding and photodissociation, both useful for optogenetic applications.

  6. Expression, one-step purification, and immobilization of HaloTag(TM) fusion proteins on chloroalkane-functionalized magnetic beads.

    Science.gov (United States)

    Motejadded, Hassan; Kranz, Bertolt; Berensmeier, Sonja; Franzreb, Matthias; Altenbuchner, Josef

    2010-11-01

    The presented work introduces a novel method to immobilize enzymes either purified or directly out of a crude extract onto magnetic particles in the micrometer range. This method is based on the creation of a fusion protein consisting of the enzyme of choice and a mutant dehalogenase. The dehalogenase gene is commercially available from the company Promega under the name HaloTag(TM). When the fusion protein is contacted with magnetic beads having chemically synthesized, chloroalkane ligands on their surface, the dehalogenase and the ligand undergo a covalent coupling leading to stable and spatially defined immobilization. The principle was proved with a lipase fused to the HaloTag(TM) gene and magnetic poly(methyl)methacrylate beads as carriers. The solubility of the tagged lipase was strongly increased by fusion of the malE gene at the N-terminal end of the HaloTag(TM) lipase gene. This tripartite protein was purified on amylose resin and used for immobilization. About 13 µg protein could be immobilized per 1 mg of beads within a few minutes. Due to the defined binding site, no activity loss was observed in the course of the immobilization. The resulting enzyme carrier was tested with the same beads up to six times for lipase activity over a storage period of 36 days at 8 °C. No loss of activity was found during this time.

  7. Enhancing the solubility of recombinant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding protein as a fusion partner.

    Science.gov (United States)

    Sun, Ping; Tropea, Joseph E; Waugh, David S

    2011-01-01

    In the field of biotechnology, fusing recombinant proteins to highly soluble partners is a common practice for overcoming aggregation in Escherichia coli. E. coli maltose-binding protein (MBP) has been recognized as one of the most effective solubilizing agents, having frequently been observed to improve the yield, enhance the solubility, and promote the proper folding of its fusion partners. The use of a dual hexahistidine-maltose-binding protein affinity tag (His(6)-MBP) has the additional advantage of allowing the fusion protein to be purified by immobilized metal affinity chromatography (IMAC) instead of or in addition to amylose affinity chromatography. This chapter describes a generic method for the overproduction of combinatorially tagged His(6)-MBP fusion proteins in E. coli, with particular emphasis on the use of recombinational cloning to construct expression vectors. In addition, simple methods for evaluating the solubility of the fusion protein and the passenger protein after it is cleaved from the dual His(6)-MBP tag are presented.

  8. Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.

    Science.gov (United States)

    Fujita-Yoshigaki, Junko; Matsuki-Fukushima, Miwako; Yokoyama, Megumi; Katsumata-Kato, Osamu

    2013-11-15

    The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a β-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.

  9. Hexahistidine-tagged maltose-binding protein as a fusion partner for the production of soluble recombinant proteins in Escherichia coli.

    Science.gov (United States)

    Austin, Brian P; Nallamsetty, Sreedevi; Waugh, David S

    2009-01-01

    Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) has emerged as one of the most effective solubilizing agents. In this chapter, we describe how to construct combinatorially-tagged His(6)MBP fusion proteins by recombinational cloning and how to evaluate their yield and solubility. We also describe a procedure to determine how efficiently a His(6)MBP fusion protein is cleaved by tobacco etch virus (TEV) protease in E. coli and a method to assess the solubility of the target protein after it has been separated from His(6)MBP.

  10. Improved blue, green, and red fluorescent protein tagging vectors for S. cerevisiae.

    Science.gov (United States)

    Lee, Sidae; Lim, Wendell A; Thorn, Kurt S

    2013-01-01

    Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging.

  11. Gene Tagging Strategies To Assess Protein Expression, Localization, and Function in Drosophila

    Science.gov (United States)

    Kanca, Oguz; Bellen, Hugo J.; Schnorrer, Frank

    2017-01-01

    Analysis of gene function in complex organisms relies extensively on tools to detect the cellular and subcellular localization of gene products, especially proteins. Typically, immunostaining with antibodies provides these data. However, due to cost, time, and labor limitations, generating specific antibodies against all proteins of a complex organism is not feasible. Furthermore, antibodies do not enable live imaging studies of protein dynamics. Hence, tagging genes with standardized immunoepitopes or fluorescent tags that permit live imaging has become popular. Importantly, tagging genes present in large genomic clones or at their endogenous locus often reports proper expression, subcellular localization, and dynamics of the encoded protein. Moreover, these tagging approaches allow the generation of elegant protein removal strategies, standardization of visualization protocols, and permit protein interaction studies using mass spectrometry. Here, we summarize available genomic resources and techniques to tag genes and discuss relevant applications that are rarely, if at all, possible with antibodies. PMID:28978772

  12. Quantification of green fluorescent protein-(GFP-) tagged membrane proteins by capillary gel electrophoresis.

    Science.gov (United States)

    Danish, Azeem; Lee, Sang-Yong; Müller, Christa E

    2017-10-07

    A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A 2A receptor (A 2A AR), and connexin 43 (Cx43). Quantification of SV2A and A 2A AR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.

  13. An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes

    OpenAIRE

    Cipak, Lubos; Spirek, Mario; Novatchkova, Maria; Chen, Zhiming; Rumpf, Cornelia; Lugmayr, Wolfgang; Mechtler, Karl; Ammerer, Gustav; Csaszar, Edina; Gregan, Juraj

    2009-01-01

    Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag. We used this technique to design strategies for TAP-tagging of predicted Schizosaccharomyces pombe genes (http://mendel.imp.ac.at/Pombe_tagging/). To validate the approach...

  14. Binding ability of a HHP-tagged protein towards Ni{sup 2+} studied by paramagnetic NMR relaxation: The possibility of obtaining long-range structure information

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, Malene Ringkjo bing [University of Copenhagen, H.C. Orsted Institute, Department of Chemistry (Denmark); Lauritzen, Conni; Dahl, Soren Weis; Pedersen, John [Unizyme Laboratories A/S (Denmark); Led, Jens J. [University of Copenhagen, H.C. Orsted Institute, Department of Chemistry (Denmark)

    2004-06-15

    The binding ability of a protein with a metal binding tag towards Ni{sup 2+} was investigated by longitudinal paramagnetic NMR relaxation, and the possibility of obtaining long-range structure information from the paramagnetic relaxation was explored. A protein with a well-defined solution structure (Escherichia coli thioredoxin) was used as the model system, and the peptide His-His-Pro (HHP) fused to the N-terminus of the protein was used as the metal binding tag. It was found that the tag forms a stable dimer complex with the paramagnetic Ni{sup 2+} ion, where each metal ion binds two HHP-tagged protein molecules. However, it was also found that additional sites in the protein compete with the HHP-tag for the binding of the metal ion. These binding sites were identified as the side chain carboxylate groups of the aspartic and glutamic acid residues. Yet, the carboxylate groups bind the Ni{sup 2+} ions considerably weaker than the HHP-tag, and only protons spatially close to the carboxylate sites are affected by the Ni{sup 2+} ions bound to these groups. As for the protons that are unaffected by the carboxylate-bound Nisupb2+bsup> ions, it was found that the long-range distances derived from the paramagnetic relaxation enhancements are in good agreement with the solution structure of thioredoxin. Specifically, the obtained long-range paramagnetic distance constraints revealed that the dimer complex is asymmetric with different orientations of the two protein molecules relative to the Ni{sup 2+} ion. Abbreviations: NOE - nuclear Overhauser enhancement; E. coli - Escherichia coli; Trx - thioredoxin; IMAC - immobilized metal ion affinity chromatography; HT-DPPI - Polyhistidine-tagged dipeptidyl peptidase I; IPTG - isopropyl-1-thio-galactopyranoside; IR - inversion recovery; RMSD - root mean square deviation.

  15. Polyionic and cysteine-containing fusion peptides as versatile protein tags.

    Science.gov (United States)

    Lilie, Hauke; Richter, Susanne; Bergelt, Sabine; Frost, Stefan; Gehle, Franziska

    2013-08-01

    In response to advances in proteomics research and the use of proteins in medical and biotechnological applications, recombinant protein production and the design of specific protein characteristics and functions has become a widely used technology. In this context, protein fusion tags have been developed as indispensable tools for protein expression, purification, and the design of functionalized surfaces or artificially bifunctional proteins. Here we summarize how positively or negatively charged polyionic fusion peptides with or without an additional cysteine can be used as protein tags for protein expression and purification, for matrix-assisted refolding of aggregated protein, and for coupling of proteins either to technologically relevant matrices or to other proteins. In this context we used cysteine-containing polyionic fusion peptides for the design of immunotoxins. In general, polyionic fusion tags can be considered as a multifunctional module in protein technology.

  16. HaloTag protein-mediated specific labeling of living cells with quantum dots

    International Nuclear Information System (INIS)

    So, Min-kyung; Yao Hequan; Rao Jianghong

    2008-01-01

    Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging

  17. Surface sieving coordinated IMAC material for purification of His-tagged proteins.

    Science.gov (United States)

    Li, Senwu; Yang, Kaiguang; Liu, Lukuan; Zhao, Baofeng; Chen, Yuanbo; Li, Xiao; Zhang, Lihua; Zhang, Yukui

    2018-01-02

    Tailor-made materials for the purification of proteins with His-tag was designed through synergizing the selectivity of surface sieving and metal ion affinity. By excluding impurity proteins out of the surface polymer network, such materials could purify His-tagged proteins from the crude cell lysis with purity up to 90%, improved by 14% compared to that obtained by the commercial metal chelating affinity materials. This study might promote the His-tagged protein purification to a new level. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588 (Japan); Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki [System Instruments Co., Ltd., 776-2 Komiya-cho, Hachioji, Tokyo 192-0031 (Japan); Noda, Mamoru; Igimi, Shizunobu [Division of Biomedical Food Research, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Ikebukuro, Kazunori, E-mail: ikebu@cc.tuat.ac.jp [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588 (Japan)

    2013-11-01

    Graphical abstract: -- Highlights: •Zif268 fused to luciferase was used for E. coli O157, Salmonella and coliform detection. •Artificial zinc finger protein fused to luciferase was constructed for Norovirus detection. •An analyzer that automatically detects PCR products by zinc finger protein fused to luciferase was developed. •Target pathogens were specifically detected by the automatic analyzer with zinc finger protein fused to luciferase. -- Abstract: An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0 × 10 to 1.0 × 10{sup 6} copies.

  19. Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

    NARCIS (Netherlands)

    Peters, Fabian; Maestre-Martinez, M.; Leonov, A.; Kovacic, L.; Becker, S.; Boelens, R.; Griesinger, C.

    2011-01-01

    Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large

  20. A chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants

    Directory of Open Access Journals (Sweden)

    Frank eSainsbury

    2016-02-01

    Full Text Available The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to rapidly purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues.

  1. Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality

    Directory of Open Access Journals (Sweden)

    Michael Bugiel

    2015-11-01

    Full Text Available Microspheres are often used as handles for protein purification or force spectroscopy. For example, optical tweezers apply forces on trapped particles to which motor proteins are attached. However, even though many attachment strategies exist, procedures are often limited to a particular biomolecule and prone to non-specific protein or surface attachment. Such interactions may lead to loss of protein functionality or microsphere clustering. Here, we describe a versatile coupling procedure for GFP-tagged proteins via a polyethylene glycol linker preserving the functionality of the coupled proteins. The procedure combines well-established protocols, is highly reproducible, reliable, and can be used for a large variety of proteins. The coupling is efficient and can be tuned to the desired microsphere-to-protein ratio. Moreover, microspheres hardly cluster or adhere to surfaces. Furthermore, the procedure can be adapted to different tags providing flexibility and a promising attachment strategy for any tagged protein.

  2. High-throughput purification of recombinant proteins using self-cleaving intein tags.

    Science.gov (United States)

    Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

    2017-01-01

    High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Iminodiacetic acid functionalized porous hydroxyapatite nanoparticles for capturing histidine-tagged proteins.

    Science.gov (United States)

    Yao, Shasha; Huang, Yanqin; Zhao, Yanbao; Zhang, Yu; Zou, Xueyan; Song, Chunpeng

    2014-06-01

    A simple strategy has been developed to synthesize hydroxyapatite (HAP) nanoparticles (NPs) in a simulated body fluid (SBF). The HAP NPs have an average diameter of 50nm and present porous structure. By taking advantage of surface hydroxyl groups, the HAP NPs are further modified with iminodiacetic acid (IDA), followed by chelating Ni(2+) ions. The HAP/IDA-Ni(2+) NPs as novel adsorbent can capture directly histidine-tagged (His-tagged) proteins from the mixture of lysed cells without sample pretreatment. Results indicated that the HAP/IDA-Ni(2+) NPs present negligible nonspecific adsorption and high protein binding ability, and their specificity and affinity toward His-tagged proteins can remain after 5 times of recycling. The HAP/IDA-Ni(2+) NPs are especially suitable for purification of His-tagged proteins with low molecule weight. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    Science.gov (United States)

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein.

    Science.gov (United States)

    Dammeyer, Thorben; Timmis, Kenneth N; Tinnefeld, Philip

    2013-05-20

    In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the

  6. Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags.

    Science.gov (United States)

    Gillies, Alison R; Hsii, Judy F; Oak, Seachol; Wood, David W

    2008-10-01

    We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.

  7. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    International Nuclear Information System (INIS)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

    2013-01-01

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways

  8. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo, E-mail: innks@khu.ac.kr

    2013-07-19

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways.

  9. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    Energy Technology Data Exchange (ETDEWEB)

    Yamakoshi, Takako [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Ur Rehman, Mati; Yoshihisa, Yoko [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Sugimori, Michiya [Department of Integrative Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan); Shimizu, Tadamichi, E-mail: shimizut@med.u-toyama.ac.jp [Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani, Toyama 930-0194 (Japan)

    2013-03-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.

  10. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    International Nuclear Information System (INIS)

    Yamakoshi, Takako; Makino, Teruhiko; Ur Rehman, Mati; Yoshihisa, Yoko; Sugimori, Michiya; Shimizu, Tadamichi

    2013-01-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes

  11. Expression plasmid vectors with convenient subcloning sites in lambda gt11 that efficiently produce detectable tagged proteins.

    Science.gov (United States)

    Takemoto, Y; Sato, M; Furuta, M; Hashimoto, Y

    1997-07-01

    We have generated cDNA expression vectors that efficiently produce tagged proteins. The newly introduced cloning site of this plasmid facilitates subcloning of cDNA in the lambda gt11 phage into the plasmid vector. Because the cDNA is inserted next to the motifs of the tagged DNA sequence, the protein produced by the tag sequence-coupled cDNA is easily detected by Western blot analysis or immunoprecipitation using commercially available antibodies. The double-tagged protein significantly enhances the efficiency of Western blot and immunoprecipitation detection as compared with the single-tagged protein.

  12. Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2017-04-01

    Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Specific and reversible immobilization of histidine-tagged proteins on functionalized silicon nanowires

    DEFF Research Database (Denmark)

    Liu, Yi-Chi; Rieben, Nathalie Ines; Iversen, Lars

    2010-01-01

    recognition motif attached to the Si NWs. In this study, we show that SiNWs can be chemically functionalized with Ni:NTA motifs, suitable for the specific immobilization of proteins via a short polyhistidine tag (His-tag) at close proximity to the SiNW surface. We demonstrate that the proteins preserve...... their function upon immobilization onto SiNWs. Importantly, the protein immobilization on the Si NWs is shown to be reversible after addition of EDTA or imidazole, thus allowing the regeneration of the bioFET when needed, such as in the case of proteins having a limited lifetime. We anticipate that our...

  14. Lanthanoid tagging via an unnatural amino acid for protein structure characterization.

    Science.gov (United States)

    Jiang, Wen-Xue; Gu, Xin-Hua; Dong, Xu; Tang, Chun

    2017-04-01

    Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. However, for proteins with native cysteines, site-specific attachment may only utilize functional groups orthogonal to sulfhydryl chemistry. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. Demonstrated with ubiquitin and cysteine-containing enzyme EIIB, we show that large PCSs of distinct profiles can be generated for each tag/lanthanoid combination. The DTTA-based lanthanoid tags are associated with large magnetic susceptibility tensors owing to the rigidity of the tags. In particular, introduction of the DTTA-C3 tag affords intermolecular PCSs and enables structural characterization of a transient protein complex between ubiquitin and a UBA domain. Together, we have expanded the repertoire of paramagnetic tags and the applicability of paramagnetic NMR.

  15. Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein

    DEFF Research Database (Denmark)

    Justesen, Sune; Lamberth, Kasper; Nielsen, Lise-Lotte B

    2009-01-01

    Fusion tags add desirable properties to recombinant proteins, but they are not necessarily acceptable in the final products. Ideally, fusion tags should be removed releasing the intact native protein with no trace of the tag. Unique endoproteinases with the ability to cleave outside their own...... recognition sequence can potentially cleave at the boundary of any native protein. Chymosin was recently shown to cleave a pro-chymosin derived fusion tag releasing native target proteins. In our hands, however, not all proteins are chymosin-resistant under the acidic cleavage conditions (pH 4.5) used...... in this system. Here, we have modified the pro-chymosin fusion tag and demonstrated that chymosin can remove this tag at more neutral pH (pH 6.2); conditions, that are less prone to compromise the integrity of target proteins. Chymosin was successfully used to produce intact native target protein both...

  16. Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

    International Nuclear Information System (INIS)

    Peters, Fabian; Maestre-Martinez, Mitcheell; Leonov, Andrei; Kovačič, Lidija; Becker, Stefan; Boelens, Rolf; Griesinger, Christian

    2011-01-01

    Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer.

  17. Overexpressing tagged proteins in plants using a modified gateway cloning strategy.

    Science.gov (United States)

    Dubin, Manu J; Bowler, Chris; Benvenuto, Giovanna

    2010-03-01

    In recent years, sequence-specific recombination cloning methods such as the Gateway system have become increasingly popular for (over)expressing tagged proteins in high-throughput investigations in many different organisms, including plants. Because of their versatility and ease of use, these methods have gained favor in low- and medium-throughput investigations as well. However, due to the recombination step, the resulting fusion proteins contain long and often highly charged polylinker sequences that can interfere with their physiological function. Furthermore, in some cases the gene of interest must be cloned twice (once with and once without a stop codon) for N- and C-terminal tagging. Here, we present a hybrid combinatorial cloning strategy that overcomes many of these limitations. In the first step, the gene of interest is cloned into an entry vector containing standardized cloning sites with the desired N- or C-terminal tag and an optimized polylinker sequence. A Gateway recombination reaction is used to transfer the protein-tag fusion from the entry clone to a Gateway destination vector with the desired promoter and selectable marker for the organism of interest. As experimental requirements evolve, constructs for expressing the protein of interest with the desired tag, promoter, and selectable marker or other features can rapidly and easily be created.

  18. Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation.

    Science.gov (United States)

    Horinouchi, Takahiro; Terada, Koji; Higashi, Tsunehito; Miwa, Soichi

    2016-01-01

    Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.

  19. A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits.

    Science.gov (United States)

    Gustavsson, Tobias; Trane, Maria; Moparthi, Vamsi K; Miklovyte, Egle; Moparthi, Lavanya; Górecki, Kamil; Leiding, Thom; Arsköld, Sindra Peterson; Hägerhäll, Cecilia

    2010-08-01

    Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c(550). Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c(550) domain in all the fusion proteins exhibited normal spectra and redox properties, with an E(m) of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.

  20. Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-02-01

    Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jarmander Johan

    2012-09-01

    Full Text Available Abstract Background The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium. Conclusions Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent

  2. Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins.

    Science.gov (United States)

    Cheng, Chung-Hsien; Lee, Wen-Chien

    2010-08-28

    Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R). Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction. Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could

  3. Effect of His(6)-tagging of anterior gradient 2 protein on its electro-oxidation

    Czech Academy of Sciences Publication Activity Database

    Ostatná, Veronika; Vargová, Veronika; Hrstka, R.; Durech, M.; Vojtešek, B.; Paleček, Emil

    2014-01-01

    Roč. 150, DEC2014 (2014), s. 218-222 ISSN 0013-4686 R&D Projects: GA ČR(CZ) GA13-00956S Institutional support: RVO:68081707 Keywords : Anterior Gradient 2?oncoprotein * His-tagged proteins * carbon electrodes Subject RIV: BO - Biophysics Impact factor: 4.504, year: 2014

  4. Facilitation of yeast-lethal membrane protein production by detoxifying with GFP tagging.

    Science.gov (United States)

    Oshikane, Hiroyuki; Watabe, Masahiko; Nakaki, Toshio

    2018-03-27

    Recombinant techniques for target protein production have been rapidly established and widely utilised in today's biological research. Nevertheless, methods for membrane protein production have yet to be developed, since membrane proteins generally tend to be expressed at low levels, easily aggregated, and/or even toxic to their host cells. Here we report that a GFP-tagging technique can be applied for the stable production of membrane proteins that are toxic to their host cells when overexpressed, paving the way for future advances in membrane protein biochemistry and drug development. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Low Resolution Structure of RAR1-GST-Tag Fusion Protein in Solution

    International Nuclear Information System (INIS)

    Taube, M.; Kozak, M.; Jarmolowski, A.

    2010-01-01

    RAR1 is a protein required for resistance mediated by many R genes and function upstream of signaling pathways leading to H 2 O 2 accumulation. The structure and conformation of RAR1-GST-Tag fusion protein from barley (Hordeum vulgare) in solution was studied by the small angle scattering of synchrotron radiation. It was found that the dimer of RAR1-GST-Tag protein is characterized in solution by radius of gyration R G = 6.19 nm and maximal intramolecular vector D max = 23 nm. On the basis of the small angle scattering of synchrotron radiation SAXS data two bead models obtained by ab initio modeling are proposed. Both models show elongated conformations. We also concluded that molecules of fusion protein form: dimers in solution via interaction of GST domains. (authors)

  6. Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system.

    Science.gov (United States)

    Costa, Sofia; Almeida, André; Castro, António; Domingues, Lucília

    2014-01-01

    Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide's immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system.

  7. Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

    Science.gov (United States)

    Costa, Sofia; Almeida, André; Castro, António; Domingues, Lucília

    2014-01-01

    Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system. PMID:24600443

  8. Subcellular localization and functional expression of the glycerol uptake protein 1 (GUP1) of Saccharomyces cerevisiae tagged with green fluorescent protein.

    Science.gov (United States)

    Bleve, Gianluca; Zacheo, Giuseppe; Cappello, Maria Stella; Dellaglio, Franco; Grieco, Francesco

    2005-08-15

    GFP (green fluorescent protein) from Aequorea victoria was used as an in vivo reporter protein when fused to the N- and C-termini of the glycerol uptake protein 1 (Gup1p) of Saccharomyces cerevisiae. The subcellular localization and functional expression of biologically active Gup1-GFP chimaeras was monitored by confocal laser scanning and electron microscopy, thus supplying the first study of GUP1 dynamics in live yeast cells. The Gup1p tagged with GFP is a functional glycerol transporter localized at the plasma membrane and endoplasmic reticulum levels of induced cells. The factors involved in proper localization and turnover of Gup1p were revealed by expression of the Gup1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaerical protein was targeted to the plasma membrane through a Sec6-dependent process; on treatment with glucose, it was endocytosed through END3 and targeted for degradation in the vacuole. Gup1p belongs to the list of yeast proteins rapidly down-regulated by changing the carbon source in the culture medium, in agreement with the concept that post-translational modifications triggered by glucose affect proteins of peripheral functions. The immunoelectron microscopy assays of cells expressing either Gup1-GFP or GFP-Gup1 fusions suggested the Gup1p membrane topology: the N-terminus lies in the periplasmic space, whereas its C-terminal tail has an intracellular location. An extra cytosolic location of the N-terminal tail is not generally predicted or determined in yeast membrane transporters.

  9. Metal ions-binding T4 lysozyme as an intramolecular protein purification tag compatible with X-ray crystallography.

    Science.gov (United States)

    Boura, Evzen; Baumlova, Adriana; Chalupska, Dominika; Dubankova, Anna; Klima, Martin

    2017-06-01

    Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X-ray crystallography. © 2017 The Protein Society.

  10. Cytoplasmic delivery and selective, multicomponent labeling with oligoarginine-linked protein tags.

    Science.gov (United States)

    Zou, Xiaoyan; Rajendran, Megha; Magda, Darren; Miller, Lawrence W

    2015-03-18

    Strategies that leverage bio-orthogonal interactions between small molecule ligands and genetically encoded amino acid sequences can be used to attach high-performance fluorophores to proteins in living cells. However, a major limitation of chemical protein labeling is that cells' plasma membranes are impermeable to many useful probes and biolabels. Here, we show that conjugation to nonaarginine, a cell penetrating peptide (CPP), enables passive cytoplasmic delivery of otherwise membrane-impermeant, small molecule protein labels. Heterodimers consisting of a luminescent Tb(3+) complex, Lumi4, linked to benzyl guanine, benzyl cytosine, and trimethoprim were conjugated to the peptide CysArg9 with a reducible disulfide linker. When added to culture medium, the peptide conjugates rapidly (cells. The benzyl guanine, benzyl cytosine, and trimethoprim derivatives bind selectively to fusion proteins tagged with SNAP-Tag, CLIP-Tag, and Escherichia coli dihydrofolate reductase (eDHFR), respectively. Furthermore, eDHFR and SNAP-Tag fusions can be labeled with Lumi4 analogues in the same cell, and this labeling can be detected using two-color, time-gated Förster resonance energy transfer (FRET) microscopy. Finally, we present quantitative data showing that cytoplasmic uptake of nonaarginine-conjugated probes occurs in multiple cell types (MDCK, HeLa, NIH 3T3), most cells in a culture (>75%) are loaded with probe, and the cellular probe concentration can be controlled by varying incubation conditions. CPP-mediated delivery of Lumi4-linked protein labels will greatly increase the utility of lanthanide-based FRET microscopy. Moreover, our results strongly suggest that this approach can be adapted to deliver a wide variety of protein-targeted fluorophores or other functional probes that were previously unavailable for intracellular imaging studies.

  11. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    Science.gov (United States)

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

  12. The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts.

    Directory of Open Access Journals (Sweden)

    Steffen Frey

    Full Text Available During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.

  13. Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis.

    Science.gov (United States)

    Chen, Jia-Liang; Zhao, Yu; Gong, Yan-Jun; Pan, Bin-Bin; Wang, Xiao; Su, Xun-Cheng

    2018-02-01

    Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein-ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.

  14. In Vivo Immobilization of Fusion Proteins on Bioplastics by the Novel Tag BioF

    Science.gov (United States)

    Moldes, Cristina; García, Pedro; García, José L.; Prieto, María A.

    2004-01-01

    A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-β-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme. PMID:15184113

  15. Specific and reversible immobilization of histidine-tagged proteins on functionalized silicon nanowires

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yi-Chi C; Rieben, Nathalie; Iversen, Lars; Martinez, Karen L [Bio-Nanotechnology Laboratory, Department of Neuroscience and Pharmacology and Nano-Science Center, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Oe (Denmark); Soerensen, Brian S; Nygaard, Jesper [Niels Bohr Institute and Nano-Science Center, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Oe (Denmark); Park, Jiwoong, E-mail: martinez@nano.ku.dk [Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853 (United States)

    2010-06-18

    Silicon nanowire (Si NW)-based field effect transistors (FETs) have shown great potential as biosensors (bioFETs) for ultra-sensitive and label-free detection of biomolecular interactions. Their sensitivity depends not only on the device properties, but also on the function of the biological recognition motif attached to the Si NWs. In this study, we show that SiNWs can be chemically functionalized with Ni:NTA motifs, suitable for the specific immobilization of proteins via a short polyhistidine tag (His-tag) at close proximity to the SiNW surface. We demonstrate that the proteins preserve their function upon immobilization onto SiNWs. Importantly, the protein immobilization on the Si NWs is shown to be reversible after addition of EDTA or imidazole, thus allowing the regeneration of the bioFET when needed, such as in the case of proteins having a limited lifetime. We anticipate that our methodology may find a generic use for the development of bioFETs exploiting functional protein assays because of its high compatibility to various types of NWs and proteins.

  16. Stable transformation of an episomal protein-tagging shuttle vector in the piscine diplomonad Spironucleus vortens

    Directory of Open Access Journals (Sweden)

    Cronembold Daniela

    2008-04-01

    Full Text Available Abstract Background Diplomonads are common free-living inhabitants of anoxic aquatic environments and are also found as intestinal commensals or parasites of a wide variety of animals. Spironucleus vortens is a putatively commensal diplomonad of angelfish that grows to high cell densities in axenic culture. Genomic sequencing of S. vortens is in progress, yet little information is available regarding molecular and cellular aspects of S. vortens biology beyond descriptive ultrastructural studies. To facilitate the development of S. vortens as an additional diplomonad experimental model, we have constructed and stably transformed an episomal plasmid containing an enhanced green fluorescent protein (GFP tag, an AU1 epitope tag, and a tandem affinity purification (TAP tag. This construct also contains selectable antibiotic resistance markers for both S. vortens and E. coli. Results Stable transformants of S. vortens grew relatively rapidly (within 7 days after electroporation and were maintained under puromycin selection for over 6 months. We expressed the enhanced GFP variant, eGFP, under transcriptional control of the S. vortens histone H3 promoter, and visually confirmed diffuse GFP expression in over 50% of transformants. Next, we generated a histone H3::GFP fusion using the S. vortens conventional histone H3 gene and its native promoter. This construct was also highly expressed in the majority of S. vortens transformants, in which the H3::GFP fusion localized to the chromatin in both nuclei. Finally, we used fluorescence in situ hybridization (FISH of the episomal plasmid to show that the transformed plasmid localized to only one nucleus/cell and was present at roughly 10–20 copies per nucleus. Because S. vortens grows to high densities in laboratory culture, it is a feasible diplomonad from which to purify native protein complexes. Thus, we also included a TAP tag in the plasmid constructs to permit future tagging and subsequent purification

  17. Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

    Science.gov (United States)

    Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

    2018-01-01

    The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

  18. Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins

    Directory of Open Access Journals (Sweden)

    Cheng Chung-Hsien

    2009-07-01

    Full Text Available Abstract Background Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase, were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D, and the other with five contiguous arginine residues (5R. Results Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac from N-acetyl-D-glucosamine (GlcNAc. Conclusion Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.

  19. Novel Techniques for Weak Alignment of Proteins in Solution Using Chemical Tags Coordinating Lanthanide Ions

    International Nuclear Information System (INIS)

    Ikegami, Takahisa; Verdier, Laurent; Sakhaii, Peyman; Grimme, Susanne; Pescatore, Barbara; Saxena, Krishna; Fiebig, Klaus M.; Griesinger, Christian

    2004-01-01

    A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual 15 N, 1 H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag

  20. The elution of certain protein affinity tags with millimolar concentrations of diclofenac.

    Science.gov (United States)

    Baliova, Martina; Juhasova, Anna; Jursky, Frantisek

    2015-12-01

    Diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid) is a sparingly soluble, nonsteroidal anti-inflammatory drug therapeutically acting at low micromolar concentrations. In pH range from 8 to 11, its aqueous solubility can be increased up to 200 times by the presence of counter ions such as sodium. Our protein interaction studies revealed that a millimolar concentration of sodium diclofenac is able to elute glutathione S-transferase (GST), cellulose binding protein (CBD), and maltose binding protein (MBP) but not histidine-tagged or PDZ-tagged proteins from their affinity resins. The elution efficiency of diclofenac is comparable with the eluting agents normally used at similar concentrations. Native gel electrophoresis of sodium diclofenac-treated proteins showed that the interaction is non-covalent and non-denaturing. These results suggest that sodium diclofenac, in addition to its pharmaceutical applications, can also be exploited as a lead for the development of new proteomics reagents. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Efficient secretion of human lysozyme fused to the Sh ble phleomycin resistance protein by the fungus Tolypocladium geodes.

    Science.gov (United States)

    Baron, M; Tiraby, G; Calmels, T; Parriche, M; Durand, H

    1992-07-01

    Tolypocladium geodes strain NC50 was transformed by different integrating vectors bearing both a synthetic gene encoding human lysozyme (HLz) and the Sh ble phleomycin resistance marker, either in separate expression cassettes or in transcriptional or translational fusion configurations. Clones derived from all vectors were able to secrete HLz. The highest productivities in shake flasks (up to 150 mg l-1 in 5 days) were obtained when HLz was fused at the C-terminal end of the Sh ble protein. The fusion protein is efficiently secreted and release of active lysozyme occurs by extracellular proteolytic cleavage in the junction peptide.

  2. An AIL/IL-based liquid/liquid extraction system for the purification of His-tagged proteins.

    Science.gov (United States)

    Xu, Weiyuan; Cao, Huazhen; Ren, Guangwei; Xie, Hujun; Huang, Jianying; Li, Shijun

    2014-06-01

    A sorbent based on affinity ionic liquid (AIL), triazacyclononane-ionic liquid, was synthesized, characterized, and applied to the extraction of histidine (His)-tagged proteins from aqueous buffer to ionic liquid (IL) phase. The adsorbed His-tagged proteins could be back-extracted from the IL phase to the aqueous buffer with an imidazole solution. The specific binding of His-tagged proteins with AIL/IL could be affected by a few factors including the ionic strength and coordinated metal ions. In the case of His-tagged enhanced green fluorescent protein (EGFP), the maximum binding capacity of Cu(2+)-AIL/IL reached 2.58 μg/μmol under the optimized adsorption conditions. The eluted His-tagged EGFP kept fluorescent and remained active through the purification process. Moreover, a tandem extraction process successively using Cu(2+)-AIL/IL and Zn(2+)-AIL/IL systems was developed, which was proven very efficient to obtain the ultimate protein with a purity of about 90 %. An effective reclamation method for the AIL/IL extraction system was further established. The sorbent could be easily regenerated by removing metal ions with EDTA and the followed reimmobilization of metal ions. Easy handling of the presented M(2+)-AIL/IL system and highly specific ability to absorb His-tagged proteins make it attractive and potentially applicable in biomolecular separation.

  3. Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins.

    Science.gov (United States)

    Hein, Birka; Willig, Katrin I; Wurm, Christian A; Westphal, Volker; Jakobs, Stefan; Hell, Stefan W

    2010-01-06

    We show far-field fluorescence nanoscopy of different structural elements labeled with an organic dye within living mammalian cells. The diffraction barrier limiting far-field light microscopy is outperformed by using stimulated emission depletion. We used the tagging protein hAGT (SNAP-tag), which covalently binds benzylguanine-substituted organic dyes, for labeling. Tetramethylrhodamine was used to image the cytoskeleton (vimentin and microtubule-associated protein 2) as well as structures located at the cell membrane (caveolin and connexin-43) with a resolution down to 40 nm. Comparison with structures labeled with the yellow fluorescent protein Citrine validates this labeling approach. Nanoscopic movies showing the movement of connexin-43 clusters across the cell membrane evidence the capability of this technique to observe structural changes on the nanoscale over time. Pulsed or continuous-wave lasers for excitation and stimulated emission depletion yield images of similar resolution in living cells. Hence fusion proteins that bind modified organic dyes expand widely the application range of far-field fluorescence nanoscopy of living cells. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS-PAGE gel.

    Science.gov (United States)

    Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Matsuda, Ayumi; Koike, Tohru

    2014-11-01

    The sensitivity of Western blotting analysis after Phos-tag SDS-PAGE is occasionally inferior to that after normal (Phos-tag-free) SDS-PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos-tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet-tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos-tag SDS-PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos-tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet-tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos-tag gels were similar to those from normal gels. In some cases involving the use of a Phos-tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli.

    Science.gov (United States)

    Malik, Ajamaluddin

    2016-06-01

    Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity. Production of recombinant proteins containing disulfide bond for therapeutic, diagnostic and various other purposes is a challenging task of research. Production of such proteins in the reducing cytosolic compartment of E. coli usually ends up in inclusion bodies formation. Refolding of inclusion bodies can be difficult, time and labor consuming and uneconomical. Translocation of these proteins into the oxidative periplasmic compartment provides correct environment to undergo proper disulfide bonds formation and thus achieving native conformation. However, not all proteins can be efficiently translocated to the periplasm with the help of bacterial signal peptides. Therefore, fusion to a small well-folded and stable periplasmic protein is more promising for periplasmic production of disulfide bonded proteins. In the past decades, several full-length proteins or domains were used for enhancing translocation and solubility. Here, protein fusion tags that significantly increase the yields of target proteins in the periplasmic space are reviewed.

  6. Peptide tag/probe pairs based on the coordination chemistry for protein labeling.

    Science.gov (United States)

    Uchinomiya, Shohei; Ojida, Akio; Hamachi, Itaru

    2014-02-17

    Protein-labeling methods serve as essential tools for analyzing functions of proteins of interest under complicated biological conditions such as in live cells. These labeling methods are useful not only to fluorescently visualize proteins of interest in biological systems but also to conduct protein and cell analyses by harnessing the unique functions of molecular probes. Among the various labeling methods available, an appropriate binding pair consisting of a short peptide and a de novo designed small molecular probe has attracted attention because of its wide utility and versatility. Interestingly, most peptide tag/probe pairs exploit metal-ligand coordination interactions as the main binding force responsible for their association. Herein, we provide an overview of the recent progress of these coordination-chemistry-based protein-labeling methods and their applications for fluorescence imaging and functional analysis of cellular proteins, while highlighting our originally developed labeling methods. These successful examples clearly exemplify the utility and versatility of metal coordination chemistry in protein functional analysis.

  7. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    Science.gov (United States)

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Stable expression of foot-and-mouth disease virus protein VP1 fused with cholera toxin B subunit in the potato (Solanum tuberosum).

    Science.gov (United States)

    He, Dong-Mei; Qian, Kai-Xian; Shen, Gui-Fang; Li, Yi-Nü; Zhang, Zhi-Fang; Su, Zhong-Liang; Shao, Hong-Bo

    2007-04-01

    The expression vector, pBI121CTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed by fused PCR and transferred into potato (Solanum tuberosum L.) by Agrobacterium-mediated transformation. Transformed plants were obtained by selecting on kanamycin-resistant medium strictly and regenerated. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results showed that the fused genes were expressed stablely under the control of specific-tuber patatin promoter. The expressed fused proteins have a certain degree of immunogenicity.

  9. Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography.

    Science.gov (United States)

    Bartnicki, Filip; Kowalska, Ewa; Pels, Katarzyna; Strzalka, Wojciech

    2015-10-30

    Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors

    Directory of Open Access Journals (Sweden)

    Godement Pierre

    2007-11-01

    Full Text Available Abstract Background Dynamic monitoring of protein expression and localization is fundamental to the understanding of biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing. Results We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving most of its functions. Our results demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type. Conclusion The GFP-tagged Otx2 mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of Otx2 gene activity. This opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between Otx2 and the mutated gene.

  11. A New Method for Immobilization of His-Tagged Proteins with the Application of Low-Frequency AC Electric Field.

    Science.gov (United States)

    Takahashi, Shunsuke; Kishi, Kazuki; Hiraga, Ryota; Hayashi, Kazuki; Mamada, Youhei; Oshige, Masahiko; Katsura, Shinji

    2018-03-05

    Continued advancement of protein array, bioelectrode, and biosensor technologies is necessary to develop methods for higher amount and highly oriented immobilization activity of proteins. In pursuit of these goals, we developed a new immobilization method by combining electrostatic transport and subsequent molecular diffusion of protein molecules. Our developed immobilization method is based on a model that transports proteins toward the substrate surface due to steep concentration gradient generated by low-frequency AC electric field. The immobilization of the maximum amounts can be obtained by the application of the AC voltage of 80 Vpp, 20 Hz both for His-tagged Green Fluorescent Protein (GFP) and Discosoma sp. Red Fluorescent Protein (DsRed), used as model proteins. The amounts of the immobilized His-tagged GFP and DsRed were approximately seven-fold higher than that in the absence of the application of low-frequency AC electric field. Furthermore, the positively and negatively charged His-tagged GFP at acidic and alkaline pH were immobilized by applying of low-frequency AC electric field, whereas the non-charged His-tagged GFP at the pH corresponding to its isoelectric point (pI) was not immobilized. Therefore, unless the pH is equal to pI, the immobilization of electrically charged proteins was strongly enhanced through electrostatic transport and subsequent molecular diffusion.

  12. A New Method for Immobilization of His-Tagged Proteins with the Application of Low-Frequency AC Electric Field

    Directory of Open Access Journals (Sweden)

    Shunsuke Takahashi

    2018-03-01

    Full Text Available Continued advancement of protein array, bioelectrode, and biosensor technologies is necessary to develop methods for higher amount and highly oriented immobilization activity of proteins. In pursuit of these goals, we developed a new immobilization method by combining electrostatic transport and subsequent molecular diffusion of protein molecules. Our developed immobilization method is based on a model that transports proteins toward the substrate surface due to steep concentration gradient generated by low-frequency AC electric field. The immobilization of the maximum amounts can be obtained by the application of the AC voltage of 80 Vpp, 20 Hz both for His-tagged Green Fluorescent Protein (GFP and Discosoma sp. Red Fluorescent Protein (DsRed, used as model proteins. The amounts of the immobilized His-tagged GFP and DsRed were approximately seven-fold higher than that in the absence of the application of low-frequency AC electric field. Furthermore, the positively and negatively charged His-tagged GFP at acidic and alkaline pH were immobilized by applying of low-frequency AC electric field, whereas the non-charged His-tagged GFP at the pH corresponding to its isoelectric point (pI was not immobilized. Therefore, unless the pH is equal to pI, the immobilization of electrically charged proteins was strongly enhanced through electrostatic transport and subsequent molecular diffusion.

  13. Ubiquitin-like prokaryotic MoaD as a fusion tag for expression of heterologous proteins in Escherichia coli.

    Science.gov (United States)

    Yuan, Sujuan; Wang, Xin; Xu, Jian; Yan, Zheng; Wang, Nan

    2014-01-21

    Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli. Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli. Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.

  14. Electrodeposition of polymer nanodots with controlled density and their reversible functionalization by polyhistidine-tag proteins.

    Science.gov (United States)

    Bazin, Damien; Chevalier, Sébastien; Saadaoui, Hassan; Santarelli, Xavier; Larpent, Chantal; Feracci, Hélène; Faure, Chrystel

    2012-10-02

    We present a simple and rapid procedure for producing polymer-coated substrates that can be easily functionalized by ion-chelating proteins. The procedure consists of depositing 18 nm metal-chelating cyclam-modified polymer nanoparticles (cyclam-nps) onto a conductive substrate (an Indium Tin Oxide (ITO) electrode) from an aqueous dispersion of Cu(2+)-loaded cyclam-nps while being subjected to a direct current (DC) field. The density of deposited nps as measured by AFM is shown to be in direct correlation to the concentration of nps in the dispersion with deposition of the particles taking less than 5 s. Because of the functionalization of the nps with cyclam groups, they can be used as anchoring sites for 6-Histidine (6-His) tagged proteins through complexation with divalent metal ions. In this work 6-His Green Fluorescent Protein (6-His GFP) is used as a model protein. The characterization by fluorescence microscopy clearly shows that the protein affinity was ion dependent and that the 6-His GFP density can be controlled by np density, which is itself easily tunable. AFM observations confirmed the immobilization of 6-His GFP onto cyclam-nps and its subsequent removal by treatment with ethylenediaminetetraacetic acid (EDTA).

  15. Kinetic Control of Histidine-Tagged Protein Surface Density on Supported Lipid Bilayers

    Energy Technology Data Exchange (ETDEWEB)

    Nye, Jeffrey A. [Univ. of California, Berkeley, CA (United States); Groves, Jay T. [Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2008-02-28

    Nickel-chelating lipids are general tools for anchoring polyhistidine-tagged proteins to supported lipid bilayers (SLBs), but controversy exists over the stability of the protein-lipid attachment. In this study, we show that chelator lipids are suitable anchors for building stable, biologically active surfaces but that a simple Langmuirian model is insufficient to describe their behavior. Desorption kinetics from chelator lipids are governed by the valency of surface binding: monovalently bound proteins desorb within minutes (t1/2 ≈ 6 min), whereas polyvalently bound species remain bound for hours (t1/2 ≈ 12 h). Evolution between surface states is slow, so equilibrium is unlikely to be reached on experimental timescales. However, by tuning incubation conditions, the populations of each species can be kinetically controlled, providing a wide range of protein densities on SLBs with a single concentration of chelator lipid. In conclusion, we propose guidelines for the assembly of SLB surfaces functionalized with specific protein densities and demonstrate their utility in the formation of hybrid immunological synapses.

  16. Construction of individual, fused, and co-expressed proteins of endoglucanase and β-glucosidase for hydrolyzing sugarcane bagasse.

    Science.gov (United States)

    Kurniasih, Sari Dewi; Alfi, Almasul; Natalia, Dessy; Radjasa, Ocky Karna; Nurachman, Zeily

    2014-01-01

    At least a combination of endoglucanase (EglII) and β-glucosidase (BglZ) is required for hydrolyzing crystalline cellulose. To understand the catalytic efficiency of combination enzymes for converting biomass to sugars, EglII and BglZ were constructed in the form of individual, fused as well as co-expression proteins, and their activities for hydrolyzing sugarcane bagasse were evaluated. The genes, eglII isolated from Bacillus amyloliquefaciens PSM3.1 earlier and bglZ from B. amyloliquefaciens ABBD, were expressed extracellularly in Bacillus megaterium MS941. EglII exhibited both exoglucanase and endoglucanase activities, and BglZ belonging to the glycoside hydrolase 1 family (GH 1) showed β-glucosidase activity. A combination of EglII and BglZ showed activity on substrates Avicel, CMC and sugarcane bagasse. Specifically for hydrolyzing sugarcane bagasse, fused protein (fus-EglII+BglZ), co-expression protein (coex-BglZ+EglII), and mixed-individual protein (mix-EglII+BglZ) produced cellobiose as the main product, along with a small amount of glucose. The amount of reducing sugars released from the hydrolyzing bleached sugarcane bagasse (BSB) using fus-EglII+BglZ and mix-EglII+BglZ was 2.7- and 4.2-fold higher, respectively, than steamed sugarcane bagasse (SSB), indicating the synergetic enzymes worked better on treated sugarcane bagasse. Compared with fus-EglII+BglZ and mix-EglII+BglZ, coex-BglZ+EglII released more mol reducing sugars from SSB, indicating the enzymes were potential for biomass conversion. Additionally, coex-BglZ+EglII acted on BSB 2.5-fold faster than fus-EglII+BglZ. Thus, coex-bglZ+eglII expression system was the best choice to produce enzymes for hydrolyzing sugarcane baggase. Copyright © 2014 Elsevier GmbH. All rights reserved.

  17. Facile fabrication of nickel immobilized on magnetic nanoparticles as an efficient affinity adsorbent for purification of his-tagged protein.

    Science.gov (United States)

    Rashid, Zahra; Naeimi, Hossein; Zarnani, Amir-Hassan; Mohammadi, Fereshteh; Ghahremanzadeh, Ramin

    2017-11-01

    In the present research, an efficient, convenient, and inexpensive method for the one-pot synthesis of Fe 3 O 4 @Histidine is developed. Histidine is readily loaded on magnetic nanoparticles by one step and simple method without any supplemental linkers. In the structure of Fe 3 O 4 @Histidine, histidine covalently immobilized on the surface of Fe 3 O 4 , magnetic nanoparticles are able to trap Ni 2+ ions through a strong interaction between nickel and histidines in protein tag. Two coordination sites of nickel are occupied with ligand on the surface of magnetic nanoparticles and four coordination sites have been remained that these sites will be occupied with histidine tag of recombinant protein A. The functionalized nanoparticles were spherical and well separated with an average diameter around 30nm. The obtained magnetic nanoparticles have a saturation magnetization of about 54emu/g. Fe 3 O 4 @Histidine-Ni was used to enrich and purify 6×histidine-tagged recombinant protein-A directly from the mixture of lysed cells. It has been found that Ni(II)-immobilized Fe 3 O 4 @Histidine magnetic nanoparticles present negligible nonspecific protein adsorption and high His-tag protein binding capacity The average binding capacity (MW 42k Da), is 700±25μg·mg -1 (protein/Fe 3 O 4 @Histidine-Ni). Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis.

    Science.gov (United States)

    Neef, Jolanda; Milder, Fin J; Koedijk, Danny G A M; Klaassens, Marindy; Heezius, Erik C; van Strijp, Jos A G; Otto, Andreas; Becher, Dörte; van Dijl, Jan Maarten; Buist, Girbe

    2015-11-01

    Recent studies have shown that the Gram-positive bacterium Lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized metal affinity chromatography was so far lacking. Here we describe three novel vectors that, respectively, facilitate the nisin-inducible production of N- or C-terminally hexa-histidine (His6)-tagged proteins in L. lactis. One of these vectors also encodes a tobacco etch virus (TEV) protease cleavage site allowing removal of the N-terminal His6-tag from expressed proteins. Successful application of the developed vectors for protein expression, purification and/or functional studies is exemplified with six different cell wall-bound or secreted proteins from Staphylococcus aureus. The results show that secretory production of S. aureus proteins is affected by the position, N- or C-terminal, of the His6-tag. This seems to be due to an influence of the His6-tag on protein stability. Intriguingly, the S. aureus IsdB protein, which is phosphorylated in S. aureus, was also found to be phosphorylated when heterologously produced in L. lactis, albeit not on the same Tyr residue. This implies that this particular post-translational protein modification is to some extent conserved in S. aureus and L. lactis. Altogether, we are confident that the present vector set combined with the L. lactis expression host has the potential to become a very useful tool in optimization of the expression, purification and functional analysis of extracytoplasmic bacterial proteins.

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

    Directory of Open Access Journals (Sweden)

    Buhrman Jason S

    2012-09-01

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

  20. Sensitive Western-Blot Analysis of Azide-Tagged Protein Post Translational Modifications Using Thermoresponsive Polymer Self-Assembly.

    Science.gov (United States)

    Liu, Tong; Zhang, Wanjun; Zhang, Zheng; Chen, Mingli; Wang, Jianhua; Qian, Xiaohong; Qin, Weijie

    2018-02-06

    Western-blot (WB) is a powerful analytical technique for protein identification in complex biological samples and has been widely used in biological studies for decades. Detection specificity and sensitivity of WB largely relies on quality of the antibodies and performance of the conjugated HRP. However, the application of WB analysis for the detection of protein post-translational modifications (PTMs) is hampered by the low abundance of protein PTMs and by the limited availability of antibodies that specifically differentiate various kinds of PTMs from their protein substrates. Therefore, new recognition mechanisms and signal amplification strategies for WB analysis of protein PTMs is in high demand. In this work, we prepared a soluble polymer that detects various azide-tagged PTM proteins in WB analysis using triarylphosphine and HRP modified thermoresponsive polymer. Specific and efficient detection of azide-tagged PTM protein is achieved via the bioorthogonal reaction between azide and triarylphosphine. More importantly, the chemiluminiscent signal in the WB analysis is largely amplified by the temperature induced self-assembly of numerous thermoresponsive polymer chains carrying multiple HRPs. As a result, approximately 100 times more sensitive detection than commercial antibodies is achieved by this method using standard PTM proteins. Though, this new reagent does not directly detect native PTMs in cell, tissue or blood samples, it still has important application potential in protein PTM studies, considering the wide availability of azide-tagging techniques to a variety of PTMs.

  1. Do-it-yourself histidine-tagged bovine enterokinase: A handy member of the protein engineer's toolbox☆

    Science.gov (United States)

    Skala, Wolfgang; Goettig, Peter; Brandstetter, Hans

    2013-01-01

    Enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its N-terminal propeptide. Due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate N-terminal affinity tags from target proteins with authentic N-termini. In order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase light chain. The purified, highly active enzyme successfully processed recombinant target proteins, while the pentahistidine-tag facilitated post-cleavage removal. Hence, we conclude that producing enterokinase in one's own laboratory is an efficient alternative to the commercial enzyme. PMID:24184090

  2. Dissection of an old protein reveals a novel application: domain D of Staphylococcus aureus Protein A (sSpAD as a secretion - tag

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    Paal Michael

    2010-11-01

    Full Text Available Abstract Background Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous. Results We discovered that domain D of the Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation. The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD. Conclusions In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.

  3. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    Science.gov (United States)

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag.

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    Hiroyuki Takeda

    Full Text Available There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1 rabbit monoclonal antibody clone Ra62 (Ra62 antibody as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence. We found that single amino acid substitution in D1CE sequence (GQHVT-COOH increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs, 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with

  5. LDRD final report on imaging self-organization of proteins in membranes by photocatalytic nano-tagging.

    Energy Technology Data Exchange (ETDEWEB)

    Zavadil, Kevin Robert; Shelnutt, John Allen; Sasaki, Darryl Yoshio; Song, Yujiang; Medforth, Craig J.

    2005-11-01

    We have developed a new nanotagging technology for detecting and imaging the self-organization of proteins and other components of membranes at nanometer resolution for the purpose of investigating cell signaling and other membrane-mediated biological processes. We used protein-, lipid-, or drug-bound porphyrin photocatalysts to grow in-situ nanometer-sized metal particles, which reveal the location of the porphyrin-labeled molecules by electron microscopy. We initially used photocatalytic nanotagging to image assembled multi-component proteins and to monitor the distribution of lipids and porphyrin labels in liposomes. For example, by exchanging the heme molecules in hemoproteins with a photocatalytic tin porphyrin, a nanoparticle was grown at each heme site of the protein. The result obtained from electron microscopy for a tagged multi-subunit protein such as hemoglobin is a symmetric constellation of a specific number of nanoparticle tags, four in the case of the hemoglobin tetramer. Methods for covalently linking photocatalytic porphyrin labels to lipids and proteins were also developed to detect and image the self-organization of lipids, protein-protein supercomplexes, and membrane-protein complexes. Procedures for making photocatalytic porphyrin-drug, porphyrin-lipid, and porphyrin-protein hybrids for non-porphyrin-binding proteins and membrane components were pursued and the first porphyrin-labeled lipids was investigated in liposomal membrane models. Our photocatalytic nanotagging technique may ultimately allow membrane self-organization and cell signaling processes to be imaged in living cells. Fluorescence and plasmonic spectra of the tagged proteins might also provide additional information about protein association and membrane organization. In addition, a porphyrin-aspirin or other NSAID hybrid may be used to grow metal nanotags for the pharmacologically important COX enzymes in membranes so that the distribution of the protein can be imaged at the

  6. Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy

    Science.gov (United States)

    Risco, Cristina; Sanmartín-Conesa, Eva; Tzeng, Wen-Pin; Frey, Teryl K.; Seybold, Volker; de Groot, Raoul J.

    2012-01-01

    Summary More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell’s interior are lagging behind. We describe a novel approach, metal-tagging TEM (METTEM) that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before. PMID:22579245

  7. A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags.

    Science.gov (United States)

    Söveges, B; Imre, T; Szende, T; Póti, Á L; Cserép, G B; Hegedűs, T; Kele, P; Németh, K

    2016-07-07

    Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.

  8. Prediction of Apoptosis Protein's Subcellular Localization by Fusing Two Different Descriptors Based on Evolutionary Information.

    Science.gov (United States)

    Liang, Yunyun; Zhang, Shengli

    2018-03-12

    The apoptosis protein has a central role in the development and the homeostasis of an organism. Obtaining information about the subcellular localization of apoptosis protein is very helpful to understand the apoptosis mechanism and the function of this protein. Prediction of apoptosis protein's subcellular localization is a challenging task, and currently the existing feature extraction methods mainly rely on the protein's primary sequence. In this paper we develop a feature extraction model based on two different descriptors of evolutionary information, which contains the 192 frequencies of triplet codons (FTC) in the RNA sequence derived from the protein's primary sequence and the 190 features from a detrended forward moving-average cross-correlation analysis (DFMCA) based on a position-specific scoring matrix (PSSM) generated by the PSI-BLAST program. Hence, this model is called FTC-DFMCA-PSSM. A 382-dimensional (382D) feature vector is constructed on the ZD98, ZW225 and CL317 datasets. Then a support vector machine is adopted as classifier, and the jackknife cross-validation test method is used for evaluating the accuracy. The overall prediction accuracies are further improved by an objective and rigorous jackknife test. Our model not only broadens the source of the feature information, but also provides a more accurate and reliable automated calculation method for the prediction of apoptosis protein's subcellular localization.

  9. Electrochemical impedance spectroscopy for black lipid membranes fused with channel protein supported on solid-state nanopore.

    Science.gov (United States)

    Khan, Muhammad S; Dosoky, Noura S; Berdiev, Bakhrom K; Williams, John D

    2016-12-01

    Black lipid membranes (BLMs) have been used for detecting single-channel activities of pore-forming peptides and ion channels. However, the short lifetimes and poor mechanical stability of suspended bilayers limit their applications in high throughput electrophysiological experiments. In this work, we present a synthetic solid-state nanopore functionalized with BLM fused with channel protein. A nanopore with diameter of ~180 nm was electrochemically fabricated in a thin silicon membrane. Folding and painting techniques were demonstrated for production of stable suspended BLMs followed by incorporation of transmembrane protein, ENaC. Membrane formation was confirmed by employing electrochemical impedance spectroscopy (EIS) in the frequency regime of 10 -2 -10 5  Hz. Results show that electrochemically fabricated solid state nanopore support resulted in excellent membrane stability, with >1 GΩ of up to 72 and 41 h for painting and folding techniques, respectively. After fusion of ENaC channel protein, the BLM exhibits the stability of ~5 h. We anticipate that such a solid-state nanopore with diameter in the range of 150-200 nm and thickness <1 µm could be a potential platform to enhance the throughput of ion-channel characterization using BLMs.

  10. A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Hikone, Yuya [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan); Hirai, Go [RIKEN, Synthetic Organic Chemistry Laboratory (Japan); Mishima, Masaki [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan); Inomata, Kohsuke [RIKEN, Quantitative Biology Center (Japan); Ikeya, Teppei; Arai, Souichiro [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan); Shirakawa, Masahiro [Japan Agency for Medical Research and Development, AMED-CREST (Japan); Sodeoka, Mikiko [RIKEN, Synthetic Organic Chemistry Laboratory (Japan); Ito, Yutaka, E-mail: ito-yutaka@tmu.ac.jp [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan)

    2016-10-15

    Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N–H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

  11. A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells

    International Nuclear Information System (INIS)

    Hikone, Yuya; Hirai, Go; Mishima, Masaki; Inomata, Kohsuke; Ikeya, Teppei; Arai, Souichiro; Shirakawa, Masahiro; Sodeoka, Mikiko; Ito, Yutaka

    2016-01-01

    Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N–H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

  12. Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos-tag SDS-PAGE migration.

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Karata, Kiyonobu; Kawano, Toshiki; Nishiyama, Atsuhiro; Yamato, Morihisa; Koike, Tohru

    2017-04-01

    We describe two unique proteins, Escherichia coli ClpX and human histone H2A, that show extremely retarded migrations relative to their molecular weights in Phos-tag SDS-PAGE, despite being nonphosphorylated. Although ClpX separated into multiple migration bands in Phos-tag gels, the separation was not due to phosphorylation. The N-terminal 47-61 region of ClpX was responsible for producing multiple phosphorylation-independent structural variants, even under denaturing conditions, and some of these variants were detected as highly up-shifted bands. By systematic Ala-scanning mutation analysis in the N-47-61 region, we concluded that the Glu-51 or Glu-54 residue was responsible for the appearance of exaggerated mobility-shifting bands. Histone H2A showed a much slower migration in Phos-tag gels in comparison with other major histones having similar molecular weights, and we found that the Glu-62 or Glu-65 residue caused the retarded migration. In addition, Phos-tag SDS-PAGE permitted us to detect a shift in the mobility of the phosphorylated form of histone H2A from that of the nonphosphorylated one. This is the first report showing that exaggerated retardation in the migration of a certain protein in Phos-tag SDS-PAGE is induced by interactions between the Phos-tag molecule and the carboxylate group of a specific Glu residue on the target. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Development of a novel fluorescent protein construct by genetically fusing green fluorescent protein to the N-terminal of aspartate dehydrogenase.

    Science.gov (United States)

    Ozyurt, Canan; Evran, Serap; Telefoncu, Azmi

    2013-01-01

    We developed a fluorescent protein construct by genetically fusing green fluorescent protein (GFP) to aspartate dehydrogenase from Thermotoga maritima. The fusion protein was cloned, heterologously expressed in Escherichia coli cells, and purified by Ni-chelate affinity chromatography. It was then introduced into a measurement cuvette to monitor its fluorescence signal. Aspartate dehydrogenase functioned as the biorecognition element, and aspartate-induced conformational change was converted to a fluorescence signal by GFP. The recombinant protein responded to l-aspartate (l-Asp) linearly within the concentration range of 1-50 mM, and it was capable of giving a fluorescence signal in 1 Min. Although a linear response was also observed for l-Glu, the fluorescence signal was 2.7 times lower than that observed for l-Asp. In the present study, we describe two novelties: development of a genetically encoded fluorescent protein construct for monitoring of l-Asp in vitro, and employment of aspartate dehydrogenase scaffold as a biorecognition element. A few genetically encoded amino-acid biosensors have been described in the literature, but to our knowledge, a protein has not been constructed solely for determination of l-Asp. Periplasmic ligand binding proteins offer high binding affinity in the micromolar range, and they are frequently used as biorecognition elements. Instead of choosing a periplasmic l-Asp binding protein, we attempted to use the substrate specificity of aspartate dehydrogenase enzyme. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  14. QuatIdent: a web server for identifying protein quaternary structural attribute by fusing functional domain and sequential evolution information.

    Science.gov (United States)

    Shen, Hong-Bin; Chou, Kuo-Chen

    2009-03-01

    Many proteins exist in vivo as oligomers with various different quaternary structural attributes rather than as single individual chains. They are the structural bases of various marvelous biological functions such as cooperative effects, allosteric mechanism, and ion-channel gating. Therefore, with the avalanche of protein sequences generated in the postgenomic era, it is very important for both basic research and drug discovery to identify their quaternary structural attributes in a timely manner. In view of this, a powerful ensemble identifier, called QuatIdent, is developed by fusing the functional domain and sequential evolution information. QuatIdent is a 2-layer predictor. The 1st layer is for identifying a query protein as belonging to which one of the following 10 main quaternary structural attributes: (1) monomer, (2) dimer, (3) trimer, (4) tetramer, (5) pentamer, (6) hexamer, (7) heptamer, (8) octamer, (9) decamer, and (10) dodecamer. If the result thus obtained turns out to be anything but monomer, the process will be automatically continued to further identify it as belonging to a homo-oligomer or hetero-oligomer. The overall success rate by QuatIdent for the 1st layer identification was 71.1% and that for the 2nd layer ranged from 84 to 96%. These rates were derived by the jackknife cross-validation tests on the stringent benchmark data sets where none of proteins has > or =60% pairwise sequence identity to any other in a same subset. QuatIdent is freely accessible to the public as a web server via the site at http://www.csbio.sjtu.edu.cn/bioinf/Quaternary/ , by which one can get the desired 2-level results for a query protein sequence in around 25 seconds. The longer the sequence is, the more time that is needed.

  15. Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

    Directory of Open Access Journals (Sweden)

    Vuento Matti

    2006-12-01

    Full Text Available Abstract Fluorescence correlation spectroscopy (FCS monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids were fused to the C-terminus of the enhanced green fluorescent protein (EGFP. The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs. The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

  16. Novel fused tetrathiocines as antivirals that target the nucleocapsid zinc finger containing protein of the feline immunodeficiency virus (FIV) as a model of HIV infection.

    Science.gov (United States)

    Asquith, Christopher R M; Meli, Marina L; Konstantinova, Lidia S; Laitinen, Tuomo; Poso, Antti; Rakitin, Oleg A; Hofmann-Lehmann, Regina; Allenspach, Karin; Hilton, Stephen T

    2015-03-15

    A novel series of fused tetrathiocines were prepared for evaluation of activity against the nucleocapsid protein of the feline immunodeficiency virus (FIV) in an in vitro cell culture approach. The results demonstrated that the compounds display potent nanomolar activity and low toxicity against this key model of HIV infection. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  17. Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag.

    Directory of Open Access Journals (Sweden)

    Minh Tan Nguyen

    Full Text Available Human vascular endothelial growth factor (VEGF is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF. We created seven N-terminal fusion tag constructs with hexahistidine (His6, thioredoxin (Trx, glutathione S-transferase (GST, maltose-binding protein (MBP, N-utilization substance protein A (NusA, human protein disulfide isomerase (PDI, and the b'a' domain of PDI (PDIb'a', and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.

  18. Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

    Science.gov (United States)

    Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

    2017-05-01

    Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

  19. Large-scale identification of odorant-binding proteins and chemosensory proteins from expressed sequence tags in insects

    Directory of Open Access Journals (Sweden)

    Zhang Yong-Jun

    2009-12-01

    Full Text Available Abstract Background Insect odorant binding proteins (OBPs and chemosensory proteins (CSPs play an important role in chemical communication of insects. Gene discovery of these proteins is a time-consuming task. In recent years, expressed sequence tags (ESTs of many insect species have accumulated, thus providing a useful resource for gene discovery. Results We have developed a computational pipeline to identify OBP and CSP genes from insect ESTs. In total, 752,841 insect ESTs were examined from 54 species covering eight Orders of Insecta. From these ESTs, 142 OBPs and 177 CSPs were identified, of which 117 OBPs and 129 CSPs are new. The complete open reading frames (ORFs of 88 OBPs and 123 CSPs were obtained by electronic elongation. We randomly chose 26 OBPs from eight species of insects, and 21 CSPs from four species for RT-PCR validation. Twenty two OBPs and 16 CSPs were confirmed by RT-PCR, proving the efficiency and reliability of the algorithm. Together with all family members obtained from the NCBI (OBPs or the UniProtKB (CSPs, 850 OBPs and 237 CSPs were analyzed for their structural characteristics and evolutionary relationship. Conclusions A large number of new OBPs and CSPs were found, providing the basis for deeper understanding of these proteins. In addition, the conserved motif and evolutionary analysis provide some new insights into the evolution of insect OBPs and CSPs. Motif pattern fine-tune the functions of OBPs and CSPs, leading to the minor difference in binding sex pheromone or plant volatiles in different insect Orders.

  20. Large-scale identification of odorant-binding proteins and chemosensory proteins from expressed sequence tags in insects

    Science.gov (United States)

    2009-01-01

    Background Insect odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play an important role in chemical communication of insects. Gene discovery of these proteins is a time-consuming task. In recent years, expressed sequence tags (ESTs) of many insect species have accumulated, thus providing a useful resource for gene discovery. Results We have developed a computational pipeline to identify OBP and CSP genes from insect ESTs. In total, 752,841 insect ESTs were examined from 54 species covering eight Orders of Insecta. From these ESTs, 142 OBPs and 177 CSPs were identified, of which 117 OBPs and 129 CSPs are new. The complete open reading frames (ORFs) of 88 OBPs and 123 CSPs were obtained by electronic elongation. We randomly chose 26 OBPs from eight species of insects, and 21 CSPs from four species for RT-PCR validation. Twenty two OBPs and 16 CSPs were confirmed by RT-PCR, proving the efficiency and reliability of the algorithm. Together with all family members obtained from the NCBI (OBPs) or the UniProtKB (CSPs), 850 OBPs and 237 CSPs were analyzed for their structural characteristics and evolutionary relationship. Conclusions A large number of new OBPs and CSPs were found, providing the basis for deeper understanding of these proteins. In addition, the conserved motif and evolutionary analysis provide some new insights into the evolution of insect OBPs and CSPs. Motif pattern fine-tune the functions of OBPs and CSPs, leading to the minor difference in binding sex pheromone or plant volatiles in different insect Orders. PMID:20034407

  1. Efficient expression of codon-adapted affinity tagged super folder green fluorescent protein for synchronous protein localization and affinity purification studies in Tetrahymena thermophila.

    Science.gov (United States)

    Yilmaz, Gürkan; Arslanyolu, Muhittin

    2015-03-25

    A superior Green Fluorescent Protein (GFP) mutant, known as superfolder GFP (sfGFP), is more soluble, faster folding, and is the brightest of the known GFP mutants. This study aimed to create a codon-adapted sfGFP tag (TtsfGFP) for simultaneous protein localization and affinity purification in Tetrahymena thermophila. In vivo fluorescence spectroscopic analyses of clones carrying a codon-adapted and 6 × His tagged TtsfGFP cassette showed approximately 2-4-fold increased fluorescence emission compared with the control groups at 3 h. Fluorescence microscopy also revealed that TtsfGFP reached its emission maxima at 100 min, which was much earlier than controls expressing EGFP and sfGFP (240 min). A T. thermophila ATP-dependent DNA ligase domain containing hypothetical gene (H) was cloned into the 3' end of 6 × His-TtsfGFP to assess the affinity/localization dual tag feature. Fluorescence microscopy of the 6 × His-TtsfGFP-H clone confirmed its localization in the macro- and micronucleus of vegetative T. thermophila. Simultaneous affinity purification of TtsfGFP and TtsfGFP-H with Ni-NTA beads was feasible, as shown by Ni-NTA purified proteins analysis by SDS-PAGE and western blotting. We successfully codon adapted the N-terminal 6 × His-TtsfGFP tag and showed that it could be used for protein localization and affinity purification simultaneously in T. thermophila. We believe that this dual tag will advance T. thermophila studies by providing strong visual traceability of the target protein in vivo and in vitro during recombinant production of heterologous and homologous proteins.

  2. Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins

    International Nuclear Information System (INIS)

    Renzi, Fabiana; Panetta, Gianna; Vallone, Beatrice; Brunori, Maurizio; Arceci, Massimo; Bozzoni, Irene; Laneve, Pietro; Caffarelli, Elisa

    2006-01-01

    Recombinant His-tagged XendoU, a eukaryotic endoribonuclease, appeared to aggregate in the presence of divalent cations. Monodisperse protein which yielded crystals diffracting to 2.2 Å was obtained by addition of EDTA. XendoU is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar RNAs. It is conserved among eukaryotes and its viral homologue is essential in SARS replication and transcription. The large-scale purification and crystallization of recombinant XendoU are reported. The tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (EDTA, imidazole): aggregation is a potential drawback when purifying and crystallizing His-tagged proteins, which are widely used, especially in high-throughput structural studies. Purified monodisperse XendoU crystallized in two different space groups: trigonal P3 1 21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution

  3. Metal ions-binding T4 lysozyme as an intramolecular protein purification tag compatible with X-ray crystallography

    Czech Academy of Sciences Publication Activity Database

    Bouřa, Evžen; Bäumlová, Adriana; Chalupská, Dominika; Dubánková, Anna; Klíma, Martin

    2017-01-01

    Roč. 26, č. 6 (2017), s. 1116-1123 ISSN 0961-8368 R&D Projects: GA ČR(CZ) GJ17-07058Y; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : phage T4 * lysozyme * endolysin * histidine tag * protein purification * crystal structure Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 2.523, year: 2016

  4. N-Terminal His-Tagged AtTPR7 Interactions with Hsp70 and Hsp90 Proteins

    Directory of Open Access Journals (Sweden)

    ANANDAYU PRADITA

    2014-12-01

    Full Text Available Post-translational protein import into organelles is an important process to maintain cellular functions. During preprotein transport in the cytosol, chaperones, such as heat shock protein 70 (Hsp70 and heat shock protein 90 (Hsp90, are functioning to prevent aggregation and to maintain the correct protein folding of preproteins. This research was conducted in order to understand the chaperone-mediated, post-translational import of preproteins into the endoplasmic reticulum of Arabidopsis thaliana. AtTPR7 (Arabidopsis thaliana Tetratrico Peptide Repeat 7 is found in the endoplasmic reticulum and contains TPR domain, which mediates protein interaction with cytosolic Hsp70 and Hsp90. In this study, recombinant AtTPR7 was expressed in E. coli BL21 (DE3-RIPL cells and purified using an N-terminal His-tag. In order to study the interactions of the protein with the chaperones, we used pulldown and Western blot assays. We could thereby show that the N-terminally His-tagged AtTPR7 protein interacted with Hsp70 and Hsp90.

  5. Towards the use of protein A-tagged gold nanoparticles for signal amplification of electrochemical immunosensors in virus detection

    International Nuclear Information System (INIS)

    Huy Tran, Quang; Thuy Nguyen, Thanh; Chung Pham, Van; Hong Hanh Nguyen, Thi; Tuan Mai, Anh

    2012-01-01

    In this paper we represent a study on the potential use of protein A-tagged gold nanoparticles applied for signal amplification of electrochemical immunosensors. Gold nanoparticles (GNPs) were synthesized by the chemical reduction of tetrachloroauric (III) acid trihydrate using sodium ascorbate, and then tagged with protein A (PrA) via ultracentrifugation. UV-Vis spectroscopy and transmission electron microscopy were used to verify the characteristics of formed GNPs/PrA complex. The analyzed results indicate that GNPs were found spherically, homogeneously, and with an average diameter of about 10 nm. Immunoelectron microscopy was then used to investigate the bioactivity of the GNPs/PrA complex in solution by the effective binding of GNPs to viral particles. Scanning electron and fluorescence microscopies were also used to investigate the distribution and the bioactivity of the GNPs/PrA complex on the surface of the interdigitated sensor. Consequently, this study provided some assumptions of the potential application of protein A-tagged gold nanoparticles for signal amplification of electrochemical immunosensors in virus detection from clinical samples

  6. Immunogenicity of recombinant proteins consisting of Plasmodium vivax circumsporozoite protein allelic variant-derived epitopes fused with Salmonella enterica Serovar Typhimurium flagellin.

    Science.gov (United States)

    Leal, Monica Teixeira Andrade; Camacho, Ariane Guglielmi Ariza; Teixeira, Laís Helena; Bargieri, Daniel Youssef; Soares, Irene Silva; Tararam, Cibele Aparecida; Rodrigues, Mauricio M

    2013-09-01

    A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax.

  7. Billfish Tagging

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The SWFSC's constituent-based Billfish Tagging Program began in 1963 and since that time has provided conventional spaghetti type tags and tagging supplies to...

  8. Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex.

    Science.gov (United States)

    Kobayashi, Wataru; Sekine, Satoshi; Machida, Shinichi; Kurumizaka, Hitoshi

    2014-01-01

    Green fluorescent protein (GFP), fused to the N or C terminus of a protein of interest, is widely used to monitor the localization and mobility of proteins in cells. RAD51 is an essential protein that functions in mitotic DNA repair and meiotic chromosome segregation by promoting the homologous recombination reaction. A previous genetic study with Arabidopsis thaliana revealed that GFP fused to the C terminus of RAD51 (RAD51-GFP) inhibits mitotic DNA repair, but meiotic homologous recombination remained unaffected. To determine how the C-terminal GFP specifically inhibits mitotic DNA repair by RAD51, we purified rice RAD51A1-GFP and RAD51A2-GFP, and performed biochemical analyses. Interestingly, purified RAD51A1-GFP and RAD51A2-GFP are proficient in DNA binding and ATP hydrolysis. However, nucleoprotein complexes containing single-stranded DNA and RAD51A1-GFP or RAD51A2-GFP are significantly defective in binding to the second DNA molecule (secondary DNA binding), and consequently fail to catalyze homologous pairing. In contrast, RAD51A1-GFP and RAD51A2-GFP efficiently stimulated homologous pairing promoted by the meiosis-specific RAD51 isoform DMC1. These biochemical characteristics are well conserved in human RAD51-GFP. Therefore, GFP fused to the C terminus of RAD51 abolishes the homologous pairing activity of RAD51 by disrupting secondary DNA binding, but does not affect its DMC1-stimulating activity.

  9. The Isotope-Coded Affinity Tag Method for Quantitative Protein Profile Comparison and Relative Quantitation of Cysteine Redox Modifications.

    Science.gov (United States)

    Chan, James Chun Yip; Zhou, Lei; Chan, Eric Chun Yong

    2015-11-02

    The isotope-coded affinity tag (ICAT) technique has been applied to measure pairwise changes in protein expression through differential stable isotopic labeling of proteins or peptides followed by identification and quantification using a mass spectrometer. Changes in protein expression are observed when the identical peptide from each of two biological conditions is identified and a difference is detected in the measurements comparing the peptide labeled with the heavy isotope to the one with a normal isotopic distribution. This approach allows the simultaneous comparison of the expression of many proteins between two different biological states (e.g., yeast grown on galactose versus glucose, or normal versus cancer cells). Due to the cysteine-specificity of the ICAT reagents, the ICAT technique has also been applied to perform relative quantitation of cysteine redox modifications such as oxidation and nitrosylation. This unit describes both protein quantitation and profiling of cysteine redox modifications using the ICAT technique. Copyright © 2015 John Wiley & Sons, Inc.

  10. Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C

    DEFF Research Database (Denmark)

    Kristensen, B; Birkelund, Svend; Jørgensen, PL

    2003-01-01

    Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent...... along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein...... chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly...

  11. Fusing two cytochromes b of Rhodobacter capsulatus cytochrome bc1 using various linkers defines a set of protein templates for asymmetric mutagenesis.

    Science.gov (United States)

    Czapla, Monika; Borek, Arkadiusz; Sarewicz, Marcin; Osyczka, Artur

    2012-01-01

    Cytochrome bc(1) (mitochondrial complex III), one of the key enzymes of biological energy conversion, is a functional homodimer in which each monomer contains three catalytic subunits: cytochrome c(1), the iron-sulfur subunit and cytochrome b. The latter is composed of eight transmembrane α-helices which, in duplicate, form a hydrophobic core of a dimer. We show that two cytochromes b can be fused into one 16-helical subunit using a number of different peptide linkers that vary in length but all connect the C-terminus of one cytochrome with the N-terminus of the other. The fusion proteins replace two cytochromes b in the dimer defining a set of available protein templates for introducing mutations that allow breaking symmetry of a dimer. A more detailed comparison of the form with the shortest, 3 amino acid, linker to the form with 12 amino acid linker established that both forms display similar level of structural plasticity to accommodate several, but not all, asymmetric patterns of mutations that knock out individual segments of cofactor chains. While the system based on a fused gene does not allow for the assessments of the functionality of electron-transfer paths in vivo, the family of proteins with fused cytochrome b offers attractive model for detailed investigations of molecular mechanism of catalysis at in vitro/reconstitution level.

  12. Subcellular localization of proteins in the anaerobic sulfate reducer Desulfovibrio vulgaris via SNAP-tag labeling and photoconversion

    Energy Technology Data Exchange (ETDEWEB)

    Gorur, A.; Leung, C. M.; Jorgens, D.; Tauscher, A.; Remis, J. P.; Ball, D. A.; Chhabra, S.; Fok, V.; Geller, J. T.; Singer, M.; Hazen, T. C.; Juba, T.; Elias, D.; Wall, J.; Biggin, M.; Downing, K. H.; Auer, M.

    2010-06-01

    Systems Biology studies the temporal and spatial 3D distribution of macromolecular complexes with the aim that such knowledge will allow more accurate modeling of biological function and will allow mathematical prediction of cellular behavior. However, in order to accomplish accurate modeling precise knowledge of spatial 3D organization and distribution inside cells is necessary. And while a number of macromolecular complexes may be identified by its 3D structure and molecular characteristics alone, the overwhelming number of proteins will need to be localized using a reporter tag. GFP and its derivatives (XFPs) have been traditionally employed for subcelllar localization using photoconversion approaches, but this approach cannot be taken for obligate anaerobic bacteria, where the intolerance towards oxygen prevents XFP approaches. As part of the GTL-funded PCAP project (now ENIGMA) genetic tools have been developed for the anaerobe sulfate reducer Desulfovibrio vulgaris that allow the high-throughput generation of tagged-protein mutant strains, with a focus on the commercially available SNAP-tag cell system (New England Biolabs, Ipswich, MA), which is based on a modified O6-alkylguanine-DNA alkyltransferase (AGT) tag, that has a dead-end reaction with a modified O6-benzylguanine (BG) derivative and has been shown to function under anaerobic conditions. After initial challenges with respect to variability, robustness and specificity of the labeling signal we have optimized the labeling. Over the last year, as a result of the optimized labeling protocol, we now obtain robust labeling of 20 out of 31 SNAP strains. Labeling for 13 strains were confirmed at least five times. We have also successfully performed photoconversion on 5 of these 13 strains, with distinct labeling patterns for different strains. For example, DsrC robustly localizes to the periplasmic portion of the inner membrane, where as a DNA-binding protein localizes to the center of the cell, where the

  13. Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins

    DEFF Research Database (Denmark)

    Lambertsen, L.; Sternberg, Claus; Molin, Søren

    2004-01-01

    the Escherichia coli lac derived promoter, P-A1/04/03, or from the growth-rate-dependent Escherichia coli promoter P-rrnB P1. The mini-Tn7 transposons were inserted and tested in the soil bacterium, Pseudomonas putida KT2440. Successful and site-specific tagging was verified by Southern blots as well as by PCR...

  14. Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-space Image Correlation Spectroscopy

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Koffman, Jennifer Skaarup; Marlar, Saw

    2014-01-01

    Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1 was developed to enable...... routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely...... to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope Then...

  15. Using Haloarcula marismortui bacteriorhodopsin as a fusion tag for enhancing and visible expression of integral membrane proteins in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Min-Feng Hsu

    Full Text Available Membrane proteins are key targets for pharmacological intervention because of their vital functions. Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins. Here we generate a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR from Haloarcula marismortui (HmBRI/D94N as a fusion partner. A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products. The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption. In this study, we applied this approach to produce two functional integral membrane proteins, undecaprenyl pyrophosphate phosphatase and carnitine/butyrobetaine antiporter with significant yield enhancement. This technology could facilitate the development of a high-throughput strategy to screen for conditions that improve the yield of correctly folded target membrane proteins. Other robust BRs can also be incorporated in this system.

  16. Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

    Science.gov (United States)

    Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

    2015-12-19

    The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

  17. Preparation of nitrilotriacetic acid/Co2+-linked, silica/boron-coated magnetite nanoparticles for purification of 6 x histidine-tagged proteins.

    Science.gov (United States)

    Liao, Yiqun; Cheng, Yangjian; Li, Qingge

    2007-03-02

    In this report, we describe the preparation of novel nitrilotriacetic acid/Co2+-linked, silica/boron-coated magnetite nanoparticles for purification of 6 x His-tagged proteins. The nanoparticles were approximately 200 nm in size and were stable against hydrochloric acid and had negligible non-specific binding for protein. Elimination of non-specific binding by nucleic acids was readily achieved by digestion of samples with DNase and RNase. The modified nanoparticles were used to purify two model proteins: one had a C-terminal 6 x His tag, and the other had an internal 6 x His tag. Both proteins were purified within one hour into single band purity on sodium dodecyl sulfate-polyacrylamide electrophoresis gel.

  18. Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

    Science.gov (United States)

    Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

    2014-07-18

    This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins : a wash step with a low concentration of EDTA

    NARCIS (Netherlands)

    Westra, DF; Welling, GW; Koedijk, DGAM; Scheffer, AJ; The, TH; Welling-Wester, S

    2001-01-01

    Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for

  20. Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins andCas9mRNA for Genome Editing in Zebrafish.

    Science.gov (United States)

    Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

    2018-01-02

    CRISPR/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N, C, or both N and C termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7E1 assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that high frequency of indels occurred in the target sites ( tyr1 >63%, tyr2 >74%, gol1 >50% and gol2 >35%), as well as various types ( > 6 ) of indel mutations observed in all four types of Cas9 injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNA have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on gRNAs and gene loci These findings may help to simplify selection of Cas9 for gene editing using the CRISPR/Cas9 system. Copyright © 2018, G3: Genes, Genomes, Genetics.

  1. Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish

    Science.gov (United States)

    Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

    2018-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites (tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. PMID:29295818

  2. Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish

    Directory of Open Access Journals (Sweden)

    Peinan Hu

    2018-03-01

    Full Text Available The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1 assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites (tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%, as well as various types (more than six of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system.

  3. The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag

    Directory of Open Access Journals (Sweden)

    Selma Djender

    2014-04-01

    Full Text Available We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag is tightly bound by a commercial antibody when expressed at the C-term end of a polypeptide. The antigen is released in the presence of 2 M MgCl2. Consequently, constructs fused to the 6xHis-C tags can be purified by two successive and orthogonal affinity steps. Single-domain antibodies were produced either in the periplasmic or in the cytoplasmic space of E. coli. Surprisingly, the first affinity purification step performed using the EPEA-binding resin already yielded homogeneous proteins. The presence of the C tag did not interfere with the binding activity of the antibodies, as assessed by FACS and SPR analyses, and the C tag was extremely effective for immunoprecipitating HER2 receptor. Finally, the Alexa488-coupled anti-C tag allowed for simplification of FACS and IF analyses. These results show that a tag of minimal dimensions can be effectively used to improve the applicability of recombinant antibodies as reagents. In our hands, C tag was superior to His-tag in affinity purification and pull-down experiments, and practical in any other standard immune technique.

  4. Classification, expression pattern and comparative analysis of sugarcane expressed sequences tags (ESTs encoding glycine-rich proteins (GRPs

    Directory of Open Access Journals (Sweden)

    Fusaro Adriana

    2001-01-01

    Full Text Available Since the isolation of the first glycine-rich proteins (GRPs in plants a wealth of new GRPs have been identified. The highly specific but diverse expression pattern of grp genes, taken together with the distinct sub-cellular localization of some GRP groups, clearly indicate that these proteins are involved in several independent physiological processes. Notwithstanding the absence of a clear definition of the role of GRPs in plant cells, studies conducted with these proteins have provided new and interesting insights into the molecular biology and cell biology of plants. Complexly regulated promoters and distinct mechanisms for the regulation of gene expression have been demonstrated and new protein targeting pathways, as well as the exportation of GRPs from different cell types have been discovered. These data show that GRPs can be useful as markers and/or models to understand distinct aspects of plant biology. In this paper, the structural and functional features of these proteins in sugarcane (Saccharum officinarum L. are summarized. Since this is the first description of GRPs in sugarcane, special emphasis has been given to the expression pattern of these GRP genes by studying their abundance and prevalence in the different cDNA-libraries of the Sugarcane Expressed Sequence Tag (SUCEST project . The comparison of sugarcane GRPs with GRPs from other species is also discussed.

  5. Three-Dimensional Protein Fold Determination from Backbone Amide Pseudocontact Shifts Generated by Lanthanide Tags at Multiple Sites

    KAUST Repository

    Yagi, Hiromasa

    2013-06-01

    Site-specific attachment of paramagnetic lanthanide ions to a protein generates pseudocontact shifts (PCS) in the nuclear magnetic resonance (NMR) spectra of the protein that are easily measured as changes in chemical shifts. By labeling the protein with lanthanide tags at four different sites, PCSs are observed for most amide protons and accurate information is obtained about their coordinates in three-dimensional space. The approach is demonstrated with the chaperone ERp29, for which large differences have been reported between X-ray and NMR structures of the C-terminal domain, ERp29-C. The results unambiguously show that the structure of rat ERp29-C in solution is similar to the crystal structure of human ERp29-C. PCSs of backbone amides were the only structural restraints required. Because these can be measured for more dilute protein solutions than other NMR restraints, the approach greatly widens the range of proteins amenable to structural studies in solution. © 2013 Elsevier Ltd. All rights reserved.

  6. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  7. Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

    Science.gov (United States)

    Bataille, Laure; Dieryck, Wilfrid; Hocquellet, Agnès; Cabanne, Charlotte; Bathany, Katell; Lecommandoux, Sébastien; Garbay, Bertrand; Garanger, Elisabeth

    2015-06-01

    Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Hansen, Line V; Jørgensen, Thomas J D

    2007-01-01

    The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor...... as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant...... proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity...

  9. New shuttle vector-based expression system to generate polyhistidine-tagged fusion proteins in Staphylococcus aureus and Escherichia coli.

    Science.gov (United States)

    Schwendener, Sybille; Perreten, Vincent

    2015-05-01

    Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

    Science.gov (United States)

    Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

    2003-01-01

    Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

  11. In vivo stability and inertness of various direct labelled and chelate-tagged protein

    International Nuclear Information System (INIS)

    Janoki, A.; Korosi, L.; Klivenyi, G.; Spett, B.

    1987-01-01

    There were looking for methods giving precise information about composition and activity distribution of protein components, both in the initial samples and serum samples after intravenous administration. It was tested the applicability of electroimmunoassay, polyacrilamide gel electrophoresis and high performance liquid chromatography for the assessment of in vivo stability and labelled proteins. The model compound was human serum albumin (HSA) labelled with 99m Tc and 125 I, respectively. Bifunctional chelate labelling was done with desferrioxamine, in this case protein was labelled with 67 Ga. Biodistribution of the labelled compounds and their elimination from the blood were studied in rabbits. Experience with various labelling proteins, especially with Tc-Sn-HSA system indicate that in vivo stability of this compounds are generally low. Following intravenous injection of proteins labelled with metal isotopes, due to dilution and to the presence of considerable amount of compatitive protein in the serum, part of the label is being detached from the carrier protein. Distribution of the detached metal is different from the original distribution of the protein. This problem arises also with radiopharmaceuticals based on monoclonal antibodies. (M.E.L.) [es

  12. RECOMBINANT FLUORESCENT SENSOR OF HYDROGEN PEROXIDE HyPer FUSED WITH ADAPTOR PROTEIN Ruk/CIN85: DESIGNING OF EXPRESSION VECTOR AND ITS FUNCTIONAL CHARACTERIZATION

    Directory of Open Access Journals (Sweden)

    А. V. Bazalii

    2015-10-01

    Full Text Available The aim of this study was to design the expression vector encoding fluorescent sensor of hydrogen peroxide HyPer fused with adaptor protein Ruk/CIN85 as well as to check its subcellular distribution and ability to sense hydrogen peroxide. It was demonstrated that in transiently transfected HEK293 and MCF-7 cells Ruk/CIN85-HyPer is concentrated in dot-like vesicular structures of different size while HyPer is diffusely distributed throughout the cell. Using live cell fluorescence microscopy we observed gradual increase in hydrogen peroxide concentration in representative vesicular structures during the time of experiment. Thus, the developed genetic construction encoding the chimeric Ruk/CIN85-HyPer fluorescent protein represents a new tool to study localized H2O2 production in living cells.

  13. Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP fused antigens: a potential tool to develop DNA vaccines against flaviviruses

    Directory of Open Access Journals (Sweden)

    Rafael Dhalia

    2009-12-01

    Full Text Available Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP. The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado

  14. Ontologies and tag-statistics

    International Nuclear Information System (INIS)

    Tibély, Gergely; Vicsek, Tamás; Pollner, Péter; Palla, Gergely

    2012-01-01

    Due to the increasing popularity of collaborative tagging systems, the research on tagged networks, hypergraphs, ontologies, folksonomies and other related concepts is becoming an important interdisciplinary area with great potential and relevance for practical applications. In most collaborative tagging systems the tagging by the users is completely ‘flat’, while in some cases they are allowed to define a shallow hierarchy for their own tags. However, usually no overall hierarchical organization of the tags is given, and one of the interesting challenges of this area is to provide an algorithm generating the ontology of the tags from the available data. In contrast, there are also other types of tagged networks available for research, where the tags are already organized into a directed acyclic graph (DAG), encapsulating the ‘is a sub-category of’ type of hierarchy between each other. In this paper, we study how this DAG affects the statistical distribution of tags on the nodes marked by the tags in various real networks. The motivation for this research was the fact that understanding the tagging based on a known hierarchy can help in revealing the hidden hierarchy of tags in collaborative tagging systems. We analyse the relation between the tag-frequency and the position of the tag in the DAG in two large sub-networks of the English Wikipedia and a protein-protein interaction network. We also study the tag co-occurrence statistics by introducing a two-dimensional (2D) tag-distance distribution preserving both the difference in the levels and the absolute distance in the DAG for the co-occurring pairs of tags. Our most interesting finding is that the local relevance of tags in the DAG (i.e. their rank or significance as characterized by, e.g., the length of the branches starting from them) is much more important than their global distance from the root. Furthermore, we also introduce a simple tagging model based on random walks on the DAG, capable of

  15. Ontologies and tag-statistics

    Science.gov (United States)

    Tibély, Gergely; Pollner, Péter; Vicsek, Tamás; Palla, Gergely

    2012-05-01

    Due to the increasing popularity of collaborative tagging systems, the research on tagged networks, hypergraphs, ontologies, folksonomies and other related concepts is becoming an important interdisciplinary area with great potential and relevance for practical applications. In most collaborative tagging systems the tagging by the users is completely ‘flat’, while in some cases they are allowed to define a shallow hierarchy for their own tags. However, usually no overall hierarchical organization of the tags is given, and one of the interesting challenges of this area is to provide an algorithm generating the ontology of the tags from the available data. In contrast, there are also other types of tagged networks available for research, where the tags are already organized into a directed acyclic graph (DAG), encapsulating the ‘is a sub-category of’ type of hierarchy between each other. In this paper, we study how this DAG affects the statistical distribution of tags on the nodes marked by the tags in various real networks. The motivation for this research was the fact that understanding the tagging based on a known hierarchy can help in revealing the hidden hierarchy of tags in collaborative tagging systems. We analyse the relation between the tag-frequency and the position of the tag in the DAG in two large sub-networks of the English Wikipedia and a protein-protein interaction network. We also study the tag co-occurrence statistics by introducing a two-dimensional (2D) tag-distance distribution preserving both the difference in the levels and the absolute distance in the DAG for the co-occurring pairs of tags. Our most interesting finding is that the local relevance of tags in the DAG (i.e. their rank or significance as characterized by, e.g., the length of the branches starting from them) is much more important than their global distance from the root. Furthermore, we also introduce a simple tagging model based on random walks on the DAG, capable of

  16. Immobilized metal affinity chromatography co-purifies TGF-β1 with histidine-tagged recombinant extracellular proteins.

    Directory of Open Access Journals (Sweden)

    Jasvir Kaur

    Full Text Available Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC. Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls.

  17. A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

    Directory of Open Access Journals (Sweden)

    C.R.R. Ramos

    2004-08-01

    Full Text Available We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET, expression of a short 6XHis tag at N-terminus (pET3-His and a high copy number of plasmid (pRSET. The small size of the vector (2.8 kb and the high copy number/cell (200-250 copies facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture. In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site. Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

  18. Ciliary neurotrophic factor fused to a protein transduction domain retains full neuroprotective activity in the absence of cytokine-like side effects.

    Science.gov (United States)

    Rezende, Alexandre C; Peroni, Daniele; Vieira, Andrè S; Rogerio, Fabio; Talaisys, Rafael L; Costa, Fabio T M; Langone, Francesco; Skaper, Stephen D; Negro, Alessandro

    2009-06-01

    Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. CNTF prevents the degeneration of motor neurons after axotomy and in mouse mutant progressive motor neuronopathy, which has encouraged trials of CNTF for human motor neuron disease. Given systemically, however, CNTF causes severe side effects, including cachexia and a marked immune response, which has limited its clinical application. The present work describes a novel approach for administering recombinant human CNTF (rhCNTF) while conserving neurotrophic activity and avoiding deleterious side effects. rhCNTF was fused to a protein transduction domain derived from the human immunodeficiency virus-1 TAT (transactivator) protein. The resulting fusion protein (TAT-CNTF) crosses the plasma membrane within minutes and displays a nuclear localization. TAT-CNTF was equipotent to rhCNTF in supporting the survival of cultured chicken embryo dorsal root ganglion neurons. Local or subcutaneous administration of TAT-CNTF, like rhCNTF rescued motor neurons from death in neonatal rats subjected to sciatic nerve transection. In contrast to subcutaneous rhCNTF, which caused a 20-30% decrease in body weight in neonatal rats between postnatal days 2 and 7 together with a considerable fat mobilization in brown adipose tissue, TAT-CNTF lacked such side effects. Together, these results indicate that rhCNTF fused with the protein transduction domain/TAT retains neurotrophic activity in the absence of CNTFs cytokine-like side effects and may be a promising candidate for the treatment of motor neuron and other neurodegenerative diseases.

  19. Exploiting the multiplexing capabilities of tandem mass tags for high-throughput estimation of cellular protein abundances by mass spectrometry.

    Science.gov (United States)

    Ahrné, Erik; Martinez-Segura, Amalia; Syed, Afzal Pasha; Vina-Vilaseca, Arnau; Gruber, Andreas J; Marguerat, Samuel; Schmidt, Alexander

    2015-09-01

    The generation of dynamic models of biological processes critically depends on the determination of precise cellular concentrations of biomolecules. Measurements of system-wide absolute protein levels are particularly valuable information in systems biology. Recently, mass spectrometry based proteomics approaches have been developed to estimate protein concentrations on a proteome-wide scale. However, for very complex proteomes, fractionation steps are required, increasing samples number and instrument analysis time. As a result, the number of full proteomes that can be routinely analyzed is limited. Here we combined absolute quantification strategies with the multiplexing capabilities of isobaric tandem mass tags to determine cellular protein abundances in a high throughput and proteome-wide scale even for highly complex biological systems, such as a whole human cell line. We generated two independent data sets to demonstrate the power of the approach regarding sample throughput, dynamic range, quantitative precision and accuracy as well as proteome coverage in comparison to existing mass spectrometry based strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

    Directory of Open Access Journals (Sweden)

    Lili Jiang

    Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

  1. Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

    Science.gov (United States)

    Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

    2013-07-01

    Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

  2. Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.

    Science.gov (United States)

    Pashkova, Anna; Chen, Hsuan-Shen; Rejtar, Tomas; Zang, Xin; Giese, Roger; Andreev, Victor; Moskovets, Eugene; Karger, Barry L

    2005-04-01

    The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.

  3. Downward vascular translocation of a green fluorescent protein-tagged strain of Dickeya sp. (Biovar 3) from stem and leaf inoculation sites on potato

    NARCIS (Netherlands)

    Czajkowski, R.L.; de Boer, W.J.; Van Veen, J.A.; Van der Wolf, J.M.

    2010-01-01

    Translocation of a green fluorescent protein (GFP)-tagged Dickeya sp. from stems or from leaves to underground parts of potato plants was studied in greenhouse experiments. Thirty days after stem inoculation, 90% of plants expressed symptoms at the stem base and 95% of plants showed browning of

  4. Downward Vascular Translocation of a Green Fluorescent Protein-Tagged Strain of Dickeya sp. (Biovar 3) from Stem and Leaf Inoculation Sites on Potato.

    NARCIS (Netherlands)

    Czajkowski, R.L.; Boer, de W.; Veen, van J.A.; Wolf, van der J.M.

    2010-01-01

    Translocation of a green fluorescent protein (GFP)-tagged Dickeya sp. from stems or from leaves to underground parts of potato plants was studied in greenhouse experiments. Thirty days after stem inoculation, 90% of plants expressed symptoms at the stem base and 95% of plants showed browning of

  5. Cysteine-containing peptide tag for site-specific conjugation of proteins

    Science.gov (United States)

    Backer, Marina V.; Backer, Joseph M.

    2008-04-08

    The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

  6. [Construction of a GFP-fused mouse PACRG baculovirus recombinant vector and expression of the fusion protein in Sf9 inset cells].

    Science.gov (United States)

    Liu, Jun-Pin; Li, Hong-Tao; Li, Wei; Liu, Hong; Zhang, Ling; Min, Jie; Zhou, Ting; Zhou, Lei; Zhang, Zhi-Bing

    2016-07-01

    To construct a GFP-fused mouse Parkin co-regulated gene (PACRG) baculovirus recombinant PACRG/GFP-pFastBac1 vector and express the fusion protein in Sf9 insect cells. Full-length mouse PACRG cDNA was amplified by PCR and cloned in frame to the vector pFastBac1 with eGFP (rpFBac-PACRG-GFP recombinant vector). The plasmid was transformed into DH10Bac cells to obtain the recombinant bacmid plasmid, the bacmid was transfected into Sf9 insect cells, and the expressed PACRG/GFP fusion protein was analyzed by Western blot and fluorescence microscopy. The construction of the PACRG/GFP-pFastBac1 baculovirus plasmid was confirmed by sequencing and restriction enzyme digestion. Western blot showed the expression of the fusion protein carrying a green fluorescence in the Sf9 insect cells. Conclusion: A PACRG/GFP-pFastBac1 recombinant baculovirus vector was successfully constructed and the fusion protein was highly expressed in the Sf9 insect cells. Our findings have provided a basis for further studies on the structure of the PACRG protein and regulation of spermatogenesis.

  7. Selective binding and magnetic separation of His-tagged proteins using Fe3O4/PAM/NTA-Ni2+ Magnetic Nanoparticles

    Science.gov (United States)

    Guo, Huiling; Li, Mengyun; Tu, Shu; Sun, Honghao

    2018-03-01

    Fe3O4 nanoparticles coated with polyacrylamide (PAM) were synthesized. The magnetic core, with an average hydrodynamic size of 235.5 nm, allowed the magnetic nanoparticles (MNPs) rapid separation from solutions under an external magnetic field. NTA-Ni2+ was modified on the surface of Fe3O4/PAM MNPs to selectively trap his-tagged green fluorescent protein (GFP). The results showed that Fe3O4/PAM/NTA-Ni2+ MNPs exhibited remarkable capability of selective binding and separating his-tagged GFP. The adsorption efficiency was 93.37%.

  8. Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hansen, Thomas Steen; Lind, Johan Ulrik

    2012-01-01

    and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre...

  9. Production of recombinant proteins GST L1, E6 and E7 tag HPV 16 ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-02-04

    goat anti-human IgG diluted 1:1000 in casein buffer) was added .... The purified recombinant proteins were separated in silver-stained sodium dodecyl sulphate polyacrylamide gels. Production of antigens in plants is known to ...

  10. Long-range effects of tag sequence on marginally stabilized structure in HIV-1 p24 capsid protein monitored using NMR.

    Science.gov (United States)

    Okazaki, Honoka; Kaneko, Chie; Hirahara, Miyuki; Watanabe, Satoru; Tochio, Naoya; Kigawa, Takanori; Nishimura, Chiaki

    2014-09-01

    N-terminal domain of HIV-1 p24 capsid protein is a globular fold composed of seven helices and two β-strands with a flexible structure including the α4-5 loop and both N- and C-terminal ends. However, the protein shows a high tendency (48%) for an intrinsically disordered structure based on the PONDR VL-XT prediction from the primary sequence. To assess the possibility of marginally stabilized structure under physiological conditions, the N-terminal domain of p24 was destabilized by the addition of an artificial flexible tag to either N- or C-terminal ends, and it was analyzed using T1, T2, hetero-nuclear NOE, and amide-proton exchange experiments. When the C-terminal tag (12 residues) was attached, the regions of the α3-4 loop and helix 6 as well as the α4-5 loop attained the flexible structures. Furthermore, in the protein containing the N-terminal tag (27 residues), helix 4 in addition to the above-mentioned area including α3-4 and α4-5 loops as well as helix 6 exhibited highly disordered structures. Thus, the long-range effects of the existence of tag sequence was observed in the stepwise manner of the appearance of disordered structures (step 1: α4-5 loop, step 2: α3-4 loop and helix 6, and step 3: helix 4). Furthermore, the disordered regions in tagged proteins were consistent with the PONDR VL-XT disordered prediction. The dynamic structure located in the middle part (α3-4 loop to helix 6) of the protein shown in this study may be related to the assembly of the viral particle. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Application of Cydia pomonella expressed sequence tags: Identification and expression of three general odorant binding proteins in codling moth.

    Science.gov (United States)

    Garczynski, Stephen F; Coates, Brad S; Unruh, Thomas R; Schaeffer, Scott; Jiwan, Derick; Koepke, Tyson; Dhingra, Amit

    2013-10-01

    The codling moth, Cydia pomonella, is one of the most important pests of pome fruits in the world, yet the molecular genetics and the physiology of this insect remain poorly understood. A combined assembly of 8 341 expressed sequence tags was generated from Roche 454 GS-FLX sequencing of eight tissue-specific cDNA libraries. Putative chemosensory proteins (12) and odorant binding proteins (OBPs) (18) were annotated, which included three putative general OBP (GOBP), one more than typically reported for other Lepidoptera. To further characterize CpomGOBPs, we cloned cDNA copies of their transcripts and determined their expression patterns in various tissues. Cloning and sequencing of the 698 nt transcript for CpomGOBP1 resulted in the prediction of a 163 amino acid coding region, and subsequent RT-PCR indicated that the transcripts were mainly expressed in antennae and mouthparts. The 1 289 nt (160 amino acid) CpomGOBP2 and the novel 702 nt (169 amino acid) CpomGOBP3 transcripts are mainly expressed in antennae, mouthparts, and female abdomen tips. These results indicate that next generation sequencing is useful for the identification of novel transcripts of interest, and that codling moth expresses a transcript encoding for a new member of the GOBP subfamily. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

  12. Plasmids for C-terminal tagging in Saccharomyces cerevisiae that contain improved GFP proteins, Envy and Ivy.

    Science.gov (United States)

    Slubowski, Christian J; Funk, Alyssa D; Roesner, Joseph M; Paulissen, Scott M; Huang, Linda S

    2015-04-01

    Green fluorescent protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP and GFPγ and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of the utmost importance. Copyright © 2015 John Wiley & Sons, Ltd.

  13. In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

    Science.gov (United States)

    Hatzenpichler, Roland; Scheller, Silvan; Tavormina, Patricia L; Babin, Brett M; Tirrell, David A; Orphan, Victoria J

    2014-01-01

    Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for l-methionine, followed by fluorescent labelling of AHA-containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano-scale secondary ion mass spectrometry (15NH3 assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and 15N enrichment. BONCAT-FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single-cell level. PMID:24571640

  14. ProClusEnsem: Predicting membrane protein types by fusing different modes of pseudo amino acid composition

    KAUST Repository

    Wang, Jim Jing-Yan

    2012-05-01

    Knowing the type of an uncharacterized membrane protein often provides a useful clue in both basic research and drug discovery. With the explosion of protein sequences generated in the post genomic era, determination of membrane protein types by experimental methods is expensive and time consuming. It therefore becomes important to develop an automated method to find the possible types of membrane proteins. In view of this, various computational membrane protein prediction methods have been proposed. They extract protein feature vectors, such as PseAAC (pseudo amino acid composition) and PsePSSM (pseudo position-specific scoring matrix) for representation of protein sequence, and then learn a distance metric for the KNN (K nearest neighbor) or NN (nearest neighbor) classifier to predicate the final type. Most of the metrics are learned using linear dimensionality reduction algorithms like Principle Components Analysis (PCA) and Linear Discriminant Analysis (LDA). Such metrics are common to all the proteins in the dataset. In fact, they assume that the proteins lie on a uniform distribution, which can be captured by the linear dimensionality reduction algorithm. We doubt this assumption, and learn local metrics which are optimized for local subset of the whole proteins. The learning procedure is iterated with the protein clustering. Then a novel ensemble distance metric is given by combining the local metrics through Tikhonov regularization. The experimental results on a benchmark dataset demonstrate the feasibility and effectiveness of the proposed algorithm named ProClusEnsem. © 2012 Elsevier Ltd.

  15. Application of an E. coli signal sequence as a versatile inclusion body tag.

    Science.gov (United States)

    Jong, Wouter S P; Vikström, David; Houben, Diane; van den Berg van Saparoea, H Bart; de Gier, Jan-Willem; Luirink, Joen

    2017-03-21

    Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form. When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins. We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.

  16. Comparative genomics evidence that only protein toxins are tagging bad bugs

    Directory of Open Access Journals (Sweden)

    Kalliopi eGeorgiades

    2011-10-01

    Full Text Available The term toxin was introduced by Roux and Yersin and describes macromolecular substances that, when produced during infection or when introduced parenterally or orally, cause an impairment of physiological functions that lead to disease or to the death of the infected organism. Long after the discovery of toxins, early genetic studies on bacterial virulence demonstrated that removing a certain number of genes from pathogenic bacteria decreases their capacity to infect hosts. Each of the removed factors was therefore referred to as a virulence factor, and it was speculated that non-pathogenic bacteria lack such supplementary factors. However, many recent comparative studies demonstrate that the specialization of bacteria to eukaryotic hosts is associated with massive gene loss. We recently demonstrated that the only features that seem to characterize 12 epidemic bacteria are toxin-antitoxin (TA modules, which are addiction molecules in host bacteria. In this study, we investigated if protein toxins are indeed the only molecules specific to pathogenic bacteria by comparing 14 epidemic bacterial killers (bad bugs with their 14 closest non-epidemic relatives (controls. We found protein toxins in significantly more elevated numbers in all of the bad bugs. For the first time, statistical principal components analysis, including genome size, GC%, TA modules, restriction enzymes and toxins, revealed that toxins are the only proteins other than TA modules that are correlated with the pathogenic character of bacteria. Moreover, intracellular toxins appear to be more correlated with the pathogenic character of bacteria than secreted toxins. In conclusion, we hypothesize that the only truly identifiable phenomena, witnessing the convergent evolution of the most pathogenic bacteria for humans are the loss of metabolic activities, i.e., the outcome of the loss of regulatory and transcription factors and the presence of protein toxins, alone or coupled as TA

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

    Czech Academy of Sciences Publication Activity Database

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

    2011-01-01

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

  18. Genetic modelling of PIM proteins in cancer: proviral tagging, cooperation with oncogenes, tumor suppressor genes and carcinogens.

    Directory of Open Access Journals (Sweden)

    Enara eAguirre

    2014-05-01

    Full Text Available The PIM proteins, which were initially discovered as proviral insertion sites in Moloney murine leukemia virus infection, are a family of highly homologous serine/threonine kinases that have been reported to be overexpressed in hematological malignancies and solid tumors. The PIM proteins have also been associated with metastasis and overall treatment responses and implicated in the regulation of apoptosis, metabolism, the cell cycle, and homing and migration, which makes these proteins interesting targets for anticancer drug discovery. The use of retroviral insertional mutagenesis and refined approaches such as complementation tagging has allowed the identification of myc, pim and a third group of genes (including bmi1 and gfi1 as complementing genes in lymphomagenesis. Moreover, mouse modeling of human cancer has provided an understanding of the molecular pathways that are involved in tumor initiation and progression at the physiological level. In particular, genetically modified mice have allowed researchers to further elucidate the role of each of the Pim isoforms in various tumor types. PIM kinases have been identified as weak oncogenes because experimental overexpression in lymphoid tissue, prostate and liver induces tumors at a relatively low incidence and with a long latency. However, very strong synergistic tumorigenicity between Pim1/2 and c-Myc and other oncogenes has been observed in lymphoid tissues. Mouse models have also been used to study whether the inhibition of specific PIM isoforms is required to prevent carcinogen-induced sarcomas, indicating that the absence of Pim2 and Pim3 greatly reduces sarcoma growth and bone invasion; the extent of this effect is similar to that observed in the absence of all 3 isoforms. This review will summarize some of the animal models that have been used to understand the isoform-specific contribution of PIM kinases to tumorigenesis.

  19. Linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with TEV protease and autonomous N-terminal cyclization with glutaminyl cyclase in vivo.

    Directory of Open Access Journals (Sweden)

    Yan-Ping Shih

    Full Text Available Overproduction of N-terminal pyroglutamate (pGlu-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I, and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.

  20. 4-Hydroxy-2-nonenal Alkylated and Peroxynitrite Nitrated Proteins Localize to the Fused Mitochondria in Malpighian Epithelial Cells of Type IV Collagen Drosophila Mutants

    Directory of Open Access Journals (Sweden)

    András A. Kiss

    2018-01-01

    Full Text Available Background. Human type IV collagenopathy is associated with mutations within the COL4A1 and to a less extent the COL4A2 genes. The proteins encoded by these genes form heterotrimers and are the highest molar ratio components of the ubiquitous basement membrane. The clinical manifestations of the COL4A1/A2 mutations are systemic affecting many tissues and organs among these kidneys. In order to uncover the cellular and biochemical alterations associated with aberrant type IV collagen, we have explored the phenotype of the Malpighian tubules, the secretory organ and insect kidney model, in col4a1 collagen gene mutants of the fruit fly Drosophila melanogaster. In Malpighian epithelial cells of col4a1 mutants, robust mitochondrial fusion indicated mutation-induced stress. Immunohistochemistry detected proteins nitrated by peroxynitrite that localized to the enlarged mitochondria and increased level of membrane peroxidation, assessed by the amount of proteins alkylated by 4-hydroxy-2-nonenal that similarly localized to the fused mitochondria. Nuclei within the Malpighian epithelium showed TUNEL-positivity suggesting cell degradation. The results demonstrated that col4a1 mutations affect the epithelia and, consequently, secretory function of the Malpighian tubules and provide mechanistic insight into col4a1 mutation-associated functional impairments not yet reported in human patients and in mouse models with mutant COL4A1.

  1. Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli

    Directory of Open Access Journals (Sweden)

    Gómez-Lim Miguel A

    2009-02-01

    Full Text Available Abstract Background Gag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB when fused to p24. Results A fusion between ricin toxin B subunit and p24 HIV (RTB/p24 was expressed in E. coli. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin. Conclusion In this work, we report the expression in E. coli of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when

  2. Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein.

    Science.gov (United States)

    Piscitelli, Alessandra; Pennacchio, Anna; Cicatiello, Paola; Politi, Jane; De Stefano, Luca; Giardina, Paola

    2017-01-15

    A fusion protein designed in order to combine the fluorescence emission of the Green Fluorescent Protein (GFP) with the adhesion ability of the class I hydrophobin Vmh2 was heterologously produced in the yeast Pichia pastoris. The Vmh2-GFP fusion protein has proven to be a smart and effective tool for the study of Vmh2 self-assembling. Since the two proteins were linked by the specific cutting site of the thrombin, the fusion protein was used as the active biological element in the realization of a thrombin biosensor. When the thrombin present in the target solution specifically hydrolyzed its cleavage sequence, a consequent decrease in the fluorescence intensity of the sample could be observed. The Vmh2-GFP based assay allowed quantification of thrombin in solution with a detection limit of 2.27aM. The specificity of the assay with respect to other proteases and proteins granted the measurement of thrombin added to healthy human plasma with same high sensitivity and a limit of detection of 2.3aM. Further advantages of the developed biosensor are the simplicity of its design and preparation, and the low requirements in terms of samples, reagents and time. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Gao, Xiaoli [Department of Biochemistry and Molecular Biology, Michigan State University (United States); Michael Garavito, R., E-mail: garavito@msu.edu [Department of Biochemistry and Molecular Biology, Michigan State University (United States)

    2011-04-22

    Highlights: {yields} Crystal structure of the intracellular domain of (pro)renin receptor (PRR-IC) as MBP fusion protein at 2.0 A (maltose-free) and 2.15 A (maltose-bound). {yields} MBP fusion protein is a dimer in crystals in the presence and absence of maltose. {yields} PRR-IC domain is responsible for the dimerization of the fusion protein. {yields} Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermolecular interactions, suggesting a role for the PRR-IC domain in PRR dimerization. -- Abstract: The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain ({approx}310 amino acids), a single transmembrane domain ({approx}20 amino acids) and an intracellular domain ({approx}19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain ({approx}30 amino acids), the IC domain is also involved in assembly of V{sub 0} portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0 A (maltose-free) and 2.15 A (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP-PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR

  4. An optimized procedure for on-tissue localized protein digestion and quantification using hydrogel discs and isobaric mass tags: analysis of cardiac myxoma.

    Science.gov (United States)

    Taverna, Domenico; Mignogna, Chiara; Gabriele, Caterina; Santise, Gianluca; Donato, Giuseppe; Cuda, Giovanni; Gaspari, Marco

    2017-04-01

    An optimized workflow for multiplexed and spatially localized on-tissue quantitative protein analysis is here presented. The method is based on the use of an enzyme delivery platform, a polymeric hydrogel disc, allowing for a localized digestion directly onto the tissue surface coupled with an isobaric mass tag strategy for peptide labeling and relative quantification. The digestion occurs within such hydrogels, followed by peptide solvent extraction and identification by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Since this is a histology-directed on-tissue analysis, multiple hydrogels were placed onto morphologically and spatially different regions of interest (ROIs) within the tissue surface, e.g., cardiac myxoma tumor vascularized region and the adjacent hypocellular area. After a microwave digestion step (2 min), enzymatically cleaved peptides were labeled using TMT reagents with isobaric mass tags, enabling analysis of multiple samples per experiment. Thus, N = 8 hydrogel-digested samples from cardiac myxoma serial tissue sections (N = 4 from the vascularized ROIs and N = 4 from the adjacent hypocellular areas) were processed and then combined before a single LC-MS/MS analysis. Regulated proteins from both cardiac myxoma regions were assayed in a single experiment. Graphical abstract The workflow for histology-guided on-tissue localized protein digestion followed by isobaric mass tagging and LC-MS/MS analysis for proteins quantification is here summarized.

  5. In Vitro Transcripts of Wild-Type and Fluorescent Protein-Tagged Triticum mosaic virus (Family Potyviridae) are Biologically Active in Wheat.

    Science.gov (United States)

    Tatineni, Satyanarayana; McMechan, Anthony J; Bartels, Melissa; Hein, Gary L; Graybosch, Robert A

    2015-11-01

    Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat. Wheat seedlings inoculated with in vitro transcripts elicited mosaic and mottling symptoms similar to the wild-type virus, and the progeny virus was efficiently transmitted by wheat curl mites, indicating that the cloned virus retained pathogenicity, movement, and wheat curl mite transmission characteristics. A series of TriMV-based expression vectors was constructed by engineering a green fluorescent protein (GFP) or red fluorescent protein (RFP) open reading frame with homologous NIa-Pro cleavage peptides between the P1 and HC-Pro cistrons. We found that GFP-tagged TriMV with seven or nine amino acid cleavage peptides efficiently processed GFP from HC-Pro. TriMV-GFP vectors were stable in wheat for more than 120 days and for six serial passages at 14-day intervals by mechanical inoculation and were transmitted by wheat curl mites similarly to the wild-type virus. Fluorescent protein-tagged TriMV was observed in wheat leaves, stems, and crowns. The availability of fluorescent protein-tagged TriMV will facilitate the examination of virus movement and distribution in cereal hosts and the mechanisms of cross protection and synergistic interactions between TriMV and Wheat streak mosaic virus.

  6. The FUSE binding proteins FBP1 and FBP3 are potential c-myc regulators in renal, but not in prostate and bladder cancer

    Directory of Open Access Journals (Sweden)

    Meyer Hellmuth-Alexander

    2008-12-01

    Full Text Available Abstract Background The three far-upstream element (FUSE binding proteins (FBP1, FBP2, and FBP3 belong to an ancient family of single-stranded DNA binding proteins which are required for proper regulation of the c-myc proto-oncogene. Whereas it is known that c-myc alterations play a completely different role in various carcinomas of the urogenital tract, the relevance of FBPs is unclear. Methods FBP1, FBP3 and c-myc expression was studied in 105 renal cell, 95 prostate and 112 urinary bladder carcinomas by immunohistochemistry using tissue microarrays. Results High rates of FBP1 and FBP3 expression were observed in all cancer types. There was a concomitant up-regulation of FBP1 and FBP3 in renal cell and prostate carcinomas (p C-myc expression was detectable in 21% of prostate, 30% of renal and 34% of urothelial carcinomas. Interestingly, strong FBP1 and FBP3 expression was associated with c-myc up-regulation in clear cell renal cell carcinomas (p Conclusion The correlation between FBP1/FBP3, c-myc and high proliferation rate in renal cell carcinoma provides strong in vivo support for the suggested role of FBP1 and FBP3 as activators of c-myc. The frequent up-regulation of FBP1 and FBP3 in urothelial and prostate carcinoma suggests that FBPs also have an important function in gene regulation of these tumors.

  7. Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans

    Science.gov (United States)

    2018-01-01

    Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying β-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater β-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the β-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall

  8. Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

    International Nuclear Information System (INIS)

    Delorme, Caroline; Joshi, Monika; Allingham, John S.

    2012-01-01

    Highlights: ► The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. ► The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. ► The MBP–Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance, we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the “ATP state” of the mechanochemical cycle. This site differs from the Kar3 neck–core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.

  9. Immunization with the Recombinant Cholera Toxin B Fused to Fimbria 2 Protein Protects against Bordetella pertussis Infection

    Directory of Open Access Journals (Sweden)

    Noelia Olivera

    2014-01-01

    Full Text Available This study examined the immunogenic properties of the fusion protein fimbria 2 of Bordetella pertussis (Fim2—cholera toxin B subunit (CTB in the intranasal murine model of infection. To this end B. pertussis Fim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2 were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside enzyme-linked immunosorbent assay (ELISA. To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 μg of CTB-Fim2. Recombinant (rFim2 or purified (BpFim2 Fim2, CTB, and phosphate-buffered saline (PBS were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (P<0.01 or P<0.001, resp.. Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen against Bordetella pertussis infection.

  10. Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

    Science.gov (United States)

    Dienerowitz, Maria; Ilchenko, Mykhailo; Su, Bertram; Deckers-Hebestreit, Gabriele; Mayer, Günter; Henkel, Thomas; Heitkamp, Thomas; Börsch, Michael

    2016-02-01

    Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap -- a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.

  11. Structural and energetic basis of ALS-causing mutations in the atypical proline-tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zi Chao; Chook, Yuh Min [UTSMC

    2012-10-02

    Mutations in the proline/tyrosine–nuclear localization signal (PY-NLS) of the Fused in Sarcoma protein (FUS) cause amyotrophic lateral sclerosis (ALS). Here we report the crystal structure of the FUS PY-NLS bound to its nuclear import receptor Karyopherinβ2 (Kapβ2; also known as Transportin). The FUS PY-NLS occupies the structurally invariant C-terminal arch of Kapβ2, tracing a path similar to that of other characterized PY-NLSs. Unlike other PY-NLSs, which generally bind Kapβ2 in fully extended conformations, the FUS peptide is atypical as its central portion forms a 2.5-turn α-helix. The Kapβ2-binding epitopes of the FUS PY-NLS consist of an N-terminal PGKM hydrophobic motif, a central arginine-rich α-helix, and a C-terminal PY motif. ALS mutations are found almost exclusively within these epitopes. Each ALS mutation site makes multiple contacts with Kapβ2 and mutations of these residues decrease binding affinities for Kapβ2 (KD for wild-type FUS PY-NLS is 9.5 nM) up to ninefold. Thermodynamic analyses of ALS mutations in the FUS PY-NLS show that the weakening of FUS-Kapβ2 binding affinity, the degree of cytoplasmic mislocalization, and ALS disease severity are correlated.

  12. Design, synthesis, and application of a hydrazide-functionalized isotope-coded affinity tag for the quantification of oxylipid-protein conjugates.

    Science.gov (United States)

    Han, Bingnan; Stevens, Jan F; Maier, Claudia S

    2007-05-01

    An isotopically coded affinity probe was developed and evaluated for the characterization and quantification of proteins adducted by 2-alkenals derived from lipid peroxidation (LPO) processes. Lipid-derived 2-alkenals, such as acrolein and 4-hydroxy-2-nonenal (HNE), have the ability to react with cysteine, histidine, and lysine residues in proteins, thus causing protein damage and loss of protein function. Such modifications of proteins are difficult to characterize in biological samples by mass spectrometry due to the complexity of protein extracts and the low abundance of adducted proteins. The novel aldehyde-reactive, hydrazide-functionalized, isotope-coded affinity tag (HICAT) described in this study was found effective for the selective isolation, detection, and quantification of Michael-type adducts of 2-alkenals with proteins using a combination of affinity isolation, nanoLC, and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS). The chemical and mass spectrometric properties of the new probe are demonstrated on a model protein treated with HNE. The efficacy of HICAT for the analysis of complex samples was tested using preparations of mitochondrial proteins that were modified in vitro with HNE. The potential of the HICAT strategy for the identification, characterization, and quantification of in vivo oxylipid-protein conjugates is demonstrated on cardiac mitochondrial protein preparations, in which, for example, the ADP/ATP translocase 1 was found adducted to the 2-alkenals, acrolein and 4-hydroxy-2-hexenal, at Cys-256.

  13. Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

    Science.gov (United States)

    Eyre, Nicholas S; Johnson, Stephen M; Eltahla, Auda A; Aloi, Maria; Aloia, Amanda L; McDevitt, Christopher A; Bull, Rowena A; Beard, Michael R

    2017-12-01

    Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing , connector , and β- ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies. IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the

  14. Dissecting the salt dependence of the Tus-Ter protein-DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus.

    Science.gov (United States)

    Moreau, Morgane J J; Schaeffer, Patrick M

    2013-12-01

    The analysis of the salt dependence of protein-DNA complexes provides useful information about the non-specific electrostatic and sequence-specific parameters driving complex formation and stability. The differential scanning fluorimetry of GFP-tagged protein (DSF-GTP) assay has been geared with an automatic Tm peak recognition system and was applied for the high-throughput (HT) determination of salt-induced effects on the GFP-tagged DNA replication protein Tus in complex with various Ter and Ter-lock sequences. The system was designed to generate two-dimensional heat map profiles of Tus-GFP protein stability allowing for a comparative study of the effect of eight increasing salt concentrations on ten different Ter DNA species at once. The data obtained with the new HT DSF-GTP allowed precise dissection of the non-specific electrostatic and sequence-specific parameters driving Tus-Ter and Tus-Ter-lock complex formation and stability. The major factor increasing the thermal resistance of Tus-Ter-lock complexes in high-salt is the formation of the TT-lock, e.g. a 10-fold higher Kspe was obtained for Tus-GFP:Ter-lockB than for Tus-GFP:TerB. It is anticipated that the system can be easily adapted for the study of other protein-DNA complexes.

  15. The FUSE binding proteins FBP1 and FBP3 are potential c-myc regulators in renal, but not in prostate and bladder cancer

    International Nuclear Information System (INIS)

    Weber, Achim; Moch, Holger; Kristiansen, Glen; Kristiansen, Ilka; Johannsen, Manfred; Oelrich, Beibei; Scholmann, Katharina; Gunia, Sven; May, Matthias; Meyer, Hellmuth-Alexander; Behnke, Silvia

    2008-01-01

    The three far-upstream element (FUSE) binding proteins (FBP1, FBP2, and FBP3) belong to an ancient family of single-stranded DNA binding proteins which are required for proper regulation of the c-myc proto-oncogene. Whereas it is known that c-myc alterations play a completely different role in various carcinomas of the urogenital tract, the relevance of FBPs is unclear. FBP1, FBP3 and c-myc expression was studied in 105 renal cell, 95 prostate and 112 urinary bladder carcinomas by immunohistochemistry using tissue microarrays. High rates of FBP1 and FBP3 expression were observed in all cancer types. There was a concomitant up-regulation of FBP1 and FBP3 in renal cell and prostate carcinomas (p < 0.001 both). C-myc expression was detectable in 21% of prostate, 30% of renal and 34% of urothelial carcinomas. Interestingly, strong FBP1 and FBP3 expression was associated with c-myc up-regulation in clear cell renal cell carcinomas (p < 0.001 and 0.09 resp.), but not in bladder or prostate cancer. The correlation between FBP1/FBP3, c-myc and high proliferation rate in renal cell carcinoma provides strong in vivo support for the suggested role of FBP1 and FBP3 as activators of c-myc. The frequent up-regulation of FBP1 and FBP3 in urothelial and prostate carcinoma suggests that FBPs also have an important function in gene regulation of these tumors

  16. CRISPR/Cas9 allows efficient and complete knock-in of a destabilization domain-tagged essential protein in a human cell line, allowing rapid knockdown of protein function.

    Directory of Open Access Journals (Sweden)

    Arnold Park

    Full Text Available Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1 in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and

  17. CRISPR/Cas9 allows efficient and complete knock-in of a destabilization domain-tagged essential protein in a human cell line, allowing rapid knockdown of protein function.

    Science.gov (United States)

    Park, Arnold; Won, Sohui T; Pentecost, Mickey; Bartkowski, Wojciech; Lee, Benhur

    2014-01-01

    Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to

  18. Sorption of his-tagged Protein G and Protein G onto chitosan/divalent metal ion sorbent used for detection of microcystin-LR.

    Science.gov (United States)

    Demey, Hary; Tria, Scherrine A; Soleri, Romain; Guiseppi-Elie, Anthony; Bazin, Ingrid

    2017-01-01

    A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR) by using strategies for oriented immobilization of functionally intact polyclonal antibodies on chitosan surface. Several physicochemical parameters such as metal ion adsorption, hexahistidine-tagged Protein G sorption, the dilution ratio polyclonal antibody concentration, and peroxidase-labeled MC-LR concentration were studied and optimized. The sorption in batch system of G-histidine and G-proteins was studied on a novel sorbent consisting of chitosan/divalent metal ions. Transition metals as Ni ++ and Zn ++ were immobilized through interaction with -NH 2 groups of chitosan in order to supply a material capable to efficiently remove the proteins from aqueous solutions. The maximum uptake of divalent metals onto the chitosan material was found to be 230 mg g -1 for Zn ++ and 62 mg g -1 for Ni ++ . Experimental data were evaluated using the Langmuir and Freundlich models; the results were well fitted with the Langmuir model; chitosan/Ni ++ foam was found to be the best sorbent for G-protein, maximum sorption capacity obtained was 17 mg g -1 , and chitosan/Zn ++ was found to be the best for G-histidine with a maximum sorption capacity of 44 mg g -1 . Kinetic data was evaluated with pseudo-first- and pseudo-second-order models; the sorption kinetics were in all cases better represented by a pseudo-second-order model. Under optimum conditions, the calibration curve obtained for MC-LR gave detection limits of 0.5 ± 0.06 μg L -1 , the 50 % inhibition concentration (IC50) was 2.75 ± 0.03 μg L -1 , and the quantitative detection range was 0.5-25 μg L -1 . The limit of detection (LOD) attained from the calibration curves and the results obtained demonstrate the potential use of CLEIA with chitosan support as a screening tool for the analysis of pollutants in environmental samples.

  19. Transmission of Mannheimia haemolytica from domestic sheep (Ovis aries) to bighorn sheep (Ovis canadensis): unequivocal demonstration with green fluorescent protein-tagged organisms.

    Science.gov (United States)

    Lawrence, Paulraj K; Shanthalingam, Sudarvili; Dassanayake, Rohana P; Subramaniam, Renuka; Herndon, Caroline N; Knowles, Donald P; Rurangirwa, Fred R; Foreyt, William J; Wayman, Gary; Marciel, Ann Marie; Highlander, Sarah K; Srikumaran, Subramaniam

    2010-07-01

    Previous studies demonstrated that bighorn sheep (Ovis canadensis) died of pneumonia when commingled with domestic sheep (Ovis aries) but did not conclusively prove that the responsible pathogens were transmitted from domestic to bighorn sheep. The objective of this study was to determine, unambiguously, whether Mannheimia haemolytica can be transmitted from domestic to bighorn sheep when they commingle. Four isolates of M. haemolytica were obtained from the pharynx of two of four domestic sheep and tagged with a plasmid carrying the genes for green fluorescent protein (GFP) and ampicillin resistance (AP(R)). Four domestic sheep, colonized with the tagged bacteria, were kept about 10 m apart from four bighorn sheep for 1 mo with no clinical signs of pneumonia observed in the bighorn sheep during that period. The domestic and bighorn sheep were then allowed to have fence-line contact for 2 mo. During that period, three bighorn sheep acquired the tagged bacteria from the domestic sheep. At the end of the 2 mo of fence-line contact, the animals were allowed to commingle. All four bighorn sheep died 2 days to 9 days following commingling. The lungs from all four bighorn sheep showed gross and histopathologic lesions characteristic of M. haemolytica pneumonia. Tagged M. haemolytica were isolated from all four bighorn sheep, as confirmed by growth in ampicillin-containing culture medium, PCR-amplification of genes encoding GFP and Ap(R), and immunofluorescent staining of GFP. These results unequivocally demonstrate transmission of M. haemolytica from domestic to bighorn sheep, resulting in pneumonia and death of bighorn sheep.

  20. Functional Use Database (FUse)

    Data.gov (United States)

    U.S. Environmental Protection Agency — There are five different files for this dataset: 1. A dataset listing the reported functional uses of chemicals (FUse) 2. All 729 ToxPrint descriptors obtained from...

  1. Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag

    Energy Technology Data Exchange (ETDEWEB)

    Sengupta, Ishita; Gao, Min; Arachchige, Rajith J.; Nadaud, Philippe S. [The Ohio State University, Department of Chemistry and Biochemistry (United States); Cunningham, Timothy F.; Saxena, Sunil [University of Pittsburgh, Department of Chemistry (United States); Schwieters, Charles D. [National Institutes of Health, Center for Information Technology (United States); Jaroniec, Christopher P., E-mail: jaroniec@chemistry.ohio-state.edu [The Ohio State University, Department of Chemistry and Biochemistry (United States)

    2015-01-15

    Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu{sup 2+} binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC–Cu{sup 2+} K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ∼10 Å of the Cu{sup 2+} center experience considerable transverse PREs leading to severely attenuated resonances in 2D {sup 15}N–{sup 13}C correlation spectra. For more distant residues, electron–nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for {sup 15}N–Cu{sup 2+} distances up to ∼20 Å.

  2. Binary polypeptide system for permanent and oriented protein immobilization

    Directory of Open Access Journals (Sweden)

    Bailes Julian

    2010-05-01

    Full Text Available Abstract Background Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag. Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case leading to the requirement for chemical coupling. Results Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. This irreversible protein attachment system (IPAS uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. Conclusions IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

  3. Binary polypeptide system for permanent and oriented protein immobilization.

    Science.gov (United States)

    Ferrari, Enrico; Darios, Frédéric; Zhang, Fan; Niranjan, Dhevahi; Bailes, Julian; Soloviev, Mikhail; Davletov, Bazbek

    2010-05-12

    Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling. Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. This irreversible protein attachment system (IPAS) uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

  4. Prokaryotic ubiquitin-like ThiS fusion enhances the heterologous protein overexpression and aggregation in Escherichia coli.

    Science.gov (United States)

    Yuan, Sujuan; Xu, Jian; Ge, Ying; Yan, Zheng; Du, Guohua; Wang, Nan

    2013-01-01

    Fusion tags are commonly employed to enhance target protein expression, improve their folding and solubility, and reduce protein degradation in expression of recombinant proteins. Ubiquitin (Ub) and SUMO are highly conserved small proteins in eukaryotes, and frequently used as fusion tags in prokaryotic expression. ThiS, a smaller sulfur-carrier protein involved in thiamin synthesis, is conserved among most prokaryotic species. The structural similarity between ThiS and Ub provoked us into expecting that the former could be used as a fusion tag. Hence, ThiS was fused to insulin A and B chains, murine Ribonuclease Inhibitor (mRI) and EGFP, respectively. When induced in Escherichia coli, ThiS-fused insulin A and B chains were overexpressed in inclusion bodies, and to higher levels in comparison to the same proteins fused with Ub. On the contrast, ThiS fusion of mRI, an unstable protein, resulted in enhanced degradation that was not alleviated in protease-deficient strains. While the degradation of Ub- and SUMO-fused mRI was less and seemed protease-dependent. Enhanced degradation of mRI did not occur for the fusions with half-molecules of ThiS. When ThiS-tag was fused to the C-terminus of EGFP, higher expression, predominantly in inclusion bodies, was observed again. It was further found that ThiS fusion of EGFP significantly retarded its refolding process. These results indicated that prokaryotic ThiS is able to promote the expression of target proteins in E. coli, but enhanced degradation may occur in case of unstable targets. Unlike eukaryotic Ub-based tags usually increase the solubility and folding of proteins, ThiS fusion enhances the expression by augmenting the formation of inclusion bodies, probably through retardation of the folding of target proteins.

  5. Effect of Recombinant alpha1-Antitrypsin Fc-Fused (AAT-Fc)Protein on the Inhibition of Inflammatory Cytokine Production and Streptozotocin-Induced Diabetes

    NARCIS (Netherlands)

    Lee, S.; Lee, Y.; Hong, K.; Hong, J.; Bae, S.; Choi, J.; Jhun, H.; Kwak, A.; Kim, E.; Jo, S.; Dinarello, C.A.; Kim, S.

    2013-01-01

    alpha1-Antitrypsin (AAT) is a member of the serine proteinase inhibitor family that impedes the enzymatic activity of serine proteinases, including human neutrophil elastase, cathepsin G and neutrophil proteinase 3. Here, we expressed recombinant AAT by fusing the intact AAT gene to the constant

  6. Cloning and expression of aequorin photoprotein using intein tag

    Directory of Open Access Journals (Sweden)

    Elah sadat Seyed Hosseini

    2015-01-01

    Full Text Available Background: Intein (INT, is the internal parts of the protein which can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. This protein sequence and their characteristic of self-cleavage by thiol induction, temperature and pH changes is used for protein purification. The advantage of this method compared to the other protein purification methods is that it doesn’t require any protease enzyme and protease removal steps that make this method important economically. In this study, aequorin photoprotein was hybridized with INT in molecular form and its expression was evaluated. Materials and Methods: In this study, aequorin coding gene that was cloned in pET21-a in the previous studies, was cloned in pTYB21 vector containing INT tag by specific primers and restriction enzymes. Then the resulting pTY-aequarin was transformed to the ER2566 expression strain and cloning accuracy was confirmed by electrophoresis, western blotting and sequencing. Results: The photoprotein aequorin was cloned into SapI/PstI restriction site of pTYB21 plasmid accurately and successfully. Aequorin- INT hybrid protein expression confirmed using traditional methods. Conclusion: The photoprotein aequorin constract in fused with INT confirmed by molecular methods. Also rate of Aequorin- INT expression determined about %25 of cell total protein.

  7. Yellowtail Tagging Data (MRDBS)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Yellowtail Flounder Tagging Program began in 2003 and works with commercial fishermen to tag and release yellowtaiI flounder with pink and yellow disc tags or...

  8. Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis

    NARCIS (Netherlands)

    Neef, Jolanda; Milder, Fin J.; Koedijk, Danny G. A. M.; Klaassens, Marindy; Heezius, Erik C.; van Strijp, Jos A. G.; Otto, Andreas; Becher, Doerte; van Dijl, Jan Maarten; Buist, Girbe

    2015-01-01

    Recent studies have shown that the Gram-positive bacterium Lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized

  9. Hepatitis C virus expressing flag-tagged envelope protein 2 has unaltered infectivity and density, is specifically neutralized by flag antibodies and can be purified by affinity chromatography

    DEFF Research Database (Denmark)

    Prentø, Jannick Cornelius; Bukh, Jens

    2011-01-01

    , flag-tagged viruses had drastically altered properties, and purification yield was low. In this study, we found that insertion of flag tag at the N-terminus of E2 in HCV recombinant J6/JFH1 did not affect viability in Huh7.5 cells, and that flag-tagged virus had physiochemical properties similar...... physiochemical properties of flag-tagged HCV is an important improvement for utilizing these viruses for imaging, virion composition analysis and possibly vaccine development....

  10. Efficient expression of a soluble lipid transfer protein (LTP) of Platanus orientalis using short peptide tags and structural comparison with the natural form.

    Science.gov (United States)

    Salari, Farhad; Vahedi, Fatemeh; Chamani, Jamshidkhan; Varasteh, Abdolreza; Ketabdar, Hanieh; Sankian, Mojtaba

    2015-01-01

    Successful recombinant allergen-based immunotherapy has drawn a great deal of attention to use recombinant allergens for new therapeutic and/or diagnostic strategies. The Escherichia coli expression system is frequently used to produce recombinant allergens; however, protein expression in E. coli often results in inclusion bodies. Here, we focused on the expression of two recombinant soluble forms of Pla or 3 using solubility-enhancing peptide tags, human immune deficiency virus type 1 transactivator of transcription core domain and poly-arginine-lysine: rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3. Structural characteristics and IgE reactivity of purified recombinant proteins were compared with natural Pla or 3 (nPla or 3) isolated from Platanus orientalis using circular dichroism spectra, fluorescence spectroscopy, and immunoblotting. Likewise, intrinsic viscosity and Stokes radius of the natural and recombinant Pla or 3 allergens were determined to analyze structural compactness in aqueous media. The results indicate high-level solubility and efficient expression of the fusion proteins (rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3) compared with the wild-type recombinant. Furthermore, the similar structural characteristics and IgE-binding activities of the fusion proteins to nPla or 3 provide a promising tool for allergy diagnosis and treatment. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  11. An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins

    Energy Technology Data Exchange (ETDEWEB)

    Barb, Adam W., E-mail: abarb@iastate.edu; Subedi, Ganesh P. [Iowa State University, Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology (United States)

    2016-01-15

    Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in X-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded within a recombinant protein expression construct. However, LBTs often exhibit significant mobility relative to the target molecule. Here we report the characterization of improved LBTs sequences for insertion into a protein loop. These LBTs were inserted to connect two parallel alpha helices of an immunoglobulin G (IgG)-binding Z domain platform. Variants A and B bound Tb{sup 3+} with high affinity (0.70 and 0.13 μM, respectively) and displayed restricted LBT motion. Compared to the parent construct, the metal-bound A experienced a 2.5-fold reduction in tag motion as measured by magnetic field-induced residual dipolar couplings and was further studied in a 72.2 kDa complex with the human IgG1 fragment crystallizable (IgG1 Fc) glycoprotein. The appearance of both pseudo-contact shifts (−0.221 to 0.081 ppm) and residual dipolar couplings (−7.6 to 14.3 Hz) of IgG1 Fc resonances in the IgG1 Fc:(variant A:Tb{sup 3+}){sub 2} complex indicated structural restriction of the LBT with respect to the Fc. These studies highlight the applicability of improved LBT sequences with reduced mobility to probe the structure of macromolecular systems.

  12. An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins

    International Nuclear Information System (INIS)

    Barb, Adam W.; Subedi, Ganesh P.

    2016-01-01

    Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in X-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded within a recombinant protein expression construct. However, LBTs often exhibit significant mobility relative to the target molecule. Here we report the characterization of improved LBTs sequences for insertion into a protein loop. These LBTs were inserted to connect two parallel alpha helices of an immunoglobulin G (IgG)-binding Z domain platform. Variants A and B bound Tb 3+ with high affinity (0.70 and 0.13 μM, respectively) and displayed restricted LBT motion. Compared to the parent construct, the metal-bound A experienced a 2.5-fold reduction in tag motion as measured by magnetic field-induced residual dipolar couplings and was further studied in a 72.2 kDa complex with the human IgG1 fragment crystallizable (IgG1 Fc) glycoprotein. The appearance of both pseudo-contact shifts (−0.221 to 0.081 ppm) and residual dipolar couplings (−7.6 to 14.3 Hz) of IgG1 Fc resonances in the IgG1 Fc:(variant A:Tb 3+ ) 2 complex indicated structural restriction of the LBT with respect to the Fc. These studies highlight the applicability of improved LBT sequences with reduced mobility to probe the structure of macromolecular systems

  13. Needles in the EST haystack: large-scale identification and analysis of excretory-secretory (ES proteins in parasitic nematodes using expressed sequence tags (ESTs.

    Directory of Open Access Journals (Sweden)

    Shivashankar H Nagaraj

    2008-09-01

    Full Text Available Parasitic nematodes of humans, other animals and plants continue to impose a significant public health and economic burden worldwide, due to the diseases they cause. Promising antiparasitic drug and vaccine candidates have been discovered from excreted or secreted (ES proteins released from the parasite and exposed to the immune system of the host. Mining the entire expressed sequence tag (EST data available from parasitic nematodes represents an approach to discover such ES targets.In this study, we predicted, using EST2Secretome, a novel, high-throughput, computational workflow system, 4,710 ES proteins from 452,134 ESTs derived from 39 different species of nematodes, parasitic in animals (including humans or plants. In total, 2,632, 786, and 1,292 ES proteins were predicted for animal-, human-, and plant-parasitic nematodes. Subsequently, we systematically analysed ES proteins using computational methods. Of these 4,710 proteins, 2,490 (52.8% had orthologues in Caenorhabditis elegans, whereas 621 (13.8% appeared to be novel, currently having no significant match to any molecule available in public databases. Of the C. elegans homologues, 267 had strong "loss-of-function" phenotypes by RNA interference (RNAi in this nematode. We could functionally classify 1,948 (41.3% sequences using the Gene Ontology (GO terms, establish pathway associations for 573 (12.2% sequences using Kyoto Encyclopaedia of Genes and Genomes (KEGG, and identify protein interaction partners for 1,774 (37.6% molecules. We also mapped 758 (16.1% proteins to protein domains including the nematode-specific protein family "transthyretin-like" and "chromadorea ALT," considered as vaccine candidates against filariasis in humans.We report the large-scale analysis of ES proteins inferred from EST data for a range of parasitic nematodes. This set of ES proteins provides an inventory of known and novel members of ES proteins as a foundation for studies focused on understanding the

  14. Needles in the EST Haystack: Large-Scale Identification and Analysis of Excretory-Secretory (ES) Proteins in Parasitic Nematodes Using Expressed Sequence Tags (ESTs)

    Science.gov (United States)

    Nagaraj, Shivashankar H.; Gasser, Robin B.; Ranganathan, Shoba

    2008-01-01

    Background Parasitic nematodes of humans, other animals and plants continue to impose a significant public health and economic burden worldwide, due to the diseases they cause. Promising antiparasitic drug and vaccine candidates have been discovered from excreted or secreted (ES) proteins released from the parasite and exposed to the immune system of the host. Mining the entire expressed sequence tag (EST) data available from parasitic nematodes represents an approach to discover such ES targets. Methods and Findings In this study, we predicted, using EST2Secretome, a novel, high-throughput, computational workflow system, 4,710 ES proteins from 452,134 ESTs derived from 39 different species of nematodes, parasitic in animals (including humans) or plants. In total, 2,632, 786, and 1,292 ES proteins were predicted for animal-, human-, and plant-parasitic nematodes. Subsequently, we systematically analysed ES proteins using computational methods. Of these 4,710 proteins, 2,490 (52.8%) had orthologues in Caenorhabditis elegans, whereas 621 (13.8%) appeared to be novel, currently having no significant match to any molecule available in public databases. Of the C. elegans homologues, 267 had strong “loss-of-function” phenotypes by RNA interference (RNAi) in this nematode. We could functionally classify 1,948 (41.3%) sequences using the Gene Ontology (GO) terms, establish pathway associations for 573 (12.2%) sequences using Kyoto Encyclopaedia of Genes and Genomes (KEGG), and identify protein interaction partners for 1,774 (37.6%) molecules. We also mapped 758 (16.1%) proteins to protein domains including the nematode-specific protein family “transthyretin-like” and “chromadorea ALT,” considered as vaccine candidates against filariasis in humans. Conclusions We report the large-scale analysis of ES proteins inferred from EST data for a range of parasitic nematodes. This set of ES proteins provides an inventory of known and novel members of ES proteins as a

  15. Synaptic Tagging, Evaluation of Memories, and the Distal Reward Problem

    Science.gov (United States)

    Papper, Marc; Kempter, Richard; Leibold, Christian

    2011-01-01

    Long-term synaptic plasticity exhibits distinct phases. The synaptic tagging hypothesis suggests an early phase in which synapses are prepared, or "tagged," for protein capture, and a late phase in which those proteins are integrated into the synapses to achieve memory consolidation. The synapse specificity of the tags is consistent with…

  16. Prime-boost therapeutic vaccination in mice with DNA/DNA or DNA/Fowlpox virus recombinants expressing the Human Papilloma Virus type 16 E6 and E7 mutated proteins fused to the coat protein of Potato virus X.

    Science.gov (United States)

    Illiano, Elena; Bissa, Massimiliano; Paolini, Francesca; Zanotto, Carlo; De Giuli Morghen, Carlo; Franconi, Rosella; Radaelli, Antonia; Venuti, Aldo

    2016-10-02

    The therapeutic antitumor potency of a prime-boost vaccination strategy was explored, based on the mutated, nontransforming forms of the E6 (E6 F47R ) and E7 (E7 GGG ) oncogenes of Human Papilloma Virus type 16 (HPV16), fused to the Potato virus X (PVX) coat protein (CP) sequence. Previous data showed that CP fusion improves the immunogenicity of tumor-associated antigens and may thus increase their efficacy. After verifying the correct expression of E6 F47R CP and E7 GGG CP inserted into DNA and Fowlpox virus recombinants by Western blotting and immunofluorescence, their combined use was evaluated for therapy in a pre-clinical mouse model of HPV16-related tumorigenicity. Immunization protocols were applied using homologous (DNA/DNA) or heterologous (DNA/Fowlpox) prime-boost vaccine regimens. The humoral immune responses were determined by ELISA, and the therapeutic efficacy evaluated by the delay in tumor appearance and reduced tumor volume after inoculation of syngeneic TC-1* tumor cells. Homologous DNA/DNA genetic vaccines were able to better delay tumor appearance and inhibit tumor growth when DNAE6 F47R CP and DNAE7 GGG CP were administered in combination. However, the heterologous DNA/Fowlpox vaccination strategy was able to delay tumor appearance in a higher number of animals when E6 F47R CP and in particular E7 GGG CP were administered alone. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. OLED panel with fuses

    Science.gov (United States)

    Levermore, Levermore; Pang, Huiqing; Rajan, Kamala

    2014-09-16

    Embodiments may provide a first device that may comprise a substrate, a plurality of conductive bus lines disposed over the substrate, and a plurality of OLED circuit elements disposed on the substrate, where each of the OLED circuit elements comprises one and only one pixel electrically connected in series with a fuse. Each pixel may further comprise a first electrode, a second electrode, and an organic electroluminescent (EL) material disposed between the first and the second electrodes. The fuse of each of the plurality of OLED circuit elements may electrically connect each of the OLED circuit elements to at least one of the plurality of bus lines. Each of the plurality of bus lines may be electrically connected to a plurality of OLED circuit elements that are commonly addressable and at least two of the bus lines may be separately addressable.

  18. Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield

    Directory of Open Access Journals (Sweden)

    Straus Suzana K

    2011-06-01

    Full Text Available Abstract Background Obtaining membrane proteins in sufficient quantity for biophysical study and biotechnological applications has been a difficult task. Use of the maltose binding protein/hexahistidine dual tag system with E.coli as an expression host is emerging as a high throughput method to enhance membrane protein yield, solubility, and purity, but fails to be effective for certain proteins. Optimizing the variables in this system to fine-tune for efficiency can ultimately be a daunting task. To identify factors critical to success in this expression system, we have selected to study U24, a novel membrane protein from Human Herpesvirus type-6 with potent immunosuppressive ability and a possible role in the pathogenesis of the disease multiple sclerosis. Results We expressed full-length U24 as a C-terminal fusion to a maltose binding protein/hexahistidine tag and examined the effects of temperature, growth medium type, cell strain type, oxidizing vs. reducing conditions and periplasmic vs. cytoplasmic expression location. Temperature appeared to have the greatest effect on yield; at 37°C full-length protein was either poorly expressed (periplasm or degraded (cytoplasm whereas at 18°C, expression was improved especially in the periplasm of C41(DE3 cells and in the cytoplasm of oxidizing Δtrx/Δgor mutant strains, Origami 2 and SHuffle. Expression of the fusion protein in these strains were estimated to be 3.2, 5.3 and 4.3 times greater, respectively, compared to commonly-used BL21(DE3 cells. We found that U24 is isolated with an intramolecular disulfide bond under these conditions, and we probed whether this disulfide bond was critical to high yield expression of full-length protein. Expression analysis of a C21SC37S cysteine-free mutant U24 demonstrated that this disulfide was not critical for full-length protein expression, but it is more likely that strained metabolic conditions favour factors which promote protein expression. This

  19. Voltammetric Detection of S100B Protein Using His-Tagged Receptor Domains for Advanced Glycation End Products (RAGE Immobilized onto a Gold Electrode Surface

    Directory of Open Access Journals (Sweden)

    Edyta Mikuła

    2014-06-01

    Full Text Available In this work we report on an electrochemical biosensor for the determination of the S100B protein. The His-tagged VC1 domains of Receptors for Advanced Glycation End (RAGE products used as analytically active molecules were covalently immobilized on a monolayer of a thiol derivative of pentetic acid (DPTA complex with Cu(II deposited on a gold electrode surface. The recognition processes between the RAGE VC1 domain and the S100B protein results in changes in the redox activity of the DPTA-Cu(II centres which were measured by Osteryoung square-wave voltammetry (OSWV. In order to verify whether the observed analytical signal originates from the recognition process between the His6–RAGE VC1 domains and the S100B protein, the electrode modified with the His6–RAGE C2 and His6–RAGE VC1 deleted domains which have no ability to bind S100B peptides were applied. The proposed biosensor was quite sensitive, with a detection limit of 0.52 pM recorded in the buffer solution. The presence of diluted human plasma and 10 nM Aβ1-40 have no influence on the biosensor performance.

  20. Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells

    DEFF Research Database (Denmark)

    Jensen, Lena Vinther; Lademann, Ulrik Axel; Andersen, Elisabeth Veyhe

    2014-01-01

    as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N......-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1......), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1...

  1. An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

    Science.gov (United States)

    Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K; Wu, Peng; Cate, Jamie H D; Marqusee, Susan

    2017-09-22

    Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational

  2. General Strategy for Direct Cytosolic Protein Delivery via Protein-Nanoparticle Co-engineering.

    Science.gov (United States)

    Mout, Rubul; Ray, Moumita; Tay, Tristan; Sasaki, Kanae; Yesilbag Tonga, Gulen; Rotello, Vincent M

    2017-06-27

    Endosomal entrapment is a key hurdle for most intracellular protein-based therapeutic strategies. We report a general strategy for efficient delivery of proteins to the cytosol through co-engineering of proteins and nanoparticle vehicles. The proteins feature an oligo(glutamate) sequence (E-tag) that binds arginine-functionalized gold nanoparticles, generating hierarchical spherical nanoassemblies. These assemblies fuse with cell membranes, releasing the E-tagged protein directly into the cytosol. Five different proteins with diverse charges, sizes, and functions were effectively delivered into cells, demonstrating the generality of our method. Significantly, the engineered proteins retained activity after cytosolic delivery, as demonstrated through the delivery of active Cre recombinase, and granzyme A to kill cancer cells.

  3. Ni(2+)-zeolite/ferrosphere and Ni(2+)-silica/ferrosphere beads for magnetic affinity separation of histidine-tagged proteins.

    Science.gov (United States)

    Vereshchagina, T A; Fedorchak, M A; Sharonova, O M; Fomenko, E V; Shishkina, N N; Zhizhaev, A M; Kudryavtsev, A N; Frank, L A; Anshits, A G

    2016-01-28

    Magnetic Ni(2+)-zeolite/ferrosphere and Ni(2+)-silica/ferrosphere beads (Ni-ferrosphere beads - NFB) of a core-shell structure were synthesized starting from coal fly ash ferrospheres having diameters in the range of 0.063-0.050 mm. The strategy of NFB fabrication is an oriented chemical modification of the outer surface preserving the magnetic core of parent beads with the formation of micro-mesoporous coverings. Two routes of ferrosphere modification were realized, such as (i) hydrothermal treatment in an alkaline medium resulting in a NaP zeolite layer and (ii) synthesis of micro-mesoporous silica on the glass surface using conventional methods. Immobilization of Ni(2+) ions in the siliceous porous shell of the magnetic beads was carried out via (i) the ion exchange of Na(+) for Ni(2+) in the zeolite layer or (ii) deposition of NiO clusters in the zeolite and silica pores. The final NFB were tested for affinity in magnetic separation of the histidine-tagged green fluorescent protein (GFP) directly from a cell lysate. Results pointed to the high affinity of the magnetic beads towards the protein in the presence of 10 mM EDTA. The sorption capacity of the ferrosphere-based Ni-beads with respect to GFP was in the range 1.5-5.7 mg cm(-3).

  4. Quantitative determination of lateral concentration and depth profile of histidine-tagged recombinant proteins probed by grazing incidence X-ray fluorescence.

    Science.gov (United States)

    Körner, Alexander; Abuillan, Wasim; Deichmann, Christina; Rossetti, Fernanda F; Köhler, Almut; Konovalov, Oleg V; Wedlich, Doris; Tanaka, Motomu

    2013-05-02

    We have demonstrated that the complementary combination of grazing incidence X-ray fluorescence (GIXF) with specular X-ray reflectivity (XRR) can be used to quantitatively determine the density profiles of Ni(2)(+) ions complexed with chelator headgroups as well as S atoms in recombinant proteins anchored to lipid monolayers at the air/water interface. First, we prepared phospholipid monolayers incorporating chelator lipid anchors at different molar fractions at the air/water interface. The fine-structures perpendicular to the global plane of monolayers were characterized by XRR in the presence of Ni(2)(+) ions, yielding the thickness, roughness, and electron density of the stratified lipid monolayers. X-ray fluorescence intensities from Ni Kα core levels recorded at the incidence angles below and above the critical angle of total reflection allow for the determination of the position and lateral density of Ni(2)(+) ions associated with chelator headgroups with a high spatial accuracy (±5 Å). The coupling of histidine-tagged Xenopus cadherin 11 (Xcad-11) can also be identified by changes in the fines-structures using XRR. Although fluorescence intensities from S Kα level were much weaker than Ni Kα signals, we could detect the location of S atoms in recombinant Xcad-11 proteins.

  5. A dual protease approach for expression and affinity purification of recombinant proteins.

    Science.gov (United States)

    Raran-Kurussi, Sreejith; Waugh, David S

    2016-07-01

    We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

  6. Comparative analysis of secreted protein evolution using expressed sequence tags from four poplar leaf rusts (Melampsora spp.

    Directory of Open Access Journals (Sweden)

    Tanguay Philippe

    2010-07-01

    Full Text Available Abstract Background Obligate biotrophs such as rust fungi are believed to establish long-term relationships by modulating plant defenses through a plethora of effector proteins, whose most recognizable feature is the presence of a signal peptide for secretion. Since the phenotypes of these effectors extend to host cells, their genes are expected to be under accelerated evolution stimulated by host-pathogen coevolutionary arms races. Recently, whole genome sequence data has allowed the prediction of secretomes, facilitating the identification of putative effectors. Results We generated cDNA libraries from four poplar leaf rust pathogens (Melampsora spp. and used computational approaches to identify and annotate putative secreted proteins with the aim of uncovering new knowledge about the nature and evolution of the rust secretome. While more than half of the predicted secretome members encoded lineage-specific proteins, similarities with experimentally characterized fungal effectors were also identified. A SAGE analysis indicated a strong stage-specific regulation of transcripts encoding secreted proteins. The average sequence identity of putative secreted proteins to their closest orthologs in the wheat stem rust Puccinia graminis f. sp. tritici was dramatically reduced compared with non-secreted ones. A comparative genomics approach based on homologous gene groups unravelled positive selection in putative members of the secretome. Conclusion We uncovered robust evidence that different evolutionary constraints are acting on the rust secretome when compared to the rest of the genome. These results are consistent with the view that these genes are more likely to exhibit an effector activity and be involved in coevolutionary arms races with host factors.

  7. Comparative analysis of secreted protein evolution using expressed sequence tags from four poplar leaf rusts (Melampsora spp.).

    Science.gov (United States)

    Joly, David L; Feau, Nicolas; Tanguay, Philippe; Hamelin, Richard C

    2010-07-08

    Obligate biotrophs such as rust fungi are believed to establish long-term relationships by modulating plant defenses through a plethora of effector proteins, whose most recognizable feature is the presence of a signal peptide for secretion. Since the phenotypes of these effectors extend to host cells, their genes are expected to be under accelerated evolution stimulated by host-pathogen coevolutionary arms races. Recently, whole genome sequence data has allowed the prediction of secretomes, facilitating the identification of putative effectors. We generated cDNA libraries from four poplar leaf rust pathogens (Melampsora spp.) and used computational approaches to identify and annotate putative secreted proteins with the aim of uncovering new knowledge about the nature and evolution of the rust secretome. While more than half of the predicted secretome members encoded lineage-specific proteins, similarities with experimentally characterized fungal effectors were also identified. A SAGE analysis indicated a strong stage-specific regulation of transcripts encoding secreted proteins. The average sequence identity of putative secreted proteins to their closest orthologs in the wheat stem rust Puccinia graminis f. sp. tritici was dramatically reduced compared with non-secreted ones. A comparative genomics approach based on homologous gene groups unravelled positive selection in putative members of the secretome. We uncovered robust evidence that different evolutionary constraints are acting on the rust secretome when compared to the rest of the genome. These results are consistent with the view that these genes are more likely to exhibit an effector activity and be involved in coevolutionary arms races with host factors.

  8. Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1

    Directory of Open Access Journals (Sweden)

    Cordula Klockenbusch

    2010-01-01

    Full Text Available Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively.

  9. Fusing Recommendations for Social Bookmarking Websites

    DEFF Research Database (Denmark)

    Bogers, Toine; van den Bosch, Antal

    2011-01-01

    Social bookmarking websites are rapidly growing in popularity. Recommender systems, a promising remedy to the information overload accompanying the explosive growth in content, are designed to identify which unseen content might be of interest to a particular user, based on his or her past...... that use tag overlap and metadata provide better results for social bookmarking data sets than the transaction patterns that are used traditionally in recommender systems research. In addition, we investigate how to fuse different recommendation approaches to further improve recommendation accuracy. We...... preferences. Most previous work in recommendation for social bookmarking suffers from a lack of comparisons between the different available approaches. In this article, we address this issue by comparing and evaluating eight recommendation approaches on four data sets from two domains. We find that approaches...

  10. Satellite Tags- Hawaii EEZ

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Satellite tagging was implemented in 2013. Satellite tagging is conducted using a Dan Inject air rifle and deployment arrows designed by Wildlife Computers. Two...

  11. Donor Tag Game

    Science.gov (United States)

    ... Donor Community > Games > Donor Tag Game Donor Tag Game This feature requires version 6 or later of ... of Needles LGBTQ+ Donors Blood Donor Community SleevesUp Games Facebook Avatars and Badges Banners eCards Make a ...

  12. Cooperative Tagging Center (CTC)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Cooperative Tagging Center (CTC) began as the Cooperative Game Fish Tagging Program (GTP) at Woods Hole Oceanographic Institute (WHOI) in 1954. The GTP was...

  13. Key mediators modulating TAG synthesis and accumulation in ...

    African Journals Online (AJOL)

    the key mediators on TAG synthesis and accumulation, among which diacylglycerol acyltransferases (DGATs) is discussed for its clear role in TAG amount and composition. Furthermore TAG-accosiated proteins called oleosins are also discussed in depth due to their determination on the amount and size of oil bodies.

  14. Fused Lasso Additive Model.

    Science.gov (United States)

    Petersen, Ashley; Witten, Daniela; Simon, Noah

    2016-01-01

    We consider the problem of predicting an outcome variable using p covariates that are measured on n independent observations, in a setting in which additive, flexible, and interpretable fits are desired. We propose the fused lasso additive model (FLAM), in which each additive function is estimated to be piecewise constant with a small number of adaptively-chosen knots. FLAM is the solution to a convex optimization problem, for which a simple algorithm with guaranteed convergence to a global optimum is provided. FLAM is shown to be consistent in high dimensions, and an unbiased estimator of its degrees of freedom is proposed. We evaluate the performance of FLAM in a simulation study and on two data sets. Supplemental materials are available online, and the R package flam is available on CRAN.

  15. Tag-gas encapsulated assembly

    International Nuclear Information System (INIS)

    Murabayashi, Hideki.

    1980-01-01

    Purpose: To ensure tag-gas release upon failure of a fuel cladding tube with no effects from viscosity and surface tension of low melting alloy, by engaging a closing valve body to the inside surface of an opening of the assembly and securing it by means of a low melting alloy. Constitution: A tag-gas capsule is fabricated from a hollow cylindrical body having an outer diameter corresponding to the inner diameter of a fuel pin cladding tube and welded with an upper end plug and a lower end plug. The upper end plug is formed with an opening of a small diameter at the center and with a concaved face at the inside surface. A valve body of a shape just fitting to the concaved face is engaged and secured by means of a low melting alloy to the inside surface of the upper end plug. When the temperature of the capsule arrives at a predetermined temperature to fuse the alloy, the self weight of the valve body overcomes the viscosity and the surface tension of the alloy and the valve body falls instantly. (Sekiya, K.)

  16. The nucleoprotein of Tomato spotted wilt virus as protein tag for easy purification and enhanced production of recombinant proteins in plants

    NARCIS (Netherlands)

    Lacorte, C.C.; Ribeiro, S.G.; Lohuis, H.; Goldbach, R.W.; Prins, M.W.

    2007-01-01

    Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These aggregates result from the homopolymerization of the N protein, and are highly stable in plant cells. These properties feature the N protein as a

  17. A new method for measuring scouring efficiency of natural fibers based on the cellulose-binding domain-beta-glucuronidase fused protein.

    Science.gov (United States)

    Degani, Ofir; Gepstein, Shimon; Dosoretz, Carlos G

    2004-02-05

    Cellulose-binding domains (CBDs) are characterized by their ability to strongly bind to different forms of cellulose. This study examined the use of a recombinant CBD fused to the reporter enzyme beta-glucuronidase (CBD-GUS) to determine the extent of removal of the water-repellent waxy component of cotton fiber cuticles following hydrolytic treatment, i.e., scouring. The CBD-GUS test displayed higher sensitivity and repeatability than conventional water absorb techniques applied in the textile industry. Increases in the levels of CBD-GUS bound to the exposed cellulose correlated to increases in the fabric's hydrophilicity as a function of the severity of the scouring treatment applied, clearly indicating that the amount of bound enzyme increases proportionally with the amount of available binding sites. The binding of CBD-GUS also gave measurable and repeatable results when used on untreated or raw fabrics in comparison with conventional water drop techniques. The quantitative response of the reaction as bound enzyme activity was optimized for fully wettable fabrics. A minimal free enzyme concentration-to-swatch weight ratio of 75:1 was found to be necessary to ensure enzyme saturation (i.e., a linear response), corresponding to a free enzyme-to-bound enzyme ratio of at least 3:5.

  18. Tissue Distribution of Kir7.1 Inwardly Rectifying K+ Channel Probed in a Knock-in Mouse Expressing a Haemagglutinin-Tagged Protein

    Directory of Open Access Journals (Sweden)

    Isabel Cornejo

    2018-04-01

    Full Text Available Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb+ currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation

  19. Colonization of resistant and susceptible lettuce cultivars by a green fluorescent protein-tagged isolate of Verticillium dahliae.

    Science.gov (United States)

    Vallad, G E; Subbarao, K V

    2008-08-01

    Interactions between lettuce and a green fluorescent protein (GFP)-expressing, race 1 isolate of Verticillium dahliae, were studied to determine infection and colonization of lettuce cultivars resistant and susceptible to Verticillium wilt. The roots of lettuce seedlings were inoculated with a conidial suspension of the GFP-expressing isolate. Colonization was studied with the aid of laser scanning confocal and epi-fluorescence microscopes. Few differences in the initial infection and colonization of lateral roots were observed between resistant and susceptible cultivars. Hyphal colonies formed on root tips and within the root elongation zones by 5 days, leading to the colonization of cortical tissues and penetration of vascular elements regardless of the lettuce cultivar by 2 weeks. By 8 to 10 weeks after inoculation, vascular discoloration developed within the taproot and crown regions of susceptible cultivars well in advance of V. dahliae colonization. Actual foliar wilt coincided with the colonization of the taproot and crown areas and the eruption of mycelia into surrounding cortical tissues. Advance colonization of stems, pedicels, and inflorescence, including developing capitula and mature achenes was observed. Seedborne infection was limited to the maternal tissues of the achene, including the pappus, pericarp, integument, and endosperm; but the embryo was never compromised. Resistant lettuce cultivars remained free of disease symptoms. Furthermore, V. dahliae colonization never progressed beyond infected lateral roots of resistant cultivars. Results indicated that resistance in lettuce may lie with the plant's ability to shed infected lateral roots or to inhibit the systemic progress of the fungus through vascular tissues into the taproot.

  20. Intracellular localization of Equine herpesvirus type 1 tegument protein VP22.

    Science.gov (United States)

    Okada, Ayaka; Kodaira, Akari; Hanyu, Sachiko; Izume, Satoko; Ohya, Kenji; Fukushi, Hideto

    2014-11-04

    Intracellular localization of Equine herpesvirus type 1 (EHV-1) tegument protein VP22 was examined by using a plasmid that expressed VP22 fused with an enhanced green fluorescent protein (EGFP). Also a recombinant EHV-1 expressing VP22 fused with a red fluorescent protein (mCherry) was constructed to observe the localization of VP22 in infected cells. When EGFP-fused VP22 was overexpressed in the cells, VP22 localized in the cytoplasm and nucleus. Live cell imaging suggested that the fluorescently tagged VP22 also localized in the cytoplasm and nucleus. These results show that VP22 localizes in the cytoplasm and nucleus independently of other viral proteins. Experiments with truncation mutants of pEGFP-VP22 suggested that 154-188 aa might be the nuclear localization signal of EHV-1 VP22. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Coupled aggregation of mitochondrial single-strand DNA-binding protein tagged with Eos fluorescent protein visualizes synchronized activity of mitochondrial nucleoids

    Czech Academy of Sciences Publication Activity Database

    Olejár, Tomáš; Pajuelo-Reguera, David; Alán, Lukáš; Dlasková, Andrea; Ježek, Petr

    2015-01-01

    Roč. 12, č. 4 (2015), s. 5185-5190 ISSN 1791-2997 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : mitochondrial nucleoid * single- strand ed DNA -binding protein * photoconvertible fluorescent protein Eos Subject RIV: EA - Cell Biology Impact factor: 1.559, year: 2015

  2. Enhancing recombinant protein solubility with ubiquitin-like small archeal modifying protein fusion partners.

    Science.gov (United States)

    Varga, Sándor; Pathare, Ganesh Ramnath; Baka, Erzsébet; Boicu, Marius; Kriszt, Balázs; Székács, András; Zinzula, Luca; Kukolya, József; Nagy, István

    2015-11-01

    A variety of protein expression tags with different biochemical properties has been used to enhance the yield and solubility of recombinant proteins. Ubiquitin, SUMO (small ubiquitin-like modifier) and prokaryotic ubiquitin like MoaD (molybdopterin synthase, small subunit) fusion tags are getting more popular because of their small size. In this paper we report on the use of ubiquitin-like small archaeal modifier proteins (SAMPs) as fusion tags since they proved to increase expression yield, stability and solubility in our experiments. Equally important, they did not co-purify with proteins of the expression host and there was information that their specific JAB1/MPN/Mov34 metalloenzyme (JAMM) protease can recognize the C-terminal VSGG sequence when SAMPs fused, either branched or linearly to target proteins, and cleave it specifically. SAMPs and JAMM proteases from Haloferax volcanii, Thermoplasma acidophilum, Methanococcoides burtonii and Nitrosopumilus maritimus were selected, cloned, expressed heterologously in Escherichia coli and tested as fusion tags and cleaving proteases, respectively. Investigated SAMPs enhanced protein expression and solubility on a wide scale. T. acidophilum SAMPs Ta0895 and Ta01019 were the best performing tags and their effect was comparable to the widely used maltose binding protein (MBP) and N utilization substance protein A (NusA) tags. Moreover, H. volcanii SAMP Hvo_2619 contribution was mediocre, whereas M. burtonii Mbur_1415 could not be expressed. Out of four investigated JAMM proteases, only Hvo_2505 could cleave fusion tags. Interestingly, it was found active not only on its own partner substrate Hvo_2619, but it also cleaved off Ta0895. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Fused salt electrolysis

    International Nuclear Information System (INIS)

    Ares, Osvaldo; Botbol, Jose.

    1989-01-01

    Working conditions for zirconium preparation by fused salt electrolysis were studied. For such purpose, a cell was built for operation under argon atmosphere. A graphite crucible served as anode, with steel cathodes. Proper design allowed cathode rechange under the inert atmosphere. Cathodic deposits of zirconium powder occluded salts from the bath. After washing with both water and hydrochloric acid, the metallic powder was consolidated by fusion. Optimum operating conditions were found to arise from an electrolyte of 12% potassium hexafluorzirconate -88% sodium chloride, at 820 deg C and 5 A/cm 2 cathodic current density. Deposits contained 35% of metal and current efficiency reached 66%. The powder contained up to 600 ppm of chlorine and 1.700 ppm of fluorine; after fusion, those amounts decreased to 2 ppm and 3 ppm respectively, with low proportion of metallic impurities. Though oxygen proportion was 4.500 ppm, it should be lowered by improving working conditions, as well as working on an ampler scale. (Author)

  4. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    Science.gov (United States)

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( GPCR, MMP2/9, hbEGF, EGFR, erbB2, and IGF-1R. However, the mechanism through which TBA mediates changes in protein degradation is different and appears to involve only the EGFR and erbB2. Furthermore, it appears the protein kinase B pathway is involved in TBA's effects on fused BSC cultures.

  5. Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging

    Directory of Open Access Journals (Sweden)

    Feng Gao

    2016-08-01

    Full Text Available Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag, by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.

  6. Hirudin as a novel fusion tag for efficient production of lunasin in Escherichia coli.

    Science.gov (United States)

    Tian, Qinghua; Zhang, Ping; Gao, Zhan; Li, Hengli; Bai, Zhengli; Tan, Shuhua

    2017-07-03

    Fusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380 mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86 mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.

  7. Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression.

    Science.gov (United States)

    Morino, K; Katsumi, H; Akahori, Y; Iba, Y; Shinohara, M; Ukai, Y; Kohara, Y; Kurosawa, Y

    2001-11-01

    We developed a system by which antibodies, fused to fluorescent proteins with different wavelengths, can be prepared within a month against various antigens. An antibody library composed of a large number of single-chain Fv-CL fragment was constructed by means of a phage-display system. The constructs were designed to facilitate changing of the protein forms by simple enzyme manipulation. In the present study, we adopted a molecular form of antibody in which a single-chain Fv-CL fragment is fused with a green fluorescent protein (GFP) or red fluorescent protein (RFP). In addition, a His-tag was inserted between CL and GFP (or RFP). We describe the utility of this system using Caenorhabditis elegans embryo as a model.

  8. Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.

    Science.gov (United States)

    Huang, Qiwei; Li, Qingxin; Joy, Joma; Chen, Angela Shuyi; Ruiz-Carrillo, David; Hill, Jeffrey; Lescar, Julien; Kang, Congbao

    2013-12-01

    Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Tagging vs. Controlled Vocabulary

    DEFF Research Database (Denmark)

    Bogers, Toine; Petras, Vivien

    2015-01-01

    elements like core bibliographic data, controlled vocabulary terms, reviews, and tags to the retrieval performance. Our comparison is done using a test collection of over 2 million book records with information elements from Amazon, the British Library, the Library of Congress, and LibraryThing. We find...... that tags and controlled vocabulary terms do not actually outperform each other consistently, but seem to provide complementary contributions: some information needs are best addressed using controlled vocabulary terms whereas other are best addressed using tags....

  10. Tags on healthcare information websites

    DEFF Research Database (Denmark)

    Lykke, Marianne; Ådland, Marit Kristine

    2018-01-01

    This paper explores tags and tagging behaviour on health information websites using an empirical, user-oriented, exploratory case study. Taggers and editors were interviewed about tags and tagging, while taggers solved tasks that included applying tags to a website. This qualitative data...... articles, request information, and value article content. Some of these show that tags are not only not only topical descriptions, but communicative by intent. This result can potentially inform the design of tagging features....

  11. In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Aytak Novinrooz

    Full Text Available E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC, and fatal hemolytic uremic syndrome (HUS and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA, and B subunit of E. coli heat labile enterotoxin (LTB which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+ expression vector and transferred to E. coli BL21(DE3 cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3 cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG. The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient

  12. Recombinant human CIS2 (SOCS2) protein: subcloning, expression, purification, and characterization.

    Science.gov (United States)

    Biener, Eva; Maurice, Sarah; Sandowski, Yael; Cohen, Yael; Gusakowsky, Eugene E; Hooghe, Robert; Yoshimura, Akihiko; Livnah, Oded; Gertler, Arieh

    2002-07-01

    The 1x myc-tagged cDNA encoding for human CIS2 protein was subcloned into a pET-29a+ vector in order to express and produce a recombinant S-peptide tagged and 1x myc-tagged protein in Escherichia coli BL21(DE3). The constitutively expressed protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by anion-exchange chromatography on Q-Sepharose. The recombinant form was found to be pure and monomeric as judged by both SDS-PAGE and gel-filtration chromatography and its biological activity was proven by its ability to bind to the tyrosine-phosphorylated cytosolic fragment of human growth hormone receptor fused to glutathione-S-transferase. Recombinant CIS2 was compared by biochemical, immunological, and molecular methods to the CIS2 protein expressed in eukaryotic cells. This report describes the first substantial production of biologically active recombinant human CIS2.

  13. DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

    Science.gov (United States)

    Jing, Chaoran; Cornish, Virginia W.

    2013-01-01

    Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live cell imaging. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

  14. Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins.

    Science.gov (United States)

    Gutiérrez, Sonia P; Saberianfar, Reza; Kohalmi, Susanne E; Menassa, Rima

    2013-05-10

    Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels

  15. QUAD TAG MODULE

    International Nuclear Information System (INIS)

    Shiino, K.; Maruyama, K.

    1981-08-01

    Electronic circuits called QUAD TAG MODULE were developed and constructed, which were of exclusive use for signals from scintillation counter hodoscope of the photon tagging system. The circuit has functions of amplifiers, discriminators, and strobed coincidences in one NIM module. A description on functions and specifications of the module is given. (author)

  16. PIT Tagging Anurans

    Science.gov (United States)

    McCreary, Brome

    2008-01-01

    The following video demonstrates a procedure to insert a passive integrated transponder (PIT) tag under the skin of an anuran (frog or toad) for research and monitoring purposes. Typically, a 12.5 mm tag (0.5 in.) is used to uniquely identify individual anurans as smal as 40 mm (1.6 in.) in length from snout to vent. Smaller tags are also available and allow smaller anurans to be tagged. The procedure does not differ for other sizes of tages or other sizes of anurans. Anyone using this procedure should ensure that the tag is small enough to fit easily behind the sacral hump of the anuran, as shown in this video.

  17. A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication.

    Science.gov (United States)

    Ohsaki, Eriko; Ueda, Keiji

    2017-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour

    Directory of Open Access Journals (Sweden)

    Altenbach Susan B

    2010-01-01

    Full Text Available Abstract Background The gamma gliadins are a complex group of proteins that together with other gluten proteins determine the functional properties of wheat flour. The proteins have unusually high levels of glutamine and proline and contain large regions of repetitive sequences. While most gamma gliadins are monomeric proteins containing eight conserved cysteine residues, some contain an additional cysteine residue that enables them to be linked with other gluten proteins into large polymers that are critical for flour quality. The ability to differentiate among the gamma gliadins is important for studies of wheat flour quality because proteins with similar sequences can have different effects on functional properties. Results The complement of gamma gliadin genes expressed in the wheat cultivar Butte 86 was evaluated by analyzing publicly available expressed sequence tag (EST data. Eleven contigs were assembled from 153 Butte 86 ESTs. Nine of the contigs encoded full-length proteins and four of the proteins contained nine cysteine residues. Only one of the encoded proteins was a perfect match with a sequence reported in NCBI. Contigs from four different publicly available EST assemblies encoded proteins that were perfect matches with some, but not all, of the Butte 86 gamma gliadins and the complement of identical proteins was different for each assembly. A specialized database that included the sequences of Butte 86 gamma gliadins was constructed for identification of flour proteins by tandem mass spectrometry (MS/MS. In a pilot experiment, proteins corresponding to six Butte 86 gamma gliadin contigs were distinguished by MS/MS, including one containing the extra cysteine residue. Two other proteins were identified as one of two closely related Butte 86 proteins but could not be distinguished unequivocally. Unique peptide tags specific for Butte 86 gamma gliadins are reported. Conclusions Inclusion of cultivar-specific gamma gliadin sequences

  19. Unbiased Selective Isolation of Protein N-Terminal Peptides from Complex Proteome Samples Using Phospho Tagging PTAG) and TiO2-based Depletion

    NARCIS (Netherlands)

    Mommen, G.P.M.; Waterbeemd, van de B.; Meiring, H.D.; Kersten, G.; Heck, A.J.R.; Jong, de A.P.J.M.

    2012-01-01

    A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO2) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with

  20. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    International Nuclear Information System (INIS)

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

    2004-01-01

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d 3 ) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d 3 . In addition, both sCD4-gp120 and sCD4-gp120-mC3d 3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d 3 or sCD4-gp120-mC3d 3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d 3 -DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d 3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d

  1. A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters.

    Science.gov (United States)

    Ma, Cheng; Hao, Zhenyu; Huysmans, Gerard; Lesiuk, Amelia; Bullough, Per; Wang, Yingying; Bartlam, Mark; Phillips, Simon E; Young, James D; Goldman, Adrian; Baldwin, Stephen A; Postis, Vincent L G

    2015-01-01

    Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a

  2. Multicolor imaging of bacterial nucleoid and division proteins with blue, orange and near-infrared fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Fabai eWu

    2015-06-01

    Full Text Available Studies of the spatiotemporal protein dynamics within live bacterial cells impose a strong demand for multi-color imaging. Despite the increasingly large collection of fluorescent-protein variants engineered to date, only a few of these were successfully applied in bacteria. Here, we explore the performance of recently engineered variants with the blue (TagBFP, orange (TagRFP-T, mKO2 and far-red (mKate2 spectral colors by tagging HU, LacI, MinD, and FtsZ for visualizing the nucleoid and the cell division process. We find that, these fluorescent proteins outperformed previous versions in terms of brightness and photostability at their respective spectral range, both when expressed as cytosolic label and when fused to native proteins. As this indicates that their folding is sufficiently fast, these proteins thus successfully expand the applicable spectra for multi-color imaging in bacteria. A near-infrared protein (eqFP670 is found to be the most red-shifted protein applicable to bacteria so far, with brightness and photostability that are advantageous for cell-body imaging, such as in microfluidic devices. Despite the multiple advantages, we also report the alarming observation that TagBFP directly interacts with TagRFP-T, causing interference of localization patterns between their fusion proteins. Our application of diverse fluorescent proteins for endogenous tagging provides guidelines for future engineering of fluorescent fusions in bacteria, specifically: 1 The performance of newly developed fluorescent proteins should be quantified in vivo for their introduction into bacteria; 2 spectral crosstalk and inter-variant interactions between fluorescent proteins should be carefully examined for multi-color imaging; and 3 successful genomic fusion to the 5’-end of a gene strongly depends on the translational read-through of the inserted coding sequence.

  3. Cryopreservation of green fluorescent protein (GFP)-labeled primordial germ cells with GFP fused to the 3' untranslated region of the nanos gene by vitrification of Japanese eel (Anguilla japonica) somite stage embryos.

    Science.gov (United States)

    Kawakami, Y; Ishihara, M; Saito, T; Fujimoto, T; Adachi, S; Arai, K; Yamaha, E

    2012-12-01

    Primordial germ cells (PGC) are the only cell type in developing embryos with the potential to transmit genetic information to the next generation. In this study, PGC of Japanese eel (Anguilla japonica) were visualized by injection of mRNA synthesized from a construct carrying the green fluorescent protein (GFP) gene fused to the 3' untranslated region of the Japanese eel nanos gene. We investigated the feasibility of cryopreserving Japanese eel PGC by vitrification of dechorionated whole somite stage embryos. The GFP-labeled PGC were rapidly cooled using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (methanol, dimethyl sulfoxide, and glycerol for 10 min and ethylene glycol for 10, 20, and 30 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose for 1, 5, and 10 min. Ethylene glycerol is an effective cryoprotectant for embryonic cells and shows no evidence of ice formation after thawing. Vitrified and thawed PGC were transplanted into blastula stage embryos from zebrafish (Danio rerio). The GFP-labeled PGC migrated toward the host gonadal ridge, suggesting maintenance of their normal migration motility. These techniques may assist in achieving inter- and intraspecies germ-line chimers using donor Japanese eel PGC.

  4. Protein bodies in leaves exchange contents through the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Reza eSaberianfar

    2016-05-01

    Full Text Available Protein bodies (PBs are organelles found in seeds whose main function is the storage of proteins that are used during germination for sustaining growth. PBs can also be induced to form in leaves when foreign proteins are produced at high levels in the endoplasmic reticulum (ER and when fused to one of three tags: Zera®, elastin-like polypeptides (ELP, or hydrophobin-I (HFBI. In this study, we investigate the differences between ELP, HFBI and Zera PB formation, packing, and communication. Our results confirm the ER origin of all three fusion-tag-induced PBs. We show that secretory pathway proteins can be sequestered into all types of PBs but with different patterns, and that different fusion tags can target a specific protein to different PBs. Zera PBs are mobile and dependent on actomyosin motility similar to ELP and HFBI PBs. We show in vivo trafficking of proteins between PBs using GFP photoconversion. We also show that protein trafficking between ELP or HFBI PBs is faster and proteins travel further when compared to Zera PBs. Our results indicate that fusion-tag-induced PBs do not represent terminally stored cytosolic organelles, but that they form in, and remain part of the ER, and dynamically communicate with each other via the ER. We hypothesize that the previously documented PB mobility along the actin cytoskeleton is associated with ER movement rather than independent streaming of detached organelles.

  5. Synthesis of a polyhistidine-bearing amphipol and its use for immobilizing membrane proteins

    DEFF Research Database (Denmark)

    Giusti, Fabrice; Kessler, Pascal; Hansen, Randi Westh

    2015-01-01

    APol, are described. Its ability to immobilize MPs on nickel ion-bearing surfaces was tested using two complementary methods, immobilized metal affinity chromatography (IMAC) and surface plasmon resonance (SPR). Compared to a single His6-tag fused at one extremity of a MP, the presence of several His6-tags carried...... by the APol belt surrounding the transmembrane domain of a MP increases remarkably the affinity of the protein/APol complex for nickel ion-bearing SPR chips, whereas it does not show such a strong effect on an IMAC resin. HistAPol-mediated immobilization, which allows reversibility of the interaction and easy...

  6. Flavour Tagging at LHCb

    CERN Document Server

    Grabalosa Gandara, M

    2009-01-01

    To do precise CP violation measurements, the most possible accurate knowledge of the flavour at production of the reconstructed B meson is required. This poster summarizes the flavour tagging performances for the LHCb experiment. We use same side an opposite side algorithms to establish wheter the meson contained a b or a b\\bar quark. The final decision is obtained through a combination of several methods. The use of control channels, decays to a flavour specific final state, will allow to determine the wrong tag fraction \\omega (the probability of a tag to be wrong), which can be used as input for the determination of CKM unitary triangle angles.

  7. High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression.

    Science.gov (United States)

    Lai, Guan-Hua; Lin, Yen-Chang; Tsai, Yi-Lun; Lien, Yi-Yang; Lin, Ming-Kuem; Chen, Hsi-Jien; Chang, Wen-Te; Tzen, Jason T C; Lee, Meng-Shiou

    2014-05-22

    Pigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap (or C1) gene of PiCV, has been shown to be responsible for not only capsid assembly, but also has been used as antigen for detecting antibody when the host is infected with PiCV. The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system. The PiCV cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to have good antigenic

  8. Super magnetic nanoparticles NiFe2O4, coated with aluminum-nickel oxide sol-gel lattices to safe, sensitive and selective purification of his-tagged proteins.

    Science.gov (United States)

    Mirahmadi-Zare, Seyede Zohreh; Allafchian, Alireza; Aboutalebi, Fatemeh; Shojaei, Pendar; Khazaie, Yahya; Dormiani, Kianoush; Lachinani, Liana; Nasr-Esfahani, Mohammad-Hossein

    2016-05-01

    Super magnetic nanoparticle NiFe2O4 with high magnetization, physical and chemical stability was introduced as a core particle which exhibits high thermal stability (>97%) during the harsh coating process. Instead of multi-stage process for coating, the magnetic nanoparticles was mineralized via one step coating by a cheap, safe, stable and recyclable alumina sol-gel lattice (from bohemite source) saturated by nickel ions. The TEM, SEM, VSM and XRD imaging and BET analysis confirmed the structural potential of NiFe2O4@NiAl2O4 core-shell magnetic nanoparticles for selective and sensitive purification of His-tagged protein, in one step. The functionality and validity of the nickel magnetic nanoparticles were attested by purification of three different bioactive His-tagged recombinant fusion proteins including hIGF-1, GM-CSF and bFGF. The bonding capacity of the nickel magnetics nanoparticles was studied by Bradford assay and was equal to 250 ± 84 μg Protein/mg MNP base on protein size. Since the metal ion leakage is the most toxicity source for purification by nickel magnetic nanoparticles, therefor the nickel leakage in purified final protein was determined by atomic absorption spectroscopy and biological activity of final purified protein was confirmed in comparison with reference. Also, in vitro cytotoxicity of nickel magnetic nanoparticles and trace metal ions were investigated by MTS assay analysis. The results confirmed that the synthesized nickel magnetic nanoparticles did not show metal ion toxicity and not affected on protein folding. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Addition of urea and thiourea to electrophoresis sample buffer improves efficiency of protein extraction from TCA/acetone-treated smooth muscle tissues for phos-tag SDS-PAGE.

    Science.gov (United States)

    Takeya, Kosuke; Kaneko, Toshiyuki; Miyazu, Motoi; Takai, Akira

    2018-01-01

    Phosphorylation analysis by using phos-tag technique has been reported to be suitable for highly sensitive quantification of smooth muscle myosin regulatory light chain (LC 20 ) phosphorylation. However, there is another factor that will affect the sensitivity of phosphorylation analysis, that is, protein extraction. Here, we optimized the conditions for total protein extraction out of trichloroacetic acid (TCA)-fixed tissues. Standard SDS sample buffer extracted less LC 20 , actin and myosin phosphatase targeting subunit 1 (MYPT1) from TCA/acetone treated ciliary muscle strips. On the other hand, sample buffer containing urea and thiourea in addition to lithium dodecyl sulfate (LDS) or SDS extracted those proteins more efficiently, and thus increased the detection sensitivity up to 4-5 fold. Phos-tag SDS-PAGE separated dephosphorylated and phosphorylated LC 20 s extracted in LDS/urea/thiourea sample buffer to the same extent as those in standard SDS buffer. We have concluded that LDS (or SDS) /urea/thiourea sample buffer is suitable for highly sensitive phosphorylation analysis in smooth muscle, especially when it is treated with TCA/acetone. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Tagged Vector Contour (TVC)

    Data.gov (United States)

    Kansas Data Access and Support Center — The Kansas Tagged Vector Contour (TVC) dataset consists of digitized contours from the 7.5 minute topographic quadrangle maps. Coverage for the state is incomplete....

  11. N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

    DEFF Research Database (Denmark)

    Dreisig, Karin; Kristensen, Nikolaj Pagh; Dommer, Maja Wallentin

    2018-01-01

    uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N......The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins...... might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye...

  12. Western Blotting Against Tagged Virulence Determinants to Study Bacterial Pathogenicity.

    Science.gov (United States)

    Aviv, Gili; Gal-Mor, Ohad

    2018-01-01

    Western blotting is a common approach to detect the presence of a target protein in biological samples or proteins mixture using specific antibodies. This method is also useful to study regulation of virulence determinants by analyzing changes in protein expression between different genetic backgrounds or under varying environmental conditions. To avoid the need to raise specific antibodies for each studied protein, commercial antibody against commonly used peptidic epitopes can be utilized if the right target tagged version is constructed. Here we describe a C-terminal fusion between a protein of interest and the two hemagglutinin A (2HA) tag. The tagged protein is cloned into a low-copy number vector and expressed under its native promoter in Salmonella enterica. Then, the expression of the tagged protein can be analyzed by Western blotting and commercially available anti-2HA antibodies.

  13. The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology.

    Science.gov (United States)

    Costa, Sofia J; Almeida, André; Castro, António; Domingues, Lucília; Besir, Hüseyin

    2013-08-01

    The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.

  14. Liquefier Dynamics in Fused Deposition

    DEFF Research Database (Denmark)

    Bellini, Anna; Guceri, Selcuk; Bertoldi, Maurizio

    2004-01-01

    Layered manufacturing (LM) is an evolution of rapid prototyping (RP) technology whereby a part is built in layers. Fused deposition modeling (FDM) is a particular LM technique in which each section is fabricated through vector style deposition of building blocks, called roads, which...

  15. ATLAS Boosted Objects tagging performance

    CERN Document Server

    Ferreira de Lima, D E; The ATLAS collaboration

    2014-01-01

    In the session on the performance of jet substructure reconstruction and boosted object tagging. This first contribution focuses on boosted object tagging performance. Material includes the shower deconstruction CONF note (missed BOOST2013, public), the Boosted boson tagging note (ATL-PHYS-PUB-2014-004, public since April). New papers on W/Z dsicrimination or top tagging are unlikely.

  16. Facets: Ersatz, Resource and Tag

    Science.gov (United States)

    Frické, Martin H.

    2013-01-01

    Introduction: Faceted classification appears to be of utmost importance. Ersatz facets, resource faceting and tag faceting: The distinctions are drawn between facets and ersatz facets, and between faceted resources and faceted tags. Single tag resource faceting and multiple tag information object faceting: The basic features are explored of single…

  17. An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph, E-mail: kappock@purdue.edu [Purdue University, 175 South University Street, West Lafayette, IN 47907-2063 (United States)

    2015-09-23

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

  18. A comparison of tagging methods and their tagging space.

    Science.gov (United States)

    Ke, Xiayi; Miretti, Marcos M; Broxholme, John; Hunt, Sarah; Beck, Stephan; Bentley, David R; Deloukas, Panos; Cardon, Lon R

    2005-09-15

    Single-nucleotide polymorphism (SNP) tagging is widely used as a way of saving genotyping costs in association studies. A number of different tagging methods have been developed to reduce the number of markers to be genotyped while maintaining power for detecting effects on non-assayed SNPs. How the different methods perform in different settings, the degree to which they overlap and share common tags and how they differ are important questions. We investigated these questions by comparing three widely used tagging methods/algorithms--one haplotype r2-based method, one pair-wise r2-based method and one method which was based on haplotype diversity but focused on major haplotypes. Tagging efficiency was defined as the number of genotyped markers divided by the number of tagging SNPs. Tagging effectiveness was defined as the proportion of un-genotyped or 'hidden' SNPs being detected (having a pair-wise or haplotype r2 with a set of tagging SNPs over a threshold, e.g. haplotype r2> or =0.80). The ENCODE regions genotyped on the HapMap CEPH individuals were examined in this study. Tagging effectiveness was generally poor for rare SNPs than for common SNPs, for all three tagging methods. Inclusion of rare SNPs into initial HapMap scheme could enhance the performance of tags on rare hidden SNPs at the expense of increased genotyping cost. At a moderate tagging efficiency, more than 90% of hidden SNPs detected by tagging SNPs selected by one method were also detected by tagging SNPs selected by another method, and this figure could be increased to 100% if tagging efficiency was allowed to drop. These results indicate that the tagging space is highly concordant between different tagging methods, despite the fact that they often involve different sets of tagging SNPs.

  19. A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli.

    Science.gov (United States)

    Cheng, Cheng; Wu, Shanshan; Cui, Lupeng; Wu, Yulu; Jiang, Tianyue; He, Bingfang

    2017-12-21

    The high level of excretion and rapid folding ability of β-fructofuranosidase (β-FFase) in Escherichia coli has suggested that β-FFase from Arthrobacter arilaitensis NJEM01 can be developed as a fusion partner. Based on the modified Wilkinson and Harrison algorithm and the preliminary verification of the solubility-enhancing ability of β-FFase truncations, three β-FFase truncations (i.e., Ffu209, Ffu217, and Ffu312) with a native signal peptide were selected as novel Ffu fusion tags. Four difficult-to-express protein models; i.e., CARDS TX, VEGFR-2, RVs and Omp85 were used in the assessment of Ffu fusion tags. The expression levels and solubility of each protein were markedly enhanced by the Ffu fusion system. Each protein had a favorable Ffu tag. The Ffu fusion tags performed preferably when compared with the well-known fusion tags MBP and NusA. Strikingly, it was confirmed that Ffu fusion proteins were secreted into the periplasm by the periplasmic analysis and N-amino acid sequence analysis. Further, efficient excretion of HV3 with defined anti-thrombin activity was obtained when it was fused with the Ffu312 tag. Moreover, HV3 remained soluble and demonstrated notable anti-thrombin activity after the removal of the Ffu312 tag by enterokinase. Observations from this work not only complements fusion technologies, but also develops a novel and effective secretory system to solve key issues that include inclusion bodies and degradation when expressing heterologous proteins in E. coli, especially for proteins that require disulfide bond formation, eukaryotic-secreted proteins, and membrane-associated proteins.

  20. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

    Energy Technology Data Exchange (ETDEWEB)

    Tykvart, J.; Sacha, P.; Barinka, C.; Knedlik, T.; Starkova, J.; Lubkowski, J.; Konvalinka, J. (Gilead); (NCI); (Czech Academy)

    2012-02-07

    Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

  1. Tagging behaviour with support from controlled vocabulary

    DEFF Research Database (Denmark)

    Lykke, Marianne; Høj, Anne Lyhne; Madsen, Line Nørgaard

    2011-01-01

    The paper investigates how knowledge structures from a controlled vocabulary affect tagging. The study is a comparative analysis of tags assigned in two tagging systems, a simple tagging system (control system) that provides suggestions from two tag clouds (all users tags and my tags) and an enha...

  2. The use of external electronic tags on fish: an evaluation of tag retention and tagging effects

    DEFF Research Database (Denmark)

    Jepsen, Niels; Thorstad, Eva B.; Havn, Torgeir

    2015-01-01

    publications, reporting effects of external tagging for 80 different fish species, which constitute the main basis for this review. External attachment holds certain benefits compared to other tagging methods, for example, speed of application, and it may be the only option for fishes with a body shape...... unsuitable for surgical implantation, or when using tags with sensors recording the external environment. The most commonly reported problems with external tags are tissue damage, premature tag loss, and decreased swimming capacity, but the effects are highly context dependent and species specific. Reduced...... growth and survival have also been recorded, but direct mortality caused by external tagging seems rare. Most of the studies reviewed evaluate tag retention, survival, and tissue reactions. There is a general need for more research on the effects of external tagging of fish with electronic tags...

  3. TagPad

    DEFF Research Database (Denmark)

    Bornoe, Nis; Barkhuus, Louise

    2013-01-01

    The area of cyberinfrastructures has looked extensively at research within the natural sciences, however, the social sciences have been largely overlooked in terms of novel data collection and analysis systems. We developed a probe tool, TagPad, to look at the process for social science data...

  4. Enhancement of solubility and yield of a β-glucan receptor Dectin-1 C-type lectin-like domain in Escherichia coli with a solubility-enhancement tag.

    Science.gov (United States)

    Dulal, Hari Prasad; Nagae, Masamichi; Ikeda, Akemi; Morita-Matsumoto, Kana; Adachi, Yoshiyuki; Ohno, Naohito; Yamaguchi, Yoshiki

    2016-07-01

    Dectin-1 is a C-type lectin-like pattern recognition receptor for β(1-3)-glucans. It plays a crucial role in protecting against fungal invasion through binding to β-glucans which are commonly present on the fungal cell wall. To probe its ligand binding mechanism by NMR, we expressed the recombinant murine Dectin-1 C-type lectin-like domain (CTLD) in E. coli using pCold vector and purified it. However, the high concentration of Dectin-1 CTLD required for NMR analysis could not be attained due to its inherent low solubility and low bacterial expression. In this study, we tried to increase expression and solubility of Dectin-1 CTLD by codon optimization and fusion of a GB1 tag (B1 domain of streptococcal Protein G). GB1 was inserted on either the N-terminal (NT) or C-terminal end as well as both terminal ends of human and mouse Dectin-1 CTLDs. A pure monomeric sample was only obtained with NT-GB1 fused mouse Dectin-1. Expression of mouse Dectin-1 CTLD yielded 0.9 ± 0.2 mg/L culture, codon optimized mouse Dectin-1 CTLD produced 1.4 ± 0.2 mg/L, and the tag-fused domain 7.1 ± 0.3 mg/L. The tag also increased solubility from 0.1 mM to 1.4 mM. The recombinant protein was correctly folded, in a monomeric state, and specifically bound β-glucan laminarin. These results indicate that fusing GB1 to the N-terminus of mouse Dectin-1 domain advantageously increases yield and solubility, allows retention of native structure, and that the site of fusion is critical. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Membrane-Based Inverse Transition Cycling: An Improved Means for Purifying Plant-Derived Recombinant Protein-Elastin-Like Polypeptide Fusions

    Directory of Open Access Journals (Sweden)

    Udo Conrad

    2011-04-01

    Full Text Available Elastin-like peptide (ELP was fused to two different avian flu H5N1 antigens and expressed in transgenic tobacco plants. The presence of the ELP tag enhanced the accumulation of the heterologous proteins in the tobacco leaves. An effective membrane-based Inverse Transition Cycling was developed to recover the ELPylated antigens and antibodies from plant material. The functionality of both the ELPylated neuraminidase and an ELPylated nanobody was demonstrated.

  6. Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells.

    Science.gov (United States)

    Xiao, Yuhong; Kwon, Kwang-Chul; Hoffman, Brad E; Kamesh, Aditya; Jones, Noah T; Herzog, Roland W; Daniell, Henry

    2016-02-01

    Targeted oral delivery of GFP fused with a GM1 receptor binding protein (CTB) or human cell penetrating peptide (PTD) or dendritic cell peptide (DCpep) was investigated. Presence of GFP(+) intact plant cells between villi of ileum confirm their protection in the digestive system from acids/enzymes. Efficient delivery of GFP to gut-epithelial cells by PTD or CTB and to M cells by all these fusion tags confirm uptake of GFP in the small intestine. PTD fusion delivered GFP more efficiently to most tissues or organs than the other two tags. GFP was efficiently delivered to the liver by all fusion tags, likely through the gut-liver axis. In confocal imaging studies of human cell lines using purified GFP fused with different tags, GFP signal of DCpep-GFP was only detected within dendritic cells. PTD-GFP was only detected within kidney or pancreatic cells but not in immune modulatory cells (macrophages, dendritic, T, B, or mast cells). In contrast, CTB-GFP was detected in all tested cell types, confirming ubiquitous presence of GM1 receptors. Such low-cost oral delivery of protein drugs to sera, immune system or non-immune cells should dramatically lower their cost by elimination of prohibitively expensive fermentation, protein purification cold storage/transportation and increase patient compliance. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Coenzyme- and His-tag-induced crystallization of octopine dehydrogenase

    International Nuclear Information System (INIS)

    Smits, Sander H. J.; Mueller, Andre; Grieshaber, Manfred K.; Schmitt, Lutz

    2008-01-01

    The crystal structure of octopine dehydrogenase revealed a specific role of the His 5 tag in inducing the crystal contacts required for successful crystallization. Over the last decade, protein purification has become more efficient and standardized through the introduction of affinity tags. The choice and position of the tag, however, can directly influence the process of protein crystallization. Octopine dehydrogenase (OcDH) without a His tag and tagged protein constructs such as OcDH-His 5 and OcDH-LEHis 6 have been investigated for their crystallizability. Only OcDH-His 5 yielded crystals; however, they were multiple. To improve crystal quality, the cofactor NADH was added, resulting in single crystals that were suitable for structure determination. As shown by the structure, the His 5 tag protrudes into the cleft between the NADH and l-arginine-binding domains and is mainly fixed in place by water molecules. The protein is thereby stabilized to such an extent that the formation of crystal contacts can proceed. Together with NADH, the His 5 tag obviously locks the enzyme into a specific conformation which induces crystal growth

  8. Tissue-specific tagging of endogenous loci in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Kate Koles

    2016-01-01

    Full Text Available Fluorescent protein tags have revolutionized cell and developmental biology, and in combination with binary expression systems they enable diverse tissue-specific studies of protein function. However these binary expression systems often do not recapitulate endogenous protein expression levels, localization, binding partners and/or developmental windows of gene expression. To address these limitations, we have developed a method called T-STEP (tissue-specific tagging of endogenous proteins that allows endogenous loci to be tagged in a tissue specific manner. T-STEP uses a combination of efficient CRISPR/Cas9-enhanced gene targeting and tissue-specific recombinase-mediated tag swapping to temporally and spatially label endogenous proteins. We have employed this method to GFP tag OCRL (a phosphoinositide-5-phosphatase in the endocytic pathway and Vps35 (a Parkinson's disease-implicated component of the endosomal retromer complex in diverse Drosophila tissues including neurons, glia, muscles and hemocytes. Selective tagging of endogenous proteins allows, for the first time, cell type-specific live imaging and proteomics in complex tissues.

  9. FUSE Mission Overview and Status

    Science.gov (United States)

    Sonneborn, G.

    The Far Ultraviolet Spectroscopic Explorer satellite observes light in the 905-1187 Å region with high spectral resolution (λ/Δλ ~ 20,000). FUSE was launched on 24 June 1999 and the early observations have yielded significant results in several areas of galactic and extragalactic astronomy. The sensitivity is sufficient to examine reddened lines of sight within the Milky Way as well as active galactic nuclei and QSOs for absorption line studies of both Milky Way and extragalactic gas clouds. This spectral region contains a number of important scientific diagnostics, including O VI, H I, D I, and the strong electronic transitions of the H2 and HD molecules. The instrument has four coaligned prime-focus telescopes and Rowland spectrographs with microchannel plate detectors. Two channels use Al:LiF coatings for optimum reflectivity from ~ 1000 to 1187 Å and the other two use SiC coatings for optimized throughput between 905 and 1105 Å. The gratings were holographically ruled to largely correct for astigmatism and to minimize scattered light. The detectors have KBr photocathodes and use photon counting to achieve good quantum efficiency with low background signal. The primary mission duration is three years; an extended mission phase is planned. A majority of the observing time is allocated to guest observers. (http://fuse.pha.jhu.edu)

  10. About a New Type of Fuse Based on the Controllable Fusing Effect

    Directory of Open Access Journals (Sweden)

    PLESCA, A.

    2009-06-01

    Full Text Available Fuses are among the best known of electrical devices and there are an extremely large number in use throughout the world. Beside of the advantageous features, the nowadays fuses have certain drawbacks. Therefore, a new type of fuse based on controllable fusing concept is proposed and a study as regards the total clearing time is done. The new concept has been validated through many experimental tests at different current values. The new type of fuse based on controllable fusing concept can be integrated within an overcurrent protection system especially to protect power semiconductors where the Joule integral criterion is better satisfied.

  11. Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Feng; Jaitly, Navdeep; Jayachandran, Hemalatha; Lou, Quanzhou; Monroe, Matthew E.; Du, Xiuxia; Gritsenko, Marina A.; Zhang, Rui; Anderson, David J.; Purvine, Samuel O.; Adkins, Joshua N.; Moore, Ronald J.; Mottaz, Heather M.; Ding, Shi-Jian; Lipton, Mary S.; Camp, David G.; Udseth, Harold R.; Smith, Richard D.; Rossie, Sandra S.

    2007-10-12

    To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative Phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.

  12. Recombinant foot-and-mouth disease virus (FMDV) non-structural protein 3A fused to enhanced green fluorescent protein (EGFP) as a candidate probe to identify FMDV-infected cattle in serosurveys.

    Science.gov (United States)

    Lotufo, Cecilia M; Bergmann, Ingrid E; Mattion, Nora M; Wilda, Maximiliano; Grigera, Pablo R

    2017-08-01

    Recombinant protein 3A-EGFP, a fusion construct between foot-and-mouth disease virus (FMDV) non-structural protein 3A and the enhanced green fluorescent protein (EGFP) was expressed in BL21-DE3 cells. The identity of the partially purified protein 3A-EGFP was confirmed by its reactivity with sera from cattle infected with FMDV and with a monoclonal antibody specific for FMDV-3ABC (MAb3H7) in Western blot assays. No reactivity was observed with sera from uninfected vaccinated animals. The performance of 3A-EGFP as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) was assessed and compared with that of a previously developed and validated capture ELISA that uses a 3ABC recombinant antigen (3ABC ELISA) and has been widely applied for serological surveys in Argentina. Parallel analysis of strongly and weakly positive reference sera from infected animals and 329 serum samples from uninfected vaccinated cattle showed that the 3A-EGFP antigen unequivocally identifies sera from FMDV-infected cattle with similar performance to its 3ABC counterpart. The 3A-EGFP ELISA is simpler and faster to perform than the 3ABC ELISA, since it does not require a capture step with a specific antibody. Moreover, the expression and storage of the recombinant 3A-EGFP is simplified by the absence of residual autoproteolytic activity associated to the 3C sequence. We conclude that the 3A-EGFP ELISA constitutes a promising screening method in serosurveys to determine whether or not animals are infected with FMDV.

  13. Tag-elese or The Language of Tags

    Directory of Open Access Journals (Sweden)

    Jan Simons

    2008-01-01

    Full Text Available The core "meme" of Web 2.0 from which almost all other memes radiated was: 'You control your own data' (O'Reilly, 2005, 3. Key instruments for this user control are tagging systems that allow users to freely assign keywords of their own choosing to Internet resources of their own making as well as to documents produced by others. Of course, freely chosen keywords tags do not necessarily follow prefixed taxonomies or classification systems. But going by the maxim that interaction creates similarity and similarity creates interaction, the idea - or hope - is, however, that the tagging practices of individual users will eventually converge into an emergent common vocabulary or folksonomy (Merholz, 2004; Shirky, 2005; Vander Wal, 2005b; Mika, 2007. It is far from clear, however, that free tagging systems will eventually yield controlled vocabularies, and there are many incentives for idiosyncratic, ambiguous, and inconsistent uses of tags. Left to themselves, free tagging systems seem to be too wild and too chaotic for any order to emerge. But are these free tagging systems really as "feral" as they seem to be, or do they only look uncontrolled because one has been looking for order in the wrong place? I have done a quick-and-dirty" analysis of Flickr's tag cloud. The concept was: if folksonomies encourage users to tap on their own vernacular, everyday natural language must somehow "guide" the tagging practices of users of tagging systems. Flickr's tag cloud has been choosen because it may teach us something about tagging systems and folksonomies, and not - or not primarily - because of what tags may tell us about pictures.

  14. Use of low fusing alloy in dentistry.

    Science.gov (United States)

    Wee, A G; Schneider, R L; Aquilino, S A

    1998-11-01

    Low fusing alloy has been used in dentistry for remount procedures in both fixed and removable prosthodontics, in implant prosthodontics for the fabrication of solid implant casts, in maxillofacial prosthetics as oral radiation shields, and in dental research for its unique properties. Previously, the use of low fusing alloy was thought to offer a high degree of dimensional accuracy. However, multiple in vitro studies have shown that its presumed dimensional accuracy may be questionable. This article reviews the physical properties, metallurgical considerations of low fusing alloy, its applications in dentistry, and a safe, simple method of using low fusing alloy.

  15. Quantitative proteomic profiling of membrane proteins from the mouse brain cortex, hippocampus, and cerebellum using the HysTag reagent: mapping of neurotransmitter receptors and ion channels

    DEFF Research Database (Denmark)

    Olsen, Jesper V; Nielsen, Peter Aa; Andersen, Jens R

    2007-01-01

    of recently developed methods for isolation of membrane proteins from 10-20 mg brain tissue [Nielsen, P.Aa., Olsen, J.V., Podtelejnokov, A.V., Andersen, J.R., Mann, M., Wisniewski, J.R., 2005. Proteomic mapping of brain plasma membrane proteins. Mol. Cell. Proteomics 4, 402--408] and the Hys...... proteins mapped in distinct brain compartments and offer a technology that allows in depth study of brain membrane proteomes, such as mouse models of neurological diseases.......Analysis of the brain proteome and studying brain diseases through clinical biopsies and animal disease models require methods of quantitative proteomics that are sensitive and allow identification and quantification of low abundant membrane proteins from minute amount of tissue. Taking advantage...

  16. Isotope-Coded Dimethyl Tagging for Differential Quantification of Posttranslational Protein Carbonylation by 4-Hydroxy-2-nonenal, an End-Product of Lipid Peroxidation

    OpenAIRE

    Rauniyar, Navin; Prokai, Laszlo

    2011-01-01

    Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, α,β-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-indu...

  17. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-Based Untargeted Quantitative Proteomic Approach To Identify Change of the Plasma Proteins by Salbutamol Abuse in Beef Cattle.

    Science.gov (United States)

    Zhang, Kai; Tang, Chaohua; Liang, Xiaowei; Zhao, Qingyu; Zhang, Junmin

    2018-01-10

    Salbutamol, a selective β 2 -agonist, endangers the safety of animal products as a result of illegal use in food animals. In this study, an iTRAQ-based untargeted quantitative proteomic approach was applied to screen potential protein biomarkers in plasma of cattle before and after treatment with salbutamol for 21 days. A total of 62 plasma proteins were significantly affected by salbutamol treatment, which can be used as potential biomarkers to screen for the illegal use of salbutamol in beef cattle. Enzyme-linked immunosorbent assay measurements of five selected proteins demonstrated the reliability of iTRAQ-based proteomics in screening of candidate biomarkers among the plasma proteins. The plasma samples collected before and after salbutamol treatment were well-separated by principal component analysis (PCA) using the differentially expressed proteins. These results suggested that an iTRAQ-based untargeted quantitative proteomic strategy combined with PCA pattern recognition methods can discriminate differences in plasma protein profiles collected before and after salbutamol treatment.

  18. Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System.

    Science.gov (United States)

    Steinmetz, Eric J; Auldridge, Michele E

    2017-11-01

    The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  19. Social Tagging of Mission Data

    Science.gov (United States)

    Norris, Jeffrey S.; Wallick, Michael N.; Joswig, Joseph C.; Powell, Mark W.; Torres, Recaredo J.; Mittman, David S.; Abramyan, Lucy; Crockett, Thomas M.; Shams, Khawaja S.; Fox, Jason M.; hide

    2010-01-01

    Mars missions will generate a large amount of data in various forms, such as daily plans, images, and scientific information. Often, there is a semantic linkage between images that cannot be captured automatically. Software is needed that will provide a method for creating arbitrary tags for this mission data so that items with a similar tag can be related to each other. The tags should be visible and searchable for all users. A new routine was written to offer a new and more flexible search option over previous applications. This software allows users of the MSLICE program to apply any number of arbitrary tags to a piece of mission data through a MSLICE search interface. The application of tags creates relationships between data that did not previously exist. These tags can be easily removed and changed, and contain enough flexibility to be specifically configured for any mission. This gives users the ability to quickly recall or draw attention to particular pieces of mission data, for example: Give a semantic and meaningful description to mission data; for example, tag all images with a rock in them with the tag "rock." Rapidly recall specific and useful pieces of data; for example, tag a plan as"driving template." Call specific data to a user s attention; for example, tag a plan as "for:User." This software is part of the MSLICE release, which was written in Java. It will run on any current Windows, Macintosh, or Linux system.

  20. Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments.

    Science.gov (United States)

    Grimm, Jonathan B; Brown, Timothy A; English, Brian P; Lionnet, Timothée; Lavis, Luke D

    2017-01-01

    The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging.

  1. Evaluation of gel electrophoresis conditions for the separation of metal-tagged proteins with subsequent laser ablation ICP-MS detection.

    Science.gov (United States)

    Raab, Andrea; Pioselli, Barbara; Munro, Caroline; Thomas-Oates, Jane; Feldmann, Jörg

    2009-01-01

    Although laser ablation (LA)-ICP-MS has been reported for the determination of metalloproteins separated by gel electrophoretic techniques (GE), systematic studies that define the conditions essential for successful measurements are still scarce. In this paper we present the results of our studies of basic conditions for the effective application of GE-LA-ICP-MS for the separation of metal-binding proteins, focusing on their stability during GE and post-separation gel treatment. The stability of metal-protein complexes (haemoglobin, myoglobin, superoxide dismutase, carbonic anhydrase, transferrin, albumin, cytochrome c) during GE is dependent on the nature of the metal-protein interaction and the principle of separation. We have observed that non-denaturing GE is a suitable separation technique for most metal-protein complexes (e.g. Zn in carbonic anhydrase and Fe in Tf and myoglobin were quantitatively recovered in a spiked liver cytosol), whereas separation by denaturing GE strongly impaired the stability of the complexes. Equally important is the post-separation treatment of the gel to enable successful detection of the metal. LA-ICP-MS requires drying of the gel without loss of protein-bound metal or cracking of the gel. This was successfully achieved using glycerol followed by heating. We demonstrate that staining of the gel prior to LA-ICP-MS using silver or Coomassie blue is not recommended, since most protein-bound metal is lost during the staining procedure. Furthermore it has been shown that only line scanning with a speed of less than 30 microm/s can reliably distinguish between lines 1 mm apart, while raster spot analysis carries the risk of misinterpretation due to contamination in/on inhomogeneous gels.

  2. Isotope-coded dimethyl tagging for differential quantification of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation.

    Science.gov (United States)

    Rauniyar, Navin; Prokai, Laszlo

    2011-10-01

    Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, α, β-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H(12)CHO) and heavy (D(13)CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ε-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography-mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress. Copyright

  3. Transplantation of Crossed Fused Renal Ectopia

    Directory of Open Access Journals (Sweden)

    Soon-Khai Lee

    2007-01-01

    Full Text Available Crossed fused renal ectopia is a type of congenital fused anomaly of the kidney. This type of kidney, when encountered, can be used as a donor organ to provide useful solution to the critical shortage of available organs for transplantation.

  4. Behavioral Tagging: A Translation of the Synaptic Tagging and Capture Hypothesis

    Directory of Open Access Journals (Sweden)

    Diego Moncada

    2015-01-01

    Full Text Available Similar molecular machinery is activated in neurons following an electrical stimulus that induces synaptic changes and after learning sessions that trigger memory formation. Then, to achieve perdurability of these processes protein synthesis is required for the reinforcement of the changes induced in the network. The synaptic tagging and capture theory provided a strong framework to explain synaptic specificity and persistence of electrophysiological induced plastic changes. Ten years later, the behavioral tagging hypothesis (BT made use of the same argument, applying it to learning and memory models. The hypothesis postulates that the formation of lasting memories relies on at least two processes: the setting of a learning tag and the synthesis of plasticity related proteins, which once captured at tagged sites allow memory consolidation. BT explains how weak events, only capable of inducing transient forms of memories, can result in lasting memories when occurring close in time with other behaviorally relevant experiences that provide proteins. In this review, we detail the findings supporting the existence of BT process in rodents, leading to the consolidation, persistence, and interference of a memory. We focus on the molecular machinery taking place in these processes and describe the experimental data supporting the BT in humans.

  5. Review on SAW RFID tags.

    Science.gov (United States)

    Plessky, Victor P; Reindl, Leonhard M

    2010-03-01

    SAW tags were invented more than 30 years ago, but only today are the conditions united for mass application of this technology. The devices in the 2.4-GHz ISM band can be routinely produced with optical lithography, high-resolution radar systems can be built up using highly sophisticated, but low-cost RF-chips, and the Internet is available for global access to the tag databases. The "Internet of Things," or I-o-T, will demand trillions of cheap tags and sensors. The SAW tags can overcome semiconductor-based analogs in many aspects: they can be read at a distance of a few meters with readers radiating power levels 2 to 3 orders lower, they are cheap, and they can operate in robust environments. Passive SAW tags are easily combined with sensors. Even the "anti-collision" problem (i.e., the simultaneous reading of many nearby tags) has adequate solutions for many practical applications. In this paper, we discuss the state-of-the-art in the development of SAW tags. The design approaches will be reviewed and optimal tag designs, as well as encoding methods, will be demonstrated. We discuss ways to reduce the size and cost of these devices. A few practical examples of tags using a time-position coding with 10(6) different codes will be demonstrated. Phase-coded devices can additionally increase the number of codes at the expense of a reduction of reading distance. We also discuss new and exciting perspectives of using ultra wide band (UWB) technology for SAW-tag systems. The wide frequency band available for this standard provides a great opportunity for SAW tags to be radically reduced in size to about 1 x 1 mm(2) while keeping a practically infinite number of possible different codes. Finally, the reader technology will be discussed, as well as detailed comparison made between SAW tags and IC-based semiconductor device.

  6. Buddy Tag CONOPS and Requirements.

    Energy Technology Data Exchange (ETDEWEB)

    Brotz, Jay Kristoffer [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Deland, Sharon M. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-12-01

    This document defines the concept of operations (CONOPS) and the requirements for the Buddy Tag, which is conceived and designed in collaboration between Sandia National Laboratories and Princeton University under the Department of State Key VerificationAssets Fund. The CONOPS describe how the tags are used to support verification of treaty limitations and is only defined to the extent necessary to support a tag design. The requirements define the necessary functions and desired non-functional features of the Buddy Tag at a high level

  7. Flavour tagging performance in LHCb

    International Nuclear Information System (INIS)

    Grabalosa Gandara, Marc

    2009-01-01

    To do precise CP violation measurements, the best possible determination of the flavour of the B-meson is necessary. This report summarizes the flavour tagging performances for the LHCb experiment. The flavour tagging is obtained through a combination of several methods, based on different signatures. The use of control channels, which are decays to flavour-specific final states, will allow to determine the wrong tag fraction ω (the probability of a tag to be wrong), which can be used as an input for the determination of CKM unitarity triangle angles.

  8. Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling

    DEFF Research Database (Denmark)

    Kristensen, Janni; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

    2005-01-01

    -His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We......We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23......) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP...

  9. Laser welding of fused quartz

    Science.gov (United States)

    Piltch, Martin S.; Carpenter, Robert W.; Archer, III, McIlwaine

    2003-06-10

    Refractory materials, such as fused quartz plates and rods are welded using a heat source, such as a high power continuous wave carbon dioxide laser. The radiation is optimized through a process of varying the power, the focus, and the feed rates of the laser such that full penetration welds may be accomplished. The process of optimization varies the characteristic wavelengths of the laser until the radiation is almost completely absorbed by the refractory material, thereby leading to a very rapid heating of the material to the melting point. This optimization naturally occurs when a carbon dioxide laser is used to weld quartz. As such this method of quartz welding creates a minimum sized heat-affected zone. Furthermore, the welding apparatus and process requires a ventilation system to carry away the silicon oxides that are produced during the welding process to avoid the deposition of the silicon oxides on the surface of the quartz plates or the contamination of the welds with the silicon oxides.

  10. LHCb Tag Collector

    CERN Document Server

    Fuente Fernàndez, P; Cousin, N

    2011-01-01

    The LHCb physics software consists of hundreds of packages, each of which is developed by one or more physicists. When the developers have some code changes that they would like released, they commit them to the version control system, and enter the revision number into a database. These changes have to be integrated into a new release of each of the physics analysis applications. Tests are then performed by a nightly build system, which rebuilds various configurations of the whole software stack and executes a suite of run-time functionality tests. A Tag Collector system has been developed using solid standard technologies to cover both the use cases of developers and integration managers. A simple Web interface, based on an AJAX-like technology, is available. Integration with software management and Nightly Build programs is possible via a Python API. Data are stored in a relational database with the help of an ORM (Object-Relational Mapping) library.

  11. Antibody tagged gold nanoparticles as scattering probes for the pico molar detection of the proteins in blood serum using nanoparticle tracking analyzer.

    Science.gov (United States)

    Kashid, Sahebrao Balaso; Tak, Rajesh D; Raut, Rajesh Warluji

    2015-09-01

    We report a rapid one-step immunoassay to detect protein using antibody conjugated gold nanoparticles (AbGNPs) where the targeted protein concentration was determined by analyzing the gold nanoparticle aggregation caused by antibody-antigen interactions using nanoparticles tracking analysis (NTA) technique. The sandwich structure constituting the binding of the targeted human IgG to the gold nanoparticle conjugates with goat anti human monoclonal IgG (AbGNPs) was confirmed by transmission electron microscopy. The binding of human IgG (antigen, mentioned hence forth as AT) induce AbGNPs to form dimers or trimers through a typical antibody-antigen-antibody sandwich structure that can be analyzed for the sensitive determination on the basis of change in hydrodynamic diameter of AbGNPs. By this method the minimum detectable concentration of AT is found to be below 2pg/ml. We expect that a significant change in the hydrodynamic diameter of AbGNP could form the basis for the rapid one-step immunoassay development. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Porphyrins Fused with Unactivated Polycyclic Aromatic Hydrocarbons

    KAUST Repository

    Diev, Vyacheslav V.

    2012-01-06

    A systematic study of the preparation of porphyrins with extended conjugation by meso,β-fusion with polycyclic aromatic hydrocarbons (PAHs) is reported. The meso-positions of 5,15-unsubstituted porphyrins were readily functionalized with PAHs. Ring fusion using standard Scholl reaction conditions (FeCl 3, dichloromethane) occurs for perylene-substituted porphyrins to give a porphyrin β,meso annulated with perylene rings (0.7:1 ratio of syn and anti isomers). The naphthalene, pyrene, and coronene derivatives do not react under Scholl conditions but are fused using thermal cyclodehydrogenation at high temperatures, giving mixtures of syn and anti isomers of the meso,β-fused porphyrins. For pyrenyl-substituted porphyrins, a thermal method gives synthetically acceptable yields (>30%). Absorption spectra of the fused porphyrins undergo a progressive bathochromic shift in a series of naphthyl (λ max = 730 nm), coronenyl (λ max = 780 nm), pyrenyl (λ max = 815 nm), and perylenyl (λ max = 900 nm) annulated porphyrins. Despite being conjugated with unsubstituted fused PAHs, the β,meso-fused porphyrins are more soluble and processable than the parent nonfused precursors. Pyrenyl-fused porphyrins exhibit strong fluorescence in the near-infrared (NIR) spectral region, with a progressive improvement in luminescent efficiency (up to 13% with λ max = 829 nm) with increasing degree of fusion. Fused pyrenyl-porphyrins have been used as broadband absorption donor materials in photovoltaic cells, leading to devices that show comparatively high photovoltaic efficiencies. © 2011 American Chemical Society.

  13. Viable, but non-culturable, state of a green fluorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water.

    Science.gov (United States)

    Cho, J C; Kim, S J

    1999-01-01

    An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescence protein (GFP) isolated from Aequorea victoria. The hybrid transposon mini Tn5 gfp was transconjugated from E. coli to S. typhi, resulting in constitutive GFP production. The survival of S. typhi GFP155 introduced into groundwater and pond water microcosms was examined by GFP-based plate counts, total cell counts, and direct viable counts. A comparison between GFP-based direct viable counts and plate counts was a good method for verifying the viable, but non-culturable (VBNC), state of S. typhi. The entry into a VBNC state of S. typhi was shown in all microcosms. S. typhi survived longer in groundwater than in pond water as both a culturable and a VBNC state.

  14. Fluorine-Based DRIE of Fused Silica

    Science.gov (United States)

    Yee, Karl; Shcheglov, Kirill; Li, Jian; Choi, Daniel

    2007-01-01

    A process of deep reactive-ion etching (DRIE) using a fluorine-based gas mixture enhanced by induction-coupled plasma (ICP) has been demonstrated to be effective in forming high-aspect-ratio three-dimensional patterns in fused silica. The patterns are defined in part by an etch mask in the form of a thick, high-quality aluminum film. The process was developed to satisfy a need to fabricate high-aspect-ratio fused-silica resonators for vibratory microgyroscopes, and could be used to satisfy similar requirements for fabricating other fused-silica components.

  15. Dynamic simulation of flywheel-type fuses

    Directory of Open Access Journals (Sweden)

    Editorial Office

    1996-07-01

    Full Text Available Rounds of ammunition are normally armed with a fuse. In this study, a fuse is developed which uses a flywheel-type mechanism controlled by time or distance. Due to its simplicity of operation and construction, the concept is expected to have high reliabil­ity. The dynamic response of all the components of this flywheel-type fuse is mathematically modelled. Simulation software was developed which connects the mathematical models of the various components. With the definition of boundary values, the response of the projectile, flywheel and other components can be determined continuously for firing and in-flight conditions.

  16. Fused Silica and Other Transparent Window Materials

    Science.gov (United States)

    Salem, Jon

    2016-01-01

    Several transparent ceramics, such as spinel and AlONs are now being produced in sufficient large areas to be used in space craft window applications. The work horse transparent material for space missions from Apollo to the International Space Station has been fused silica due in part to its low coefficient of expansion and optical quality. Despite its successful use, fused silica exhibits anomalies in its crack growth behavior, depending on environmental preconditioning and surface damage. This presentation will compare recent optical ceramics to fused silica and discuss sources of variation in slow crack growth behavior.

  17. Semiotic dynamics and collaborative tagging.

    Science.gov (United States)

    Cattuto, Ciro; Loreto, Vittorio; Pietronero, Luciano

    2007-01-30

    Collaborative tagging has been quickly gaining ground because of its ability to recruit the activity of web users into effectively organizing and sharing vast amounts of information. Here we collect data from a popular system and investigate the statistical properties of tag cooccurrence. We introduce a stochastic model of user behavior embodying two main aspects of collaborative tagging: (i) a frequency-bias mechanism related to the idea that users are exposed to each other's tagging activity; (ii) a notion of memory, or aging of resources, in the form of a heavy-tailed access to the past state of the system. Remarkably, our simple modeling is able to account quantitatively for the observed experimental features with a surprisingly high accuracy. This points in the direction of a universal behavior of users who, despite the complexity of their own cognitive processes and the uncoordinated and selfish nature of their tagging activity, appear to follow simple activity patterns.

  18. Engineering the ATLAS TAG Browser

    CERN Document Server

    Zhang, Q; The ATLAS collaboration

    2011-01-01

    ELSSI is a web-based event metadata (TAG) browser and event-level selection service for ATLAS. TAGs from all ATLAS physics and Monte Carlo data sets are routinely loaded into Oracle databases as an integral part of event processing. As data volumes increase, more and more sites are joining the distributed TAG data hosting topology. Meanwhile, TAG content and database schemata continue to evolve as new user requirements and additional sources of metadata emerge. All of this has posed many challenges to the development of ELSSI, which must support vast amounts of TAG data while source, content, geographic locations, and user query patterns may change over time. In this paper, we describe some of the challenges encountered in the process of developing ELSSI, and the software engineering strategies adopted to address those challenges. Approaches to management of access to data, browsing, data rendering, query building, query validation, execution, connection management, and communication with auxiliary services a...

  19. The Evaluation of the Impact of Age, Skin Tags, Metabolic Syndrome, Body Mass Index, and Smoking on Homocysteine, Endothelin-1, High-sensitive C-reactive Protein, and on the Heart.

    Science.gov (United States)

    El Safoury, Omar Soliman; Ezzat, Marwa; Abdelhamid, Mahmoud F; Shoukry, Nadia; Badawy, Ehssan

    2013-07-01

    Skin tags (STs) are small, pedunculated skin-colored or brown papules that occur around any site where skin folds occur. The literature is short of comprehensive and controlled clinical studies aimed to evaluate the atherogenic risk factors in patients with STs. The aim of this study is to evaluate the impact of age, STs, metabolic syndrome (METs), body mass index (BMI), and smoking on homocysteine (Hcy), endothelin-1 (ET-1), high-sensitive C-reactive protein (Hs-CRP), and on cardiovascular diseases. This study included 30 cardiac patients with STs, 30 non-cardiac patients with STs, and 30 healthy controls with neither heart disease nor STs. History of smoking, measurement of height, weight, BMI, waist circumference (WC), blood pressure, STs number, color, acanthosis nigricans, estimation of serum level of fasting glucose, triglycerides (TGs), cholesterol, high-dense lipoproteins (HDL), Hcy, ET-1, Hs-CRP, and the presence of the METs were elicited in the three groups. Regarding the Hcy, ET-1, and Hs-CRP, the cardiac-STs group showed the highest levels and the control group showed the least (P Bermuda Triangle that act against the heart.

  20. Controlling macromolecular topology with genetically encoded SpyTag-SpyCatcher chemistry.

    Science.gov (United States)

    Zhang, Wen-Bin; Sun, Fei; Tirrell, David A; Arnold, Frances H

    2013-09-18

    Control of molecular topology constitutes a fundamental challenge in macromolecular chemistry. Here we describe the synthesis and characterization of artificial elastin-like proteins (ELPs) with unconventional nonlinear topologies including circular, tadpole, star, and H-shaped proteins using genetically encoded SpyTag-SpyCatcher chemistry. SpyTag is a short polypeptide that binds its protein partner SpyCatcher and forms isopeptide bonds under physiological conditions. Sequences encoding SpyTag and SpyCatcher can be strategically placed into ELP genes to direct post-translational topological modification in situ. Placement of SpyTag at the N-terminus and SpyCatcher at the C-terminus directs formation of circular ELPs. Induction of expression at 16 °C with 10 μM IPTG yields 80% monomeric cyclic protein. When SpyTag is placed in the middle of the chain, it exhibits an even stronger tendency toward cyclization, yielding up to 94% monomeric tadpole proteins. Telechelic ELPs containing either SpyTag or SpyCatcher can be expressed, purified, and then coupled spontaneously upon mixing in vitro. Block proteins, 3-arm or 4-arm star proteins, and H-shaped proteins have been prepared, with the folded CnaB2 domain that results from the SpyTag-SpyCatcher reaction as the molecular core or branch junction. The modular character of the SpyTag-SpyCatcher strategy should make it useful for preparing nonlinear macromolecules of diverse sequence and structure.

  1. Analysis of common bean expressed sequence tags identifies sulfur metabolic pathways active in seed and sulfur-rich proteins highly expressed in the absence of phaseolin and major lectins

    Directory of Open Access Journals (Sweden)

    Sharpe Andrew

    2011-05-01

    Full Text Available Abstract Background A deficiency in phaseolin and phytohemagglutinin is associated with a near doubling of sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris, particularly cysteine, elevated by 70%, and methionine, elevated by 10%. This mostly takes place at the expense of an abundant non-protein amino acid, S-methyl-cysteine. The deficiency in phaseolin and phytohemagglutinin is mainly compensated by increased levels of the 11S globulin legumin and residual lectins. Legumin, albumin-2, defensin and albumin-1 were previously identified as contributing to the increased sulfur amino acid content in the mutant line, on the basis of similarity to proteins from other legumes. Results Profiling of free amino acid in developing seeds of the BAT93 reference genotype revealed a biphasic accumulation of gamma-glutamyl-S-methyl-cysteine, the main soluble form of S-methyl-cysteine, with a lag phase occurring during storage protein accumulation. A collection of 30,147 expressed sequence tags (ESTs was generated from four developmental stages, corresponding to distinct phases of gamma-glutamyl-S-methyl-cysteine accumulation, and covering the transitions to reserve accumulation and dessication. Analysis of gene ontology categories indicated the occurrence of multiple sulfur metabolic pathways, including all enzymatic activities responsible for sulfate assimilation, de novo cysteine and methionine biosynthesis. Integration of genomic and proteomic data enabled the identification and isolation of cDNAs coding for legumin, albumin-2, defensin D1 and albumin-1A and -B induced in the absence of phaseolin and phytohemagglutinin. Their deduced amino acid sequences have a higher content of cysteine than methionine, providing an explanation for the preferential increase of cysteine in the mutant line. Conclusion The EST collection provides a foundation to further investigate sulfur metabolism and the differential accumulation of

  2. Expression of GA733-Fc Fusion Protein as a Vaccine Candidate for Colorectal Cancer in Transgenic Plants

    Directory of Open Access Journals (Sweden)

    Zhe Lu

    2012-01-01

    Full Text Available The tumor-associated antigen GA733 is a cell-surface glycoprotein highly expressed in colorectal carcinomas. In this study, 3 recombinant genes were constructed as follows: GA733 tagged to the ER retention sequence KDEL (GA733K, GA733 fused to the immunoglobulin Fc fragment (GA733-Fc, and GA733-Fc fused to the ER retention sequence (GA733-FcK. Agrobacterium-mediated transformation was used to generate transgenic plants expressing recombinant genes. The presence of transgenes was confirmed by genomic PCR. Western blot, confocal immunofluorescence, and sandwich ELISA showed the expression of recombinant proteins. The stability, flexibility, and bioactivity of recombinant proteins were analyzed and demonstrated through N-glycosylation analysis, animal trials, and sera ELISA. Our results suggest that the KDEL retained proteins in ER with oligomannose glycan structure and enhanced protein accumulation level. The sera of mice immunized with GA733-FcK purified from plants contained immunoglobulins which were at least as efficient as the mammalian-derived GA733-Fc at recognizing human colorectal cancer cell lines. Thus, a plant system can be used to express the KDEL fusion protein with oligomannose glycosylation, and this protein induces an immune response which is comparable to non-KDEL-tagged, mammalian-derived proteins.

  3. Expression of GA733-Fc fusion protein as a vaccine candidate for colorectal cancer in transgenic plants.

    Science.gov (United States)

    Lu, Zhe; Lee, Kyung-Jin; Shao, Yingxue; Lee, Jeong-Hwan; So, Yangkang; Choo, Young-Kug; Oh, Doo-Byoung; Hwang, Kyung-A; Oh, Seung Han; Han, Yeon Soo; Ko, Kisung

    2012-01-01

    The tumor-associated antigen GA733 is a cell-surface glycoprotein highly expressed in colorectal carcinomas. In this study, 3 recombinant genes were constructed as follows: GA733 tagged to the ER retention sequence KDEL (GA733K), GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), and GA733-Fc fused to the ER retention sequence (GA733-FcK). Agrobacterium-mediated transformation was used to generate transgenic plants expressing recombinant genes. The presence of transgenes was confirmed by genomic PCR. Western blot, confocal immunofluorescence, and sandwich ELISA showed the expression of recombinant proteins. The stability, flexibility, and bioactivity of recombinant proteins were analyzed and demonstrated through N-glycosylation analysis, animal trials, and sera ELISA. Our results suggest that the KDEL retained proteins in ER with oligomannose glycan structure and enhanced protein accumulation level. The sera of mice immunized with GA733-FcK purified from plants contained immunoglobulins which were at least as efficient as the mammalian-derived GA733-Fc at recognizing human colorectal cancer cell lines. Thus, a plant system can be used to express the KDEL fusion protein with oligomannose glycosylation, and this protein induces an immune response which is comparable to non-KDEL-tagged, mammalian-derived proteins.

  4. Coordination chemistry in fused-salt solutions

    Science.gov (United States)

    Gruen, D. M.

    1969-01-01

    Spectrophotometric work on structural determinations with fused-salt solutions is reviewed. Constraints placed on the method, as well as interpretation of the spectra, are discussed with parallels drawn to aqueous spectrophotometric curves of the same materials.

  5. Comparative evaluation of fiber fuse models

    International Nuclear Information System (INIS)

    Davis, D.D.; Mettler, S.C.; DiGiovanni, D.J.

    1997-01-01

    A phenomenon which results in the catastrophic destruction of the guiding properties of an optical fiber has been observed at laser power densities on the order of 3 x 10 6 watts/cm 2 in the core. This phenomenon is characterized by the propagation of a bright visible light from the point of initiation toward the laser source. The term 'fiber fuse' has been used because of the similarity in appearance to a burning fuse. The fiber fuse has been shown to start when the end of the fiber is contacted. It has also been initiated spontaneously from mechanical splices. This paper reports experimental data gathered on the fiber fuse and discusses their relationship to proposed physical mechanisms

  6. A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

    Science.gov (United States)

    Levoye, Angélique; Zwier, Jurriaan M; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

    2015-01-01

    Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

  7. Metal-metallothioneins like proteins investigation by heteroatom-tagged proteomics in two different snails as possible sentinel organisms of metal contamination in freshwater ecosystems

    Energy Technology Data Exchange (ETDEWEB)

    Franca Maltez, Heloisa [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julian Claveria 8, 33006 Oviedo (Spain); Villanueva Tagle, Margarita [Faculty of Chemistry, University of La Habana (Cuba); Rosario Fernandez de la Campa, Maria del [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julian Claveria 8, 33006 Oviedo (Spain); Sanz-Medel, Alfredo, E-mail: asm@uniovi.es [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julian Claveria 8, 33006 Oviedo (Spain)

    2009-09-21

    Metal speciation analysis in MLPs was carried out in two snails, Marisa cornuarietis and Pomacea bridgesi, in order to investigate them as possible sentinel organisms of heavy metal contamination. To carry out this study snails born in a non-contaminated environment were divided into two groups: a control group and a contaminated one with cadmium administered for 40 days. Subsequently, we investigated the speciation of the induced MLPs in exposed animals in relation to controls. In order to obtain the MLP fraction, cytosols from both snail species where subjected to size-exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. MLP fraction was then separated by anion-exchange (AE)-FPLC using optimal chromatographic conditions for the separation of the different MLP isoforms in both snail species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with ICP-MS. The determination of the amount of metal bound to MLPs was carried out by post-column isotope dilution analysis ICP-MS, finding that the snail M. cornuarietis accumulated higher concentrations of cadmium than P. bridgesi. Thus this first snail could therefore be a better candidate sentinel organism of pollution in natural waters. Identification and characterization of the isoforms separated in M. cornuarietis was carried out for the entire or intact isoforms by MALDI-TOF and then conventional triptic digestion was also carried out to identify the nature of the formed peptides. The presence identification of a MLP isoform of relatively low molecular weight in M. cornuarietis is reported.

  8. Metal-metallothioneins like proteins investigation by heteroatom-tagged proteomics in two different snails as possible sentinel organisms of metal contamination in freshwater ecosystems

    International Nuclear Information System (INIS)

    Franca Maltez, Heloisa; Villanueva Tagle, Margarita; Rosario Fernandez de la Campa, Maria del; Sanz-Medel, Alfredo

    2009-01-01

    Metal speciation analysis in MLPs was carried out in two snails, Marisa cornuarietis and Pomacea bridgesi, in order to investigate them as possible sentinel organisms of heavy metal contamination. To carry out this study snails born in a non-contaminated environment were divided into two groups: a control group and a contaminated one with cadmium administered for 40 days. Subsequently, we investigated the speciation of the induced MLPs in exposed animals in relation to controls. In order to obtain the MLP fraction, cytosols from both snail species where subjected to size-exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. MLP fraction was then separated by anion-exchange (AE)-FPLC using optimal chromatographic conditions for the separation of the different MLP isoforms in both snail species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with ICP-MS. The determination of the amount of metal bound to MLPs was carried out by post-column isotope dilution analysis ICP-MS, finding that the snail M. cornuarietis accumulated higher concentrations of cadmium than P. bridgesi. Thus this first snail could therefore be a better candidate sentinel organism of pollution in natural waters. Identification and characterization of the isoforms separated in M. cornuarietis was carried out for the entire or intact isoforms by MALDI-TOF and then conventional triptic digestion was also carried out to identify the nature of the formed peptides. The presence identification of a MLP isoform of relatively low molecular weight in M. cornuarietis is reported.

  9. Tagged library approach to chemical genomics and proteomics.

    Science.gov (United States)

    Mitsopoulos, Gus; Walsh, Daniel P; Chang, Young-Tae

    2004-02-01

    Proteomics and chemical genomics face great challenges in the form of molecular libraries of ever increasing size and diversity requiring rapid screening, coupled with a growing number of target proteins for which complimentary molecular ligands are sought. Proteomics and chemical genomics are at a stage that requires techniques which can dramatically accelerate the discovery process. One technique that has shown great promise in accomplishing this is the tagged library approach. It entails the synthetic inclusion of an internal tag from the beginning of the synthesis. This tag adds another degree of functionality to the molecule, in addition to mere ligation, that eliminates the need for time-consuming steps downstream in the process. The tag's functional possibilities span a variety of uses including internal fluorophores, intrinsic binding motifs that enable compound identification, functionalities that play the major role in the synthesis of the ligand itself, and internal linkers that eliminate the need for lengthy 'tether effect' structure-activity relationship studies.

  10. Quantum tagging for tags containing secret classical data

    International Nuclear Information System (INIS)

    Kent, Adrian

    2011-01-01

    Various authors have considered schemes for quantum tagging, that is, authenticating the classical location of a classical tagging device by sending and receiving quantum signals from suitably located distant sites, in an environment controlled by an adversary whose quantum information processing and transmitting power is potentially unbounded. All of the schemes proposed elsewhere in the literature assume that the adversary is able to inspect the interior of the tagging device. All of these schemes have been shown to be breakable if the adversary has unbounded predistributed entanglement. We consider here the case in which the tagging device contains a finite key string shared with distant sites but kept secret from the adversary, and show this allows the location of the tagging device to be authenticated securely and indefinitely. Our protocol relies on quantum key distribution between the tagging device and at least one distant site, and demonstrates a new practical application of quantum key distribution. It also illustrates that the attainable security in position-based cryptography can depend crucially on apparently subtle details in the security scenario considered.

  11. Fuse Modeling for Reliability Study of Power Electronic Circuits

    DEFF Research Database (Denmark)

    Bahman, Amir Sajjad; Iannuzzo, Francesco; Blaabjerg, Frede

    2017-01-01

    This paper describes a comprehensive modeling approach on reliability of fuses used in power electronic circuits. When fuses are subjected to current pulses, cyclic temperature stress is introduced to the fuse element and will wear out the component. Furthermore, the fuse may be used in a large v...

  12. Construction of multiple-epitope tag sequence by PCR for sensitive Western blot analysis.

    OpenAIRE

    Nakajima, K; Yaoita, Y

    1997-01-01

    Epitope tagging is a powerful technique to characterize a recombinantly expressed protein encoded by cDNA without the purification of the protein and the immunization of animals. In some cases, however, the expression of a tagged protein is too low to analyze by Western blot. We have developed a simple method to generate tandem repetitive nucleotide sequence by PCR, which allows us to label a protein of interest with a multiple-epitope tag. When five myc epitopes were attached to vaccinia vir...

  13. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

    Directory of Open Access Journals (Sweden)

    Shweta A Raina

    Full Text Available Fluorescent protein (FP insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET measurements were used to localize green fluorescent protein (GFP insertions within the ryanodine receptor type 1 (RyR1, a large intracellular Ca(2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  14. Endodontic and post-endodontic management of a fused molar

    Directory of Open Access Journals (Sweden)

    Ruchi Gupta

    2013-01-01

    Full Text Available Treatment of fused teeth needs special care and attention to the bizarre anatomy. This paper describes root canal treatment of a fused carious tooth presenting with apical periodontitis. It is a rare case of fusion of the mandibular second molar with a paramolar. There is no literature regarding placement of crown over endodontically treated fused teeth. In this case, the fused teeth were endodontically treated and restored by a porcelain fused to metal crown.

  15. Endodontic and post-endodontic management of a fused molar.

    Science.gov (United States)

    Gupta, Ruchi; Prakash, Vijay; Sharma, Mohit

    2013-01-01

    Treatment of fused teeth needs special care and attention to the bizarre anatomy. This paper describes root canal treatment of a fused carious tooth presenting with apical periodontitis. It is a rare case of fusion of the mandibular second molar with a paramolar. There is no literature regarding placement of crown over endodontically treated fused teeth. In this case, the fused teeth were endodontically treated and restored by a porcelain fused to metal crown.

  16. Editorial Tag Endogeneity for News Websites

    OpenAIRE

    Bruno Ribeiro; Ricardo Morla; Amílcar Correia

    2013-01-01

    Editors and journalists at some news websites label their articles with structure and content-related editorial tags. Each article can have more than one tag and each tag can be used in more than one article. A network of tags can be defined whose edges are all possible pairs of tags in each article. Because editorial tags relate to structure and content rather than individual articles, the analysis of a network of editorial tags could assist editorial decisions to prioritize types of content...

  17. Tempting To Tag: An Experimental Comparison Of Four Tagging Input Mechanisms

    Directory of Open Access Journals (Sweden)

    Mark Melenhorst

    2010-01-01

    Full Text Available Tagging helps achieve improved indexing and recommendation of resources (e.g., videos or pictures in large data collections. In order to reap the benefits of tagging, people must be persuaded to label the resources they consume. This paper reports on a study in which four different tagging input mechanisms and their effect on users' motivation to tag were compared. The mechanisms consisted of a standard tag input box, a chatbot-like environment, a bookmarking mechanism, and a "tag and vote" game. The results of our experiment show that the use of the nonstandard tagging input mechanisms does not affect users' motivation to tag. In some instances tagging mechanisms were found to distract users from their primary task: consuming resources. Persuading people to tag might be accomplished more effectively by using other motivating tagging mechanisms (e.g., tagging games, or motivation could be created by explaining the usefulness of tagging.

  18. Evaluation of Tag Attachments on Small Cetaceans

    Science.gov (United States)

    2013-09-30

    tag on the dorsal fin trailing edge. Dr. Randall Wells of CZS managed the project and coordinated tag production and field tests. All of these...modeling demonstrated that horizontally oriented tags created significantly less drag than vertically oriented tags. Undesirable turbulence was identified...successfully calved in 2013. Follow-up assessments in July found no indication of health problems associated with the tags. The anti-fouling

  19. Topology Engineering of Proteins in Vivo Using Genetically Encoded, Mechanically Interlocking SpyX Modules for Enhanced Stability.

    Science.gov (United States)

    Liu, Dong; Wu, Wen-Hao; Liu, Ya-Jie; Wu, Xia-Ling; Cao, Yang; Song, Bo; Li, Xiaopeng; Zhang, Wen-Bin

    2017-05-24

    Recombinant proteins are traditionally limited to linear configuration. Herein, we report in vivo protein topology engineering using highly efficient, mechanically interlocking SpyX modules named AXB and BXA. SpyX modules are protein domains composed of p53dim (X), SpyTag (A), and SpyCatcher (B). The p53dim guides the intertwining of the two nascent protein chains followed by autocatalytic isopeptide bond formation between SpyTag and SpyCatcher to fulfill the interlocking, leading to a variety of backbone topologies. Direct expression of AXB or BXA produces protein catenanes with distinct ring sizes. Recombinant proteins containing SpyX modules are obtained either as mechanically interlocked obligate dimers if the protein of interest is fused to the N- or C-terminus of SpyX modules, or as star proteins if the protein is fused to both N- and C-termini. As examples, cellular syntheses of dimers of (GB1) 2 (where GB1 stands for immunoglobulin-binding domain B1 of streptococcal protein G) and of four-arm elastin-like star proteins were demonstrated. Comparison of the catenation efficiencies in different constructs reveals that BXA is generally much more effective than AXB, which is rationalized by the arrangement of three domains in space. Mechanical interlocking induces considerable stability enhancement. Both AXB and BXA have a melting point ∼20 °C higher than the linear controls and the BXA catenane has a melting point ~2 °C higher than the cyclic control BX'A. Notably, four-arm elastin-like star proteins demonstrate remarkable tolerance against trypsin digestion. The SpyX modules provide a convenient and versatile approach to construct unconventional protein topologies via the "assembly-reaction" synergy, which opens a new horizon in protein science for stability enhancement and function reinforcement via topology engineering.

  20. Crystallization of small proteins assisted by green fluorescent protein.

    Science.gov (United States)

    Suzuki, Nobuhiro; Hiraki, Masahiko; Yamada, Yusuke; Matsugaki, Naohiro; Igarashi, Noriyuki; Kato, Ryuichi; Dikic, Ivan; Drew, David; Iwata, So; Wakatsuki, Soichi; Kawasaki, Masato

    2010-10-01

    The generation of crystal lattice contacts by proteinaceous tags fused to target proteins is an attractive approach to aid in the crystallization of otherwise intractable proteins. Here, the use of green fluorescent protein (GFP) fusions for this purpose is demonstrated, using ubiquitin and the ubiquitin-binding motif (UBM) of Y-family polymerase ι as examples. The structure of the GFP-ubiquitin fusion protein revealed that the crystal lattice was formed by GFP moieties. Ubiquitin was accommodated in the lattice through interactions with the peripheral loops of GFP. However, in the GFP-UBM fusion crystal UBM formed extensive interactions with GFP and these interactions, together with UBM dimerization, mediated the crystal packing. Interestingly, the tyrosine residues that are involved in mediating crystal contacts in both GFP-ubiquitin and GFP-UBM crystals are arranged in a belt on the surface of the β-barrel structure of GFP. Therefore, it is likely that GFP can assist in the crystallization of small proteins and of protein domains in general.

  1. Neutral B meson flavor tagging

    CERN Document Server

    Wilson, R J

    2001-01-01

    We present an investigation of the use of net charge and kaon identification to tag the flavor of neutral B mesons. The net charge of the neutral B meson decay products is zero if all charged particles are used and slightly non-zero if only undiscriminated hadronic final states are used. The net charge of the kaons alone correctly tags the identity of the neutral meson in at least a third of all decays. We have parametrized the particle identification capability of several techniques, such as dE/dx in time projection chambers, LEP/SLC ring-imaging chambers and an enhanced BaBar DIRC. Using these parametrisations we compare the relative tagging power of each technique to that of an ideal detector. (8 refs).

  2. Purification of recombinant C-terminus polyhistidine tagged human ...

    African Journals Online (AJOL)

    Dell

    2012-05-03

    . ... E. coli TOP 10F' and P. pastoris KM71H (arg4 aox1::ARG4) strains. E. coli TOP 10F' and P. pastoris KM71H ..... I-guided inquiry-purification and characterization of a fusion protein: Histidine tag, malate dehydrogenase, and ...

  3. 30 CFR 56.6502 - Safety fuse.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Safety fuse. 56.6502 Section 56.6502 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-SURFACE METAL AND NONMETAL MINES Explosives Nonelectric Blasting § 56...

  4. 30 CFR 57.6502 - Safety fuse.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Safety fuse. 57.6502 Section 57.6502 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Explosives Nonelectric Blasting...

  5. Fungal Systematics and Evolution: FUSE 1

    NARCIS (Netherlands)

    Crous, Pedro W; Schumacher, René K; Wingfield, Michael J; Lombard, Lorenzo; Giraldo, Alejandra; Christensen, Martha; Gardiennet, Alain; Nakashima, Chiharu; Pereira, Olinto L; Smith, Alexander J; Groenewald, Johannes Z

    2015-01-01

    Fungal Systematics and Evolution (FUSE) is introduced as a new series to expedite the publication of issues relating to the epitypification of formerly described species, report new sexual-asexual connections, the merging of sexual and asexual gen¬era following the end of dual nomenclature, and to

  6. Radiation hardness study on fused silica

    NARCIS (Netherlands)

    Hoek, M.; Bennet, E. D.; Branford, D.; Cowie, E. N.; Dueren, M.; Foehl, K.; Glazier, D.; Kaiser, R.; Lehmann, A.; Lu, S.; Marton, J.; Ostendorf, R.; Schepers, G.; Schwarz, C.; Seitz, B.; Teufel, A.; Watts, D.

    2008-01-01

    Radiation hardness tests have been carried out on fused silica samples of the highest optical grade from three different manufacturers (Suprasil, Lithosil and Coming). The samples were irradiated with protons in order to emulate the expected accumulated radiation damage over the entire life span of

  7. Renovating a Fusee Ceramique Barrel Vault

    NARCIS (Netherlands)

    Kamerling, M.W.

    2015-01-01

    This paper describes a proposal to renovate a Fusee Ceramique Barrel Vault with steel diagonals In 1956 two workshops, varying in height and span, were built in Wormerveer, The Netherlands. Both workshops were roofed with a concrete barrel vault with a thickness of 110 mm. The cylindrical vaults

  8. Fused aromatic thienopyrazines: structure, properties and function

    KAUST Repository

    Mondal, Rajib

    2010-01-01

    Recent development of a fused aromatic thieno[3.4-b]pyrazine system and their application in optoelectronic devices are reviewed. Introduction of a fused aromatic unit followed by side chain engineering, dramatically enhanced the charge carrier mobility in thin film transistor devices and mobilities up to 0.2 cm2/Vs were achieved. The optoelectronic properties of these fused aromatic thienopyrazine polymers (Eg = 1.3 to 1.6 eV, HOMO = -4.9 to -5.2 V) were tuned by introduction of various fused aromatic rings within thienopyrazine. By balancing the fundamental properties of these polymers, both high charge carrier mobilities and moderate PCEs in solar cells were achieved. Further, effects of copolymerizing units are discussed. Low band gap semiconducting polymer (Eg ∼ 1 eV) with high field effect mobility (0.044 cm2/Vs) was obtained using cyclopentadithiophene as copolymerizing unit. Finally, a molecular design approach to enhance the absorption coefficients is discussed, which resulted in improved power conversion efficiency in bulk heterojunction solar cells. © 2010 The Royal Society of Chemistry.

  9. WebTag: Web browsing into sensor tags over NFC.

    Science.gov (United States)

    Echevarria, Juan Jose; Ruiz-de-Garibay, Jonathan; Legarda, Jon; Alvarez, Maite; Ayerbe, Ana; Vazquez, Juan Ignacio

    2012-01-01

    Information and Communication Technologies (ICTs) continue to overcome many of the challenges related to wireless sensor monitoring, such as for example the design of smarter embedded processors, the improvement of the network architectures, the development of efficient communication protocols or the maximization of the life cycle autonomy. This work tries to improve the communication link of the data transmission in wireless sensor monitoring. The upstream communication link is usually based on standard IP technologies, but the downstream side is always masked with the proprietary protocols used for the wireless link (like ZigBee, Bluetooth, RFID, etc.). This work presents a novel solution (WebTag) for a direct IP based access to a sensor tag over the Near Field Communication (NFC) technology for secure applications. WebTag allows a direct web access to the sensor tag by means of a standard web browser, it reads the sensor data, configures the sampling rate and implements IP based security policies. It is, definitely, a new step towards the evolution of the Internet of Things paradigm.

  10. WebTag: Web Browsing into Sensor Tags over NFC

    Directory of Open Access Journals (Sweden)

    Juan Jose Echevarria

    2012-06-01

    Full Text Available Information and Communication Technologies (ICTs continue to overcome many of the challenges related to wireless sensor monitoring, such as for example the design of smarter embedded processors, the improvement of the network architectures, the development of efficient communication protocols or the maximization of the life cycle autonomy. This work tries to improve the communication link of the data transmission in wireless sensor monitoring. The upstream communication link is usually based on standard IP technologies, but the downstream side is always masked with the proprietary protocols used for the wireless link (like ZigBee, Bluetooth, RFID, etc.. This work presents a novel solution (WebTag for a direct IP based access to a sensor tag over the Near Field Communication (NFC technology for secure applications. WebTag allows a direct web access to the sensor tag by means of a standard web browser, it reads the sensor data, configures the sampling rate and implements IP based security policies. It is, definitely, a new step towards the evolution of the Internet of Things paradigm.

  11. The evaluation of the impact of age, skin tags, metabolic syndrome, body mass index, and smoking on homocysteine, endothelin-1, high-sensitive C-reactive protein, and on the heart

    Directory of Open Access Journals (Sweden)

    Omar Soliman El Safoury

    2013-01-01

    Full Text Available Background: Skin tags (STs are small, pedunculated skin-colored or brown papules that occur around any site where skin folds occur. The literature is short of comprehensive and controlled clinical studies aimed to evaluate the atherogenic risk factors in patients with STs. Aim of Work: The aim of this study is to evaluate the impact of age, STs, metabolic syndrome (METs, body mass index (BMI, and smoking on homocysteine (Hcy, endothelin-1 (ET-1, high-sensitive C-reactive protein (Hs-CRP, and on cardiovascular diseases. Materials and Methods: This study included 30 cardiac patients with STs, 30 non-cardiac patients with STs, and 30 healthy controls with neither heart disease nor STs. History of smoking, measurement of height, weight, BMI, waist circumference (WC, blood pressure, STs number, color, acanthosis nigricans, estimation of serum level of fasting glucose, triglycerides (TGs, cholesterol, high-dense lipoproteins (HDL, Hcy, ET-1, Hs-CRP, and the presence of the METs were elicited in the three groups. Results: Regarding the Hcy, ET-1, and Hs-CRP, the cardiac-STs group showed the highest levels and the control group showed the least ( P < 0.001. The percents of patients with METs were 56.7% in the cardiac-STs, 40% in the non-cardiac-STs, and 0% in the control group ( P < 0.001. Mean BMI exceeded the limit of obesity in the cardiac-STs group (30.9 ± 3.9 and the non-cardiac-STs group (32.6 ± 6 and was normal in the control group (24.7 ± 2.8. Hyperpigmented STs were present in 66.7% of the cardiac-STs group. Multivariate regression analysis for the independent effectors on Hcy level were the presence of STs ( P < 0.001, METs ( P = 0.001, and BMI ( P = 0.024. Regarding ET-1, the effectors were the presence of STs and METs ( P = 0.032. For Hs-CRP, effectors were the presence of STs ( P < 0.001 and smoking ( P = 0.040. Multivariate logistic regression of the predictors of cardiac disease showed that the independent predictors of the occurrence

  12. Changes in protein abundance between tender and tough meat from bovine Longissimus thoracis muscle assessed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis analysis

    DEFF Research Database (Denmark)

    Bjarnadóttir, S G; Hollung, K; Høy, M

    2012-01-01

    The aim of this study was to find potential biomarkers for meat tenderness in bovine Longissimus thoracis muscle and to compare results from isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis (2-DE) analysis. The experiment included 4 tender and 4...

  13. A novel fusion partner for enhanced secretion of recombinant proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Bae, Jung-Hoon; Sung, Bong Hyun; Seo, Jeong-Woo; Kim, Chul Ho; Sohn, Jung-Hoon

    2016-12-01

    Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.

  14. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    Science.gov (United States)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors and , and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.

  15. Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Hyo-Deok; Lee, Joong-jae [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of); Kim, Yu Jung [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Hantschel, Oliver [School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne (Switzerland); Lee, Seung-Goo [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Kim, Hak-Sung, E-mail: hskim76@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of)

    2017-01-15

    Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL{sup −1} for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays. - Highlights: • A human TNF-α (hTNF-α)-specific repebody was selected using a phage display. • A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody. • mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy. • mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays.

  16. Dihydropyridine-fused and pyridine-fused coumarins: Reduction on a glassy carbon electrode in dimethylformamide

    International Nuclear Information System (INIS)

    Nuñez-Vergara, Luis J.; Pardo-Jiménez, V.; Barrientos, C.; Olea-Azar, C.A.; Navarrete-Encina, P.A.; Squella, J.A.

    2012-01-01

    In this study, two series of dihydropyridine-fused and pyridine-fused coumarins were synthesised and electrochemically characterised in aprotic medium. In both series, the most easily reducible groups were the endocyclic carbonyl groups. The electrochemical mechanism for both types of compounds is strongly dependent on the experimental time-scale. Cyclic voltammetric (CV) reduction on a glassy carbon electrode (GCE) of the endocyclic carbonyl group of dihydropyridine-fused coumarins involves an ECEC mechanism with two electron transfer steps that are coupled with chemical reactions to produce the corresponding hemiacetal derivative. In the case of pyridine-fused coumarins, CV reduction of the endocyclic carbonyl group involves an EEC mechanism. ESR studies revealed the presence of a stabilised intermediate only for the pyridine-fused derivatives. Our theoretical study showed a spin density map of radical species delocalised mainly within the coumarin ring, indicating the reduction of the endocyclic carbonyl group. In the case of the dihydropyridine-fused derivatives, the mildly acid hydrogen of the dihydropyridine ring destabilises the radical via a father–son type reaction.

  17. Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells.

    Science.gov (United States)

    Kwon, Kwang-Chul; Daniell, Henry

    2016-08-01

    Plants cells are now approved by the FDA for cost-effective production of protein drugs (PDs) in large-scale current Good Manufacturing Practice (cGMP) hydroponic growth facilities. In lyophilized plant cells, PDs are stable at ambient temperature for several years, maintaining their folding and efficacy. Upon oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery. When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. Unique tags to deliver PDs to human immune or nonimmune cells have been developed recently. After crossing the epithelium, ubiquitous proteases cleave off tags at engineered sites. PDs are also delivered to the brain or retina by crossing the blood-brain or retinal barriers. This review highlights recent advances in PD delivery to treat Alzheimer's disease, diabetes, hypertension, Gaucher's or ocular diseases, as well as the development of affordable drugs by eliminating prohibitively expensive purification, cold chain and sterile delivery.

  18. Quantifying Visual-Representativeness of Social Image Tags Using Image Tag Clarity

    Science.gov (United States)

    Sun, Aixin; Bhowmick, Sourav S.

    Tags associated with images in various social media sharing web sites are valuable information source for superior image retrieval experiences. Due to the nature of tagging, many tags associated with images are not visually descriptive. In this chapter, we propose Image Tag Clarity to evaluate the effectiveness of a tag in describing the visual content of its annotated images, which is also known as the image tag visual-representativeness. It is measured by computing the zero-mean normalized distance between the tag language model estimated from the images annotated by the tag and the collection language model. The tag/collection language models are derived from the bag of visual-word local content features of the images. The visual-representative tags that are commonly used to annotate visually similar images are given high tag clarity scores. Evaluated on a large real-world dataset containing more than 269K images and their associated tags, we show that the image tag clarity score can effectively identify the visual-representative tags from all tags contributed by users. Based on the tag clarity scores, we have made a few interesting observations that could be used to support many tag-based applications.

  19. Identifying novel genes in C. elegans using SAGE tags

    Directory of Open Access Journals (Sweden)

    Chen Nansheng

    2010-12-01

    Full Text Available Abstract Background Despite extensive efforts devoted to predicting protein-coding genes in genome sequences, many bona fide genes have not been found and many existing gene models are not accurate in all sequenced eukaryote genomes. This situation is partly explained by the fact that gene prediction programs have been developed based on our incomplete understanding of gene feature information such as splicing and promoter characteristics. Additionally, full-length cDNAs of many genes and their isoforms are hard to obtain due to their low level or rare expression. In order to obtain full-length sequences of all protein-coding genes, alternative approaches are required. Results In this project, we have developed a method of reconstructing full-length cDNA sequences based on short expressed sequence tags which is called sequence tag-based amplification of cDNA ends (STACE. Expressed tags are used as anchors for retrieving full-length transcripts in two rounds of PCR amplification. We have demonstrated the application of STACE in reconstructing full-length cDNA sequences using expressed tags mined in an array of serial analysis of gene expression (SAGE of C. elegans cDNA libraries. We have successfully applied STACE to recover sequence information for 12 genes, for two of which we found isoforms. STACE was used to successfully recover full-length cDNA sequences for seven of these genes. Conclusions The STACE method can be used to effectively reconstruct full-length cDNA sequences of genes that are under-represented in cDNA sequencing projects and have been missed by existing gene prediction methods, but their existence has been suggested by short sequence tags such as SAGE tags.

  20. HMSRP Hawaiian Monk Seal Tag Data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set contains records for all tags applied to Hawaiian monk seals since 1981. These tags were applied by PSD personnel and cooperating scientists as part of...

  1. Satellite Tags- Guam/CNMI EEZ

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Satellite tagging was implemented in 2013. Satellite tagging is conducted using a Dan Inject air rifle and deployment arrows designed by Wildlife Computers. Two...

  2. A Novel Security Method For RFID Tags

    Directory of Open Access Journals (Sweden)

    Imran Ali Jokhio

    2012-07-01

    Full Text Available RFID (Radio Frequency Identification tags use light weighted security methods because of cost constraints. In this paper a lightweight security method is investigated and proved that it significantly lacks in protecting RFID tags against simple cloning attack. In order to protect RFID tag from cloning attacks a novel security method is proposed in this paper. The proposed security method provides high level computational difficulty against the three basic attacking techniques, i.e. eavesdropping, replay and man in the middle. In order to clone a tag, attacker eavesdrops the tag responses and creates a replica of the tag. The novel security method presented in this paper increases the hardness to avoid guessing tag secrets resulting in a conditional none clone able tags. The proposed security method is also evaluated using propositional logic proofs to demonstrate the level of security it can provide.

  3. The Complex Dynamics of Collaborative Tagging

    NARCIS (Netherlands)

    H. Halpin; V. Robu (Valentin); H. Shepherd

    2007-01-01

    textabstractThe debate within the Web community over the optimal means by which to organize information often pits formalized classifications against distributed collaborative tagging systems. A number of questions remain unanswered, however, regarding the nature of collaborative tagging systems

  4. 30 CFR 57.12036 - Fuse removal or replacement.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Fuse removal or replacement. 57.12036 Section 57.12036 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND... Electricity Surface and Underground § 57.12036 Fuse removal or replacement. Fuses shall not be removed or...

  5. 30 CFR 56.12036 - Fuse removal or replacement.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Fuse removal or replacement. 56.12036 Section 56.12036 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND... § 56.12036 Fuse removal or replacement. Fuses shall not be removed or replaced by hand in an energized...

  6. Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin.

    Science.gov (United States)

    Gabe, Claire M; Brookes, Steven J; Kirkham, Jennifer

    2017-01-01

    Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli ) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic

  7. Semantic image segmentation with fused CNN features

    Science.gov (United States)

    Geng, Hui-qiang; Zhang, Hua; Xue, Yan-bing; Zhou, Mian; Xu, Guang-ping; Gao, Zan

    2017-09-01

    Semantic image segmentation is a task to predict a category label for every image pixel. The key challenge of it is to design a strong feature representation. In this paper, we fuse the hierarchical convolutional neural network (CNN) features and the region-based features as the feature representation. The hierarchical features contain more global information, while the region-based features contain more local information. The combination of these two kinds of features significantly enhances the feature representation. Then the fused features are used to train a softmax classifier to produce per-pixel label assignment probability. And a fully connected conditional random field (CRF) is used as a post-processing method to improve the labeling consistency. We conduct experiments on SIFT flow dataset. The pixel accuracy and class accuracy are 84.4% and 34.86%, respectively.

  8. Electrolysis of uranium tetrafluorure fused salts

    International Nuclear Information System (INIS)

    Perillo, P.M.; Botbol, J.

    1991-01-01

    Electrolytic preparation of U has been unsuccessful because the metal formed is in easily oxidized state. Electrolytic depositions were made under various conditions from fused NaCl-KCl baths containing UF 4 . X-ray diffraction studies were made of the products. The results indicate that mixed U with several oxides phases are produced. It was concluded that the method was unlikely to be efficient for the production of U metal. (Author) [es

  9. Titanium metal obtention by fused salts electrolysis

    International Nuclear Information System (INIS)

    Perillo, P.M.; Ares, Osvaldo; Botbol, Jose.

    1989-01-01

    Potassium fluorotitanate dissolved in fused sodium chloride or potassium chloride may be electrolyzed under an inert gas atmosphere. Solid electrolysis products are formed on the cathode which contains titanium metal, sodium chloride, lower fluorotitanates and small quantities of alkali metal fluorotitanate. The extraction of titanium from the electrolysis products may be carried out by aqueous leaching (removal of chloride salts of alkali metals and a certain amount of fluorotitanates). Titanium metal obtained is relatively pure. (Author)

  10. A comparative clinical, pathological, biochemical and genetic study of fused in sarcoma proteinopathies

    DEFF Research Database (Denmark)

    Lashley, Tammaryn; Rohrer, Jonathan D; Bandopadhyay, Rina

    2011-01-01

    Neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration are rare diseases characterized by ubiquitin-positive inclusions lacking transactive response DNA-binding protein-43 and tau. Recently, mutations in the fused in sarcoma gene have been shown to cause...... familial amyotrophic lateral sclerosis and fused in sarcoma-positive neuronal inclusions have subsequently been demonstrated in neuronal intermediate filament inclusion disease and atypical frontotemporal lobar degeneration with ubiquitinated inclusions. Here we provide clinical, imaging, morphological...... findings, as well as genetic and biochemical data in 14 fused in sarcoma proteinopathy cases. In this cohort, the age of onset was variable but included cases of young-onset disease. Patients with atypical frontotemporal lobar degeneration with ubiquitinated inclusions all presented with behavioural...

  11. Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling.

    Science.gov (United States)

    Yin, Jun; Lin, Alison J; Buckett, Peter D; Wessling-Resnick, Marianne; Golan, David E; Walsh, Christopher T

    2005-09-01

    Fluorescence imaging of living cells depends on an efficient and specific method for labeling the target cellular protein with fluorophores. Here we show that Sfp phosphopantetheinyl transferase-catalyzed protein labeling is suitable for fluorescence imaging of membrane proteins that spend at least part of their membrane trafficking cycle at the cell surface. In this study, transferrin receptor 1 (TfR1) was fused to peptide carrier protein (PCP), and the TfR1-PCP fusion protein was specifically labeled with fluorophore Alexa 488 by Sfp. The trafficking of transferrin-TfR1-PCP complex during the process of transferrin-mediated iron uptake was imaged by fluorescence resonance energy transfer between the fluorescently labeled transferrin ligand and TfR1 receptor. We thus demonstrated that Sfp-catalyzed small molecule labeling of the PCP tag represents a practical and efficient tool for molecular imaging studies in living cells.

  12. Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers. I. Theoretical studies.

    Science.gov (United States)

    Hjertén, Stellan; Mohabbati, Sheila; Westerlund, Douglas

    2004-10-22

    II). The difference between the experimentally determined total variance and the sum of the calculated variances originating from the width of the starting zone, longitudinal diffusion, Joule heating, sedimentation in the vertical section of the capillary, curvature of the capillary (i.e., the sum of all other variances) was in our most successful experiments about 28% of the variance of diffusion. The zone broadening, 2sigma, caused by diffusion was estimated at 0.77 mm. The total zone width (2sigma) calculated from the experimentally determined plate number was as small as 1 mm when the migration distance was 40 cm. Accordingly, the only efficient way to reduce drastically the total zone width is to decrease the analysis time and, thereby, the diffusional broadening. An important finding was that the variance originating from the loops of the capillary is not always negligible in high-performance runs. Therefore, one should employ straight capillaries and avoid CE apparatus with cartridges that require a strong curvature of the capillary, common in most commercial instruments. Mathematical formulas have been derived for the sedimentation of the solute zone, the enrichment factor, and the migration time in experiments where the solute is dissolved in a dilute running buffer. This zone sharpening method gave very narrow starting zones (0.04-0.4 mm). However, upon high dilution of the buffer the enrichment becomes so strong that part of the sample zone probably sediments out of the capillary; the almost inevitable change in pH may decrease the mobility of the proteins and, thus, cause the enrichment factor to become still lower than expected. Diffusion of the protein in the very narrow starting zone (located close to the tip of the capillary) and sometimes the thermal expansion of the buffer in the capillary contributes to additional loss of protein in the enrichment step. In some buffers, the interaction between the protein and the buffer constituents is so slow that

  13. A Fluorogenic TMP-tag for High Signal-to-Background Intracellular Live Cell Imaging

    OpenAIRE

    Jing, Chaoran; Cornish, Virginia W.

    2013-01-01

    Developed to compliment the use of fluorescent proteins in live cell imaging, chemical tags enjoy the benefit of modular incorporation of organic fluorophores, opening the possibility of high photon output and special photophysical properties. However, the theoretical challenge in using chemical tags as opposed to fluorescent proteins for high-resolution imaging is background noise from unbound and/or non-specifically bound ligand-fluorophore. We envisioned we could overcome this limit by eng...

  14. Transposon tagging and the study of root development in Arabidopsis

    Science.gov (United States)

    Tsugeki, R.; Olson, M. L.; Fedoroff, N. V.

    1998-01-01

    The maize Ac-Ds transposable element family has been used as the basis of transposon mutagenesis systems that function in a variety of plants, including Arabidopsis. We have developed modified transposons and methods which simplify the detection, cloning and analysis of insertion mutations. We have identified and are analyzing two plant lines in which genes expressed either in the root cap cells or in the quiescent cells, cortex/endodermal initial cells and columella cells of the root cap have been tagged with a transposon carrying a reporter gene. A gene expressed in root cap cells tagged with an enhancer-trap Ds was isolated and its corresponding EST cDNA was identified. Nucleotide and deduced amino acid sequences of the gene show no significant similarity to other genes in the database. Genetic ablation experiments have been done by fusing a root cap-specific promoter to the diphtheria toxin A-chain gene and introducing the fusion construct into Arabidopsis plants. We find that in addition to eliminating gravitropism, root cap ablation inhibits elongation of roots by lowering root meristematic activities.

  15. Modelling and Analysis of Proximity Effect in IGBT Fuses

    DEFF Research Database (Denmark)

    Iov, Florin; Blaabjerg, Frede; Rasmussen, Henrik

    2005-01-01

    . Even with an active protection, a high power IGBT still has a risk of exhibiting a violent rupture in the case of a fault if e.g. IGBT fuses are not protecting it. By introducing fuses into voltage source converters a better protection of IGBT's can be achieved. However, skin and proximity effects...... affect the current distribution in a fuse due to the high frequency currents and thus a need for de-rating the fuse. This paper shows an analytical model for studying the proximity effect into a fuse. The results obtained using this model are compared with experiments....

  16. ATLAS boosted object tagging 2

    CERN Document Server

    Caudron, Julien; The ATLAS collaboration

    2015-01-01

    A detailed study into the optimal techniques for identifying boosted hadronically decaying W or Z bosons is presented. Various algorithms for reconstructing, grooming and tagging bosonic jets are compared for W bosons with a wide range of transverse momenta using 8 TeV data and 8 TeV and 13 TeV MC simulations. In addition, given that a hadronic jet has been identified as resulting from the hadronic decay of a W or Z, a technique is developed to discriminate between W and Z bosons. The modeling of the tagging variables used in this technique is studied using 8 TeV pp collision data and systematic uncertainties for the tagger efficiency and fake rates are evaluated.

  17. Outbursts In Symbiotic Binaries (FUSE 2000)

    Science.gov (United States)

    Kenyon, Scott J.; Sonneborn, George (Technical Monitor)

    2002-01-01

    During the past year, we made good progress on analysis of FUSE observations of the symbiotic binary Z And. For background, Z And is a binary system composed of a red giant and a hot component of unknown status. The orbital period is roughly 750 days. The hot component undergoes large-scale eruptions every 10-20 yr. An outburst began several years ago, triggering this FUSE opportunity. First, we obtained an excellent set of ground-based optical data in support, of the FUSE observations. We used FAST, a high throughput low resolution spectrograph on the 1.5-m telescope at Mt. Hopkins, Arizona. A 300 g/ mm grating blazed at 4750 A, a 3 in. slit, and a thinned Loral 512 x 2688 CCD gave us spectra covering 3800-7500 A at a resolution of 6 A. The wavelength solution for each spectrum has a probable error of +/- 0.5 A or better. Most of the resulting spectra have moderate signal-to-noise, S/.N approx. greater than 30 per pixel. The time coverage for these spectra is excellent. Typically, we acquired spectra every 1-2 nights during dark runs at Mt. Hopkins. These data cover most of the rise and all of the decline of the recent outburst. The spectra show a wealth of emission lines, including H I, He I, He II, [Fe V11], and the Raman scattering bands at 6830 A and 7088 A. The Raman bands and other high ionization features vary considerably throughout the outburst. These features will enable us to correlate variations in the FUSE spectra with variations in the optical spectra. Second, we began an analysis of FUSE spectra of Z And. We have carefully examined the spectra, identifying real features and defects. We have identified and measured fluxes for all strong emission lines, including the O VI doublet at 1032 A and 1038 A. These and several other strong emission lines display pronounced P Cygni absorption components indicative of outgrowing gas. We will attempt to correlate these velocities with similar profiles observed on optical spectra. The line velocities - together

  18. Investigation of TtrD, an expressing recombinant fusion tag, in Escherichia coli.

    Science.gov (United States)

    Chen, Anqi; Zhang, Li; Gu, Shaohua; Tang, Rong; Xie, Yi; Ji, Chaoneng

    2016-04-01

    Escherichia coli is widely used for expressing recombinant proteins, and several tags have been developed to improve protein solubility. However, expressing and purifying protein from other organisms is not always successful. In this study, we investigated the possibility of using TtrD as an expressing fusion tag in E. coli. Twenty RING finger domain containing human genes were expressed in E. coli grown at 37 °C and 18 °C and tested with four other fusion tags, namely His, SUMO, GST and MBP, for comparison. The results indicated that the soluble expressing ability of the tags was MBP, GST, TtrD, SUMO, and His in descending order. A one-column refolding process was used to purify the expressed proteins in inclusion bodies, and TtrD showed the strongest refolding ability. The results suggested that the TtrD tag enhanced recombinant protein solubility and refolding ability and might be a useful tag for protein expression in E. coli. Copyright © 2015. Published by Elsevier Inc.

  19. Bag3-induced autophagy is associated with degradation of JCV oncoprotein, T-Ag.

    Directory of Open Access Journals (Sweden)

    Ilker Kudret Sariyer

    Full Text Available JC virus, JCV, is a human neurotropic polyomavirus whose replication in glial cells causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML. In addition, JCV possesses oncogenic activity and expression of its transforming protein, large T-antigen (T-Ag, in several experimental animals induces tumors of neural origin. Further, the presence of JCV DNA and T-Ag have been repeatedly observed in several human malignant tissues including primitive neuroectodermal tumors and glioblastomas. Earlier studies have demonstrated that Bag3, a member of the Bcl-2-associated athanogene (Bag family of proteins, which is implicated in autophagy and apoptosis, is downregulated upon JCV infection of glial cells and that JCV T-Ag is responsible for suppressing the activity of the BAG3 promoter. Here, we investigated the possible impact of Bag3 on T-Ag expression in JCV-infected human primary glial cells as well as in cells derived from T-Ag-induced medulloblastoma in transgenic animals. Results from these studies revealed that overexpression of Bag3 drastically decreases the level of T-Ag expression by inducing the autophagic degradation of the viral protein. Interestingly, this event leads to the inhibition of JCV infection of glial cells, suggesting that the reduced levels of T-antigen seen upon the overexpression of Bag3 has a biological impact on the viral lytic cycle. Results from protein-protein interaction studies showed that T-Ag and Bag3 physically interact with each other through the zinc-finger of T-Ag and the proline rich domains of Bag3, and this interaction is important for the autophagic degradation of T-Ag. Our observations open a new avenue of research for better understanding of virus-host interaction by investigating the interplay between T-Ag and Bag3, and their impact on the development of JCV-associated diseases.

  20. Tag Based Audio Search Engine

    OpenAIRE

    Parameswaran Vellachu; Sunitha Abburu

    2012-01-01

    The volume of the music database is increasing day by day. Getting the required song as per the choice of the listener is a big challenge. Hence, it is really hard to manage this huge quantity, in terms of searching, filtering, through the music database. It is surprising to see that the audio and music industry still rely on very simplistic metadata to describe music files. However, while searching audio resource, an efficient "Tag Based Audio Search Engine" is necessary. The current researc...

  1. Infrared tag and track technique

    Science.gov (United States)

    Partin, Judy K.; Stone, Mark L.; Slater, John; Davidson, James R.

    2007-12-04

    A method of covertly tagging an object for later tracking includes providing a material capable of at least one of being applied to the object and being included in the object, which material includes deuterium; and performing at least one of applying the material to the object and including the material in the object in a manner in which in the appearance of the object is not changed, to the naked eye.

  2. Role of fused Mycobacterium tuberculosis immunogens and adjuvants in modern tuberculosis vaccines

    Directory of Open Access Journals (Sweden)

    Ana Paula eJunqueira-Kipnis

    2014-04-01

    Full Text Available Several approaches have been developed to improve or replace the only available vaccine for tuberculosis (TB, BCG (Bacille Calmette Guerin. The development of subunit protein vaccines is a promising strategy because it combines specificity and safety. In addition, subunit protein vaccines can be designed to have selected immune epitopes associated with immunomodulating components to drive the appropriate immune response. However, the limited antigens present in subunit vaccines reduce their capacity to stimulate a complete immune response compared with vaccines composed of live attenuated or killed microorganisms. This deficiency can be compensated by the incorporation of adjuvants in the vaccine formulation. The fusion of adjuvants with Mycobacterium tuberculosis (Mtb proteins or immune epitopes has the potential to become the new frontier in the TB vaccine development field. Researchers have addressed this approach by fusing the immune epitopes of their vaccines with molecules such as interleukins, lipids, lipoproteins, and immune stimulatory peptides, which have the potential to enhance the immune response. The fused molecules are being tested as subunit vaccines alone or within live attenuated vector contexts. Therefore, the objectives of this review are to discuss the association of Mtb fusion proteins with adjuvants; Mtb immunogens fused with adjuvants; and cytokine fusion with Mtb proteins and live recombinant vectors expressing cytokines. The incorporation of adjuvant molecules in a vaccine can be complex, and developing a stable fusion with proteins is a challenging task. Overall, the fusion of adjuvants with Mtb epitopes, despite the limited number of studies, is a promising field in vaccine development.

  3. Approximation properties of haplotype tagging

    Directory of Open Access Journals (Sweden)

    Dreiseitl Stephan

    2006-01-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are locations at which the genomic sequences of population members differ. Since these differences are known to follow patterns, disease association studies are facilitated by identifying SNPs that allow the unique identification of such patterns. This process, known as haplotype tagging, is formulated as a combinatorial optimization problem and analyzed in terms of complexity and approximation properties. Results It is shown that the tagging problem is NP-hard but approximable within 1 + ln((n2 - n/2 for n haplotypes but not approximable within (1 - ε ln(n/2 for any ε > 0 unless NP ⊂ DTIME(nlog log n. A simple, very easily implementable algorithm that exhibits the above upper bound on solution quality is presented. This algorithm has running time O((2m - p + 1 ≤ O(m(n2 - n/2 where p ≤ min(n, m for n haplotypes of size m. As we show that the approximation bound is asymptotically tight, the algorithm presented is optimal with respect to this asymptotic bound. Conclusion The haplotype tagging problem is hard, but approachable with a fast, practical, and surprisingly simple algorithm that cannot be significantly improved upon on a single processor machine. Hence, significant improvement in computatational efforts expended can only be expected if the computational effort is distributed and done in parallel.

  4. Predicting floods with Flickr tags.

    Science.gov (United States)

    Tkachenko, Nataliya; Jarvis, Stephen; Procter, Rob

    2017-01-01

    Increasingly, user generated content (UGC) in social media postings and their associated metadata such as time and location stamps are being used to provide useful operational information during natural hazard events such as hurricanes, storms and floods. The main advantage of these new sources of data are twofold. First, in a purely additive sense, they can provide much denser geographical coverage of the hazard as compared to traditional sensor networks. Second, they provide what physical sensors are not able to do: By documenting personal observations and experiences, they directly record the impact of a hazard on the human environment. For this reason interpretation of the content (e.g., hashtags, images, text, emojis, etc) and metadata (e.g., keywords, tags, geolocation) have been a focus of much research into social media analytics. However, as choices of semantic tags in the current methods are usually reduced to the exact name or type of the event (e.g., hashtags '#Sandy' or '#flooding'), the main limitation of such approaches remains their mere nowcasting capacity. In this study we make use of polysemous tags of images posted during several recent flood events and demonstrate how such volunteered geographic data can be used to provide early warning of an event before its outbreak.

  5. b-tagging in DELPHI at LEP

    CERN Document Server

    Abdallah, J; Adam, W; Adye, T; Adzic, P; Albrecht, T; Alderweireld, T; Alemany-Fernandez, R; Allmendinger, T; Allport, P P; Almehed, S; Amaldi, Ugo; Amapane, N; Amato, S; Anashkin, E; Andreazza, A; Andringa, S; Anjos, N; Antilogus, P; Apel, W D; Arnoud, Y; Ask, S; Åsman, B; Augustin, J E; Augustinus, A; Baillon, Paul; Ballestrero, A; Bambade, P; Barbier, R; Bardin, Dimitri Yuri; Barker, G; Baroncelli, A; Bates, M; Battaglia, Marco; Baubillier, M; Becks, K H; Begalli, M; Behrmann, A; Benekos, N C; Benvenuti, Alberto C; Bérat, C; Berggren, M; Berntzon, L; Bertrand, D; Besançon, M; Besson, N; Bibby, J; Biffi, P; Bloch, D; Blom, M; Bonesini, M; Boonekamp, M; Booth, P S L; Borisov, G; Botner, O; Bouquet, B; Bowcock, T J V; Boyko, I; Bracko, M; Branchini, P; Brenner, R; Brodet, E; Brückman, P; Brunet, J M; Bugge, L; Buschmann, P; Caccia, M; Calvi, M; Camporesi, T; Canale, V; Carena, F; Castro, N; Cavallo, F R; Chabaud, V; Chapkin, M M; Charpentier, P; Checchia, P; Chierici, R; Shlyapnikov, P; Chudoba, J; Chung, S U; Cieslik, K; Collins, P; Contri, R; Cosme, G; Cossutti, F; Costa, M J; Couchot, F; Crawley, B; Crennell, D J; Cuevas-Maestro, J; D'Almagne, B; D'Hondt, J; Dalmau, J; Da Silva, T; Da Silva, W; Della Ricca, G; De Angelis, A; de Boer, Wim; De Clercq, C; De Lotto, B; De Maria, N; De Min, A; De Paula, L S; Di Ciaccio, Lucia; Dijkstra, H; Di Simone, A; Doroba, K; Drees, J; Dris, M; Eigen, G; Ekelöf, T J C; Ellert, M; Elsing, M; Espirito-Santo, M C; Fanourakis, G K; Fassouliotis, D; Feindt, M; Fernández, J; Ferrer, A; Ferro, F; Flagmeyer, U; Föth, H; Fokitis, E; Fulda-Quenzer, F; Fuster, J A; Gandelman, M; García, C; Gavillet, P; Gazis, E N; Geralis, T; Gokieli, R; Golob, B; Gómez-Cadenas, J J; Gómez-Ceballos, G; Gonçalves, P; Graziani, E; Grosdidier, G; Grzelak, K; Guy, J; Haag, C; Hallgren, A; Hamacher, K; Hamilton, K; Hansen, J; Haug, S; Hauler, F; Hedberg, V; Hennecke, M; Hernando, J A; Herr, H; Heuser, J M; Holmgren, S O; Holt, P J; Houlden, M A; Hultqvist, K; Jackson, J N; Jalocha, P; Jarlskog, C; Jarlskog, G; Jarry, P; Jeans, D; Johansson, E K; Johansson, P D; Jonsson, P; Joram, C; Jungermann, L; Kapusta, F; Karlsson, M; Katsanevas, S; Katsoufis, E C; Keränen, R; Kernel, G; Kersevan, Borut P; Kiiskinen, A P; King, B T; Kjaer, N J; Kluit, P; Kokkinias, P; Kourkoumelis, C; Kuznetsov, O; Krumshtein, Z; Kucharczyk, M; Kucewicz, W; Kurowska, J; Lamsa, J; Leder, G; Ledroit, F; Leinonen, L; Leitner, R; Lemonne, J; Lepeltier, V; Lesiak, T; Liebig, W; Liko, D; Lipniacka, A; Lopes, J H; López, J M; Loukas, D; Lutz, P; Lyons, L; MacNaughton, J; Malek, A; Maltezos, S; Mandl, F; Marco, J; Marco, R; Maréchal, B; Margoni, M; Marin, J C; Mariotti, C; Markou, A; Martínez-Rivero, C; Martínez-Vidal, F; Masik, J; Mastroyiannopoulos, N; Matorras, F; Matteuzzi, C; Mazzucato, F; Mazzucato, M; McNulty, R; Meroni, C; Meyer, W T; Migliore, E; Mitaroff, W A; Mjörnmark, U; Moa, T; Moch, M; Mönig, K; Monge, R; Montenegro, J; Moraes, D; Moreno, S; Morettini, P; Müller, U; Münich, K; Mulders, M; Mundim, L; Murray, W; Muryn, B; Myatt, Gerald; Myklebust, T; Nassiakou, M; Navarria, Francesco Luigi; Nawrocki, K; Nicolaidou, R; Niezurawski, P; Nikolenko, M; Nomerotski, A; Norman, A; Nygren, A; Oblakowska-Mucha, A; Obraztsov, V F; Olshevskii, A G; Onofre, A; Orava, Risto; Österberg, K; Ouraou, A; Oyanguren, A; Paganoni, M; Paiano, S; Palacios, J P; Palka, H; Papadopoulou, T D; Pape, L; Parkes, C; Parodi, F; Parzefall, U; Passeri, A; Passon, O; Peralta, L; Perepelitsa, V F; Perrotta, A; Petrolini, A; Piedra, J; Pieri, L; Pierre, F; Pimenta, M; Piotto, E; Podobnik, T; Poireau, V; Pol, M E; Polok, G; Poropat, P; Pozdnyakov, V; Pukhaeva, N; Pullia, Antonio; Rames, J; Ramler, L; Read, A; Rebecchi, P; Rehn, J; Reid, D; Reinhardt, R; Renton, P B; Richard, F; Rídky, J; Rivero, M; Rodríguez, D; Romero, A; Ronchese, P; Rosenberg, E I; Roudeau, Patrick; Rovelli, T; Ruhlmann-Kleider, V; Ryabtchikov, D; Sadovskii, A; Salmi, L; Salt, J; Savoy-Navarro, A; Schwickerath, U; Segar, A; Sekulin, R L; Siebel, M; Sissakian, A N; Smadja, G; Smirnova, O G; Sokolov, A; Sopczak, A; Sosnowski, R; Spassoff, Tz; Stanitzki, M; Stavitski, I; Stocchi, A; Strauss, J; Stugu, B; Szczekowski, M; Szeptycka, M; Szumlak, T; Tabarelli de Fatis, T; Taffard, A C; Tegenfeldt, F; Timmermans, J; Tinti, N; Tkatchev, L G; Tobin, M; Todorovova, S; Tomaradze, A G; Tomé, B; Tonazzo, A; Tortosa, P; Travnicek, P; Treille, D; Trischuk, W; Tristram, G; Trochimczuk, M; Troncon, C; Turluer, M L; Tyapkin, I A; Tyapkin, P; Tyndel, M; Tzamarias, S; Uvarov, V; Valenti, G; van Dam, P; Van Eldik, J; Van Lysebetten, A; Van Remortel, N; Van Vulpen, I B; Vegni, G; Veloso, F; Venus, W A; Verbeure, F; Verdier, P; Verzi, V; Vilanova, D; Vitale, L; Vrba, V; Wahlen, H; Washbrook, A J; Weilhammer, Peter; Weiser, C; Wicke, D; Wickens, J H; Wilkinson, G; Winter, M; Witek, M; Yushchenko, O P; Zalewska-Bak, A; Zalewski, Piotr; Zavrtanik, D; Zimin, N I; Zinchenko, A I; Zupan, M

    2004-01-01

    The standard method used for tagging b-hadrons in the DELPHI experiment at the CERN LEP Collider is discussed in detail. The main ingredient of b-tagging is the impact parameters of tracks, which relies mostly on the vertex detector. Additional information, such as the mass of particles associated to a secondary vertex, significantly improves the selection efficiency and the background suppression. The paper describes various discriminating variables used for the tagging and the procedure of their combination. In addition, applications of b-tagging to some physics analyses, which depend crucially on the performance and reliability of b-tagging, are described briefly.

  6. Evaluation of rice tetraticopeptide domain-containing thioredoxin as a novel solubility-enhancing fusion tag in Escherichia coli.

    Science.gov (United States)

    Xiao, Wenjun; Jiang, Li; Wang, Weiyu; Wang, Ruyue; Fan, Jun

    2018-02-01

    Fusion of solubility-enhancing tag is frequently used for improving soluble production of target protein in Escherichia coli. The Arabidopsis tetraticopeptide domain-containing thioredoxin (TDX) has been documented to exhibit functions of disulfide reductase, foldase chaperone, and holdase chaperone. Here, we identified that fusion of rice TDX with the smaller size increased soluble expression levels of three fluorescent proteins with different fluorophores in the E. coli strain BL21(DE3) or the Rosetta (DE3) strain with coexpression of six rare tRNAs, but decreased conformational quality of certain fluorescent proteins, as comparison with the His6-tagged ones. Among five maize proteins, the rice TDX fusion carrier displayed higher solubility-enhancing activity than the yeast SUMO3 tag toward three proteins in both E. coli strains. Five fusion constructs were cleaved with the co-expressed TEV protease variant, but the released target proteins were partly insolubly aggregated in vivo. Attachment of the His6-tag to the TDX tagged proteins had little impact on protein solubility. After Ni-NTA purification, five His6-TDX tagged proteins displayed different apparent purities. Taken together, our work presents that rice TDX tag is a novel solubility enhancer. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Secure passive RFID tag with seal

    Energy Technology Data Exchange (ETDEWEB)

    Nekoogar, Faranak; Reynolds, Matthew; Lefton, Scott; Dowla, Farid; Twogood, Richard

    2017-11-14

    A secure passive RFID tag system comprises at least one base station and at least one passive RFID tag. The tag includes a fiber optic cable with the cable ends sealed within the tag and the middle portion forming an external loop. The loop may be secured to at least portions of an object. The tag transmits and receives an optical signal through the fiber optic cable, and the cable is configured to be damaged or broken in response to removal or tampering attempts, wherein the optical signal is significantly altered if the cable is damaged or broken. The tag transmits the optical signal in response to receiving a radio signal from the base station and compares the transmitted optical signal to the received optical signal. If the transmitted optical signal and the received optical signal are identical, the tag transmits an affirmative radio signal to the base station.

  8. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    International Nuclear Information System (INIS)

    Hu, Wen-Ta; Li, Hui-Chun; Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling; Lo, Shih-Yen

    2013-01-01

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway

  9. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wen-Ta [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Li, Hui-Chun [Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China); Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Lo, Shih-Yen, E-mail: losylo@mail.tcu.edu.tw [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (China)

    2013-05-24

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

  10. Spatial distribution and temporal evolution of DRONPA-fused SNAP25 clusters in adrenal chromaffin cells

    DEFF Research Database (Denmark)

    Antoku, Yasuko; Dedecker, Peter; da Silva Pinheiro, Paulo César

    2015-01-01

    fluorescence bursts of DRONPA-fused SNAP-25 molecules in live chromaffin cells by Total Internal Reflection Fluorescence (TIRF) imaging. We find that this method allows tracking protein cluster dynamics over relatively long times (∼20 min.), partly due to the diffusion into the TIRF field of fresh molecules......Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized...

  11. Quartz/fused silica chip carriers

    Science.gov (United States)

    1992-01-01

    The primary objective of this research and development effort was to develop monolithic microwave integrated circuit (MMIC) packaging which will operate efficiently at millimeter-wave frequencies. The packages incorporated fused silica as the substrate material which was selected due to its favorable electrical properties and potential performance improvement over more conventional materials for Ka-band operation. The first step towards meeting this objective is to develop a package that meets standard mechanical and thermal requirements using fused silica and to be compatible with semiconductor devices operating up to at least 44 GHz. The second step is to modify the package design and add multilayer and multicavity capacity to allow for application specific integrated circuits (ASIC's) to control multiple phase shifters. The final step is to adapt the package design to a phased array module with integral radiating elements. The first task was a continuation of the SBIR Phase 1 work. Phase 1 identified fused silica as a viable substrate material by demonstrating various plating, machining, and adhesion properties. In Phase 2 Task 1, a package was designed and fabricated to validate these findings. Task 2 was to take the next step in packaging and fabricate a multilayer, multichip module (MCM). This package is the predecessor to the phased array module and demonstrates the ability to via fill, circuit print, laminate, and to form vertical interconnects. The final task was to build a phased array module. The radiating elements were to be incorporated into the package instead of connecting to it with wire or ribbon bonds.

  12. Purification of recombinant tissue plasminogen activator (rtPA) protein from transplastomic tobacco plants.

    Science.gov (United States)

    Abdoli Nasab, Maryam; Jalali Javaran, Mokhtar; Cusido, Rosa M; Palazon, Javier

    2016-11-01

    Plants are low cost platforms for the production of recombinant proteins, but their complexity renders the purification of plant recombinant proteins more difficult than proteins expressed in yeast or bacteria. Plastid transformation enables high-level expression of foreign genes and the accumulation of recombinant proteins in plastid organelles. Histidine (His) tags are widely used for affinity purification of recombinant proteins in a nickel column. The human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of the body. The truncated form of the tissue plasminogen activator (K2S) has a longer plasma half-life, better diffusion into the clot, and higher fibrinolytic activity. In a construct designed to insert the K2S gene in the tobacco chloroplast, the sequence of six histidines and a factor Xa protease site was fused to the C-terminus of the K2S protein. The presence and amount of tPA recombinant protein in transplastomic tobacco plants was estimated by ELISA analysis using a specific antibody. The protein was purified from total soluble protein, insoluble protein aggregates and the protein was extracted from the isolated chloroplast using nickel resin and a chromatography column. After digestion of the purified protein with factor Xa, the presence of the purified tPA protein was confirmed by western blot analysis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

    Directory of Open Access Journals (Sweden)

    Qi He

    Full Text Available The endocytosis of transferrin receptor (TfR has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

  14. A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

    Science.gov (United States)

    Ardisson-Araújo, Daniel Mendes Pereira; Rocha, Juliana Ribeiro; da Costa, Márcio Hedil Oliveira; Bocca, Anamélia Lorenzetti; Dusi, André Nepomuceno; de Oliveira Resende, Renato; Ribeiro, Bergmann Morais

    2013-08-15

    Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in

  15. Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

    Science.gov (United States)

    Liu, Fang

    Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium

  16. Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin

    Directory of Open Access Journals (Sweden)

    Claire M. Gabe

    2017-06-01

    Full Text Available Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We

  17. Fused Reality for Enhanced Flight Test Capabilities

    Science.gov (United States)

    Bachelder, Ed; Klyde, David

    2011-01-01

    The feasibility of using Fused Reality-based simulation technology to enhance flight test capabilities has been investigated. In terms of relevancy to piloted evaluation, there remains no substitute for actual flight tests, even when considering the fidelity and effectiveness of modern ground-based simulators. In addition to real-world cueing (vestibular, visual, aural, environmental, etc.), flight tests provide subtle but key intangibles that cannot be duplicated in a ground-based simulator. There is, however, a cost to be paid for the benefits of flight in terms of budget, mission complexity, and safety, including the need for ground and control-room personnel, additional aircraft, etc. A Fused Reality(tm) (FR) Flight system was developed that allows a virtual environment to be integrated with the test aircraft so that tasks such as aerial refueling, formation flying, or approach and landing can be accomplished without additional aircraft resources or the risk of operating in close proximity to the ground or other aircraft. Furthermore, the dynamic motions of the simulated objects can be directly correlated with the responses of the test aircraft. The FR Flight system will allow real-time observation of, and manual interaction with, the cockpit environment that serves as a frame for the virtual out-the-window scene.

  18. Oriented Immobilization of His-Tagged Protein on a Redox Active Thiol Derivative of DPTA-Cu(II Layer Deposited on a Gold Electrode—The Base of Electrochemical Biosensors

    Directory of Open Access Journals (Sweden)

    Hanna Radecka

    2013-09-01

    Full Text Available This paper concerns the development of an electrochemical biosensor for the determination of Aβ16–23' and Aβ1–40 peptides. The His-tagged V and VC1 domains of Receptor for Advanced Glycation end Products (RAGE immobilized on a gold electrode surface were used as analytically active molecules. The immobilization of His6–RAGE domains consists of: (i formation of a mixed layer of N-acetylcysteamine (NAC and the thiol derivative of pentetic acid (DPTA; (ii complexation of Cu(II by DPTA; (iii oriented immobilization of His6–RAGE domains via coordination bonds between Cu(II sites from DPTA–Cu(II complex and imidazole nitrogen atoms of a histidine tag. Each modification step was controlled by cyclic voltammetry (CV, Osteryoung square-wave voltammetry (OSWV, and atomic force microscopy (AFM. The applicability of the proposed biosensor was tested in the presence of human plasma, which had no influence on its performance. The detection limits for Aβ1–40 determination were 1.06 nM and 0.80 nM, in the presence of buffer and human plasma, respectively. These values reach the concentration level of Aβ1–40 which is relevant for determination of its soluble form in human plasma, as well as in brain. This indicates the promising future application of biosensor presented for early diagnosis of neurodegenerative diseases.

  19. A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy

    International Nuclear Information System (INIS)

    Hass, Mathias A. S.; Liu, Wei-Min; Agafonov, Roman V.; Otten, Renee; Phung, Lien A.; Schilder, Jesika T.; Kern, Dorothee; Ubbink, Marcellus

    2015-01-01

    NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements

  20. Analyses of expressed sequence tags from apple.

    Science.gov (United States)

    Newcomb, Richard D; Crowhurst, Ross N; Gleave, Andrew P; Rikkerink, Erik H A; Allan, Andrew C; Beuning, Lesley L; Bowen, Judith H; Gera, Emma; Jamieson, Kim R; Janssen, Bart J; Laing, William A; McArtney, Steve; Nain, Bhawana; Ross, Gavin S; Snowden, Kimberley C; Souleyre, Edwige J F; Walton, Eric F; Yauk, Yar-Khing

    2006-05-01

    The domestic apple (Malus domestica; also known as Malus pumila Mill.) has become a model fruit crop in which to study commercial traits such as disease and pest resistance, grafting, and flavor and health compound biosynthesis. To speed the discovery of genes involved in these traits, develop markers to map genes, and breed new cultivars, we have produced a substantial expressed sequence tag collection from various tissues of apple, focusing on fruit tissues of the cultivar Royal Gala. Over 150,000 expressed sequence tags have been collected from 43 different cDNA libraries representing 34 different tissues and treatments. Clustering of these sequences results in a set of 42,938 nonredundant sequences comprising 17,460 tentative contigs and 25,478 singletons, together representing what we predict are approximately one-half the expressed genes from apple. Many potential molecular markers are abundant in the apple transcripts. Dinucleotide repeats are found in 4,018 nonredundant sequences, mainly in the 5'-untranslated region of the gene, with a bias toward one repeat type (containing AG, 88%) and against another (repeats containing CG, 0.1%). Trinucleotide repeats are most common in the predicted coding regions and do not show a similar degree of sequence bias in their representation. Bi-allelic single-nucleotide polymorphisms are highly abundant with one found, on average, every 706 bp of transcribed DNA. Predictions of the numbers of representatives from protein families indicate the presence of many genes involved in disease resistance and the biosynthesis of flavor and health-associated compounds. Comparisons of some of these gene families with Arabidopsis (Arabidopsis thaliana) suggest instances where there have been duplications in the lineages leading to apple of biosynthetic and regulatory genes that are expressed in fruit. This resource paves the way for a concerted functional genomics effort in this important temperate fruit crop.

  1. A fluorogenic TMP-tag for high signal-to-background intracellular live cell imaging.

    Science.gov (United States)

    Jing, Chaoran; Cornish, Virginia W

    2013-08-16

    Developed to complement the use of fluorescent proteins in live cell imaging, chemical tags enjoy the benefit of modular incorporation of organic fluorophores, opening the possibility of high photon output and special photophysical properties. However, the theoretical challenge in using chemical tags as opposed to fluorescent proteins for high-resolution imaging is background noise from unbound and/or nonspecifically bound ligand-fluorophore. We envisioned we could overcome this limit by engineering fluorogenic trimethoprim-based chemical tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR)-tagged protein displaces the quencher. Thus, we began by building a nonfluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here we take the final step and render the covalent TMP-tag fluorogenic. In brief, we designed a trimeric TMP-fluorophore-quencher molecule (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. We present the in vitro experiments showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only our initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins but also less abundant, more dynamic cytoplasmic proteins. These results suggest that the fluorogenic TMP-tag can significantly impact high-resolution live cell imaging and further establish the potential of proximity-induced reactivity and organic chemistry more broadly as part of the growing toolbox for synthetic biology and cell engineering.

  2. A Fluorogenic TMP-tag for High Signal-to-Background Intracellular Live Cell Imaging

    Science.gov (United States)

    Jing, Chaoran

    2013-01-01

    Developed to compliment the use of fluorescent proteins in live cell imaging, chemical tags enjoy the benefit of modular incorporation of organic fluorophores, opening the possibility of high photon output and special photophysical properties. However, the theoretical challenge in using chemical tags as opposed to fluorescent proteins for high-resolution imaging is background noise from unbound and/or non-specifically bound ligand-fluorophore. We envisioned we could overcome this limit by engineering fluorogenic trimethoprim-based chemical tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR) tagged protein displaces the quencher. Thus, we began by building a non-fluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here we take the final step and render the covalent TMP-tag fluorogenic. In brief, we designed a trimeric TMP-fluorophore-quencher molecule (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. We present the in vitro experiments showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only our initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins, but also less abundant, more dynamic cytoplasmic proteins. These results suggest that fluorogenic TMP-tag can significantly impact highresolution live cell imaging and further establish the potential of proximity-induced reactivity and organic chemistry more broadly as part of the growing toolbox for synthetic biology and cell engineering. PMID:23745575

  3. A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.

    Science.gov (United States)

    Sharapova, Olga A; Yurkova, Maria S; Fedorov, Alexey N

    2016-02-01

    We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Learner Corpora without Error Tagging

    Directory of Open Access Journals (Sweden)

    Rastelli, Stefano

    2009-01-01

    Full Text Available The article explores the possibility of adopting a form-to-function perspective when annotating learner corpora in order to get deeper insights about systematic features of interlanguage. A split between forms and functions (or categories is desirable in order to avoid the "comparative fallacy" and because – especially in basic varieties – forms may precede functions (e.g., what resembles to a "noun" might have a different function or a function may show up in unexpected forms. In the computer-aided error analysis tradition, all items produced by learners are traced to a grid of error tags which is based on the categories of the target language. Differently, we believe it is possible to record and make retrievable both words and sequence of characters independently from their functional-grammatical label in the target language. For this purpose at the University of Pavia we adapted a probabilistic POS tagger designed for L1 on L2 data. Despite the criticism that this operation can raise, we found that it is better to work with "virtual categories" rather than with errors. The article outlines the theoretical background of the project and shows some examples in which some potential of SLA-oriented (non error-based tagging will be possibly made clearer.

  5. Fused upper central incisors: management of two clinical cases

    OpenAIRE

    Sfasciotti, Gian Luca; Marini, Roberta; Bossù, Maurizio; Ierardo, Gaetano; Annibali, Susanna

    2012-01-01

    This paper reports the management of two clinical cases, in which the upper right central incisor was fused with a supernumerary tooth and the upper left central incisor was macrodontic. A radiographic examination revealed that the fused teeth had two separate roots. Hemisectioning of the fused teeth was performed, the supernumerary portion was extracted and the remaining part was reshaped to remove any sharp margins and to achieve a normal morphology. The macrodontic central incisors were no...

  6. Energy calibration of the photon tagging system

    International Nuclear Information System (INIS)

    Endo, Satoru; Niki, Kazuaki; Suzuki, Akira

    1989-07-01

    There is a photon tagging system at the 1.3 GeV Electron Synchrotron (ES), and the system is designed to produce a monochromatic photon beam with an energy resolution of ±5 MeV. To calibrate the energies of tagged photons produced by this system, electron beams of various energies from ES are injected directly into this system. As the result of this calibration, we conclude that the photon tagging system has the performance of designed values. (author)

  7. B-tagging in CMS at LHC

    CERN Document Server

    Cucciarelli, S

    2003-01-01

    This report provides a review of the main algorithms for offline inclusive b-tagging developed within the CMS community. Two b-tag algorithms, one based on the impact parameter measurement and the other based on the secondary vertices are discussed. The performance of these algorithms are presented for several jet transverse energies and pseudorapidity regions. An additional decay length based b-tag is also described and its preliminary performance is presented. (4 refs) .

  8. FUSE: a profit maximization approach for functional summarization of biological networks.

    Science.gov (United States)

    Seah, Boon-Siew; Bhowmick, Sourav S; Dewey, C Forbes; Yu, Hanry

    2012-03-21

    The availability of large-scale curated protein interaction datasets has given rise to the opportunity to investigate higher level organization and modularity within the protein interaction network (PPI) using graph theoretic analysis. Despite the recent progress, systems level analysis of PPIS remains a daunting task as it is challenging to make sense out of the deluge of high-dimensional interaction data. Specifically, techniques that automatically abstract and summarize PPIS at multiple resolutions to provide high level views of its functional landscape are still lacking. We present a novel data-driven and generic algorithm called FUSE (Functional Summary Generator) that generates functional maps of a PPI at different levels of organization, from broad process-process level interactions to in-depth complex-complex level interactions, through a pro t maximization approach that exploits Minimum Description Length (MDL) principle to maximize information gain of the summary graph while satisfying the level of detail constraint. We evaluate the performance of FUSE on several real-world PPIS. We also compare FUSE to state-of-the-art graph clustering methods with GO term enrichment by constructing the biological process landscape of the PPIS. Using AD network as our case study, we further demonstrate the ability of FUSE to quickly summarize the network and identify many different processes and complexes that regulate it. Finally, we study the higher-order connectivity of the human PPI. By simultaneously evaluating interaction and annotation data, FUSE abstracts higher-order interaction maps by reducing the details of the underlying PPI to form a functional summary graph of interconnected functional clusters. Our results demonstrate its effectiveness and superiority over state-of-the-art graph clustering methods with GO term enrichment.

  9. FUSE: a profit maximization approach for functional summarization of biological networks

    Directory of Open Access Journals (Sweden)

    Seah Boon-Siew

    2012-03-01

    Full Text Available Abstract Background The availability of large-scale curated protein interaction datasets has given rise to the opportunity to investigate higher level organization and modularity within the protein interaction network (PPI using graph theoretic analysis. Despite the recent progress, systems level analysis of PPIS remains a daunting task as it is challenging to make sense out of the deluge of high-dimensional interaction data. Specifically, techniques that automatically abstract and summarize PPIS at multiple resolutions to provide high level views of its functional landscape are still lacking. We present a novel data-driven and generic algorithm called FUSE (Functional Summary Generator that generates functional maps of a PPI at different levels of organization, from broad process-process level interactions to in-depth complex-complex level interactions, through a pro t maximization approach that exploits Minimum Description Length (MDL principle to maximize information gain of the summary graph while satisfying the level of detail constraint. Results We evaluate the performance of FUSE on several real-world PPIS. We also compare FUSE to state-of-the-art graph clustering methods with GO term enrichment by constructing the biological process landscape of the PPIS. Using AD network as our case study, we further demonstrate the ability of FUSE to quickly summarize the network and identify many different processes and complexes that regulate it. Finally, we study the higher-order connectivity of the human PPI. Conclusion By simultaneously evaluating interaction and annotation data, FUSE abstracts higher-order interaction maps by reducing the details of the underlying PPI to form a functional summary graph of interconnected functional clusters. Our results demonstrate its effectiveness and superiority over state-of-the-art graph clustering methods with GO term enrichment.

  10. A Radiographic Study of Fused and Geminated Tooth

    Energy Technology Data Exchange (ETDEWEB)

    Park, Chul Jae; Lee, Sang Rae [Dept. of Oral Radiology, College of Dentistry, Kyunhee University, Seoul (Korea, Republic of)

    1990-02-15

    The incidence and several characteristic features of fused and geminated teeth were studied radiographically, with full mouth periapical radiogram and pantomogram, in 4201 patients of mixed dentition and 5358 patients of permanent dentition. The obtained results were as follows: 1. The prevalence was revealed to 2.86%, 0.32%, 0.33%, and 0.06% in deciduous fused tooth, permanent fused tooth, deciduous geminated tooth and permanent geminated tooth respectively, and these anomalies were occurred in female more than male. 2. Fused teeth were observed predominantly in lower anterior teeth area, especially in lateral incisor and canine region, and many cases of deciduous geminated tooth were observed in upper central incisor region. 3. Congenital missing rates of succedaneous tooth in deciduous fused teeth were 57.1%, 85.7%, 71.0%, 69.0% in upper right and left central-lateral incisor regions, lower right and left lateral incisor-canine regions, respectively. 4. Prevalence of dental caries was 42.3%, 18.8% and 5.6% in deciduous fused, deciduous geminated and permanent fused tooth, respectively. 5. In classifying of fused and geminated teeth into 9 type, by following appearance such as number of crown, root, pulp chamber and pulp canal of those teeth, it was more favorable that Type I (2 crown, 2 root, 2 pulp chamber, 2 pulp canal) in deciduous fused tooth and Type IX (1 crown, 1 root, 1 pulp chamber, 1 pulp canal) in permanent used tooth, deciduous and permanent geminated tooth.

  11. A Radiographic Study of Fused and Geminated Tooth

    International Nuclear Information System (INIS)

    Park, Chul Jae; Lee, Sang Rae

    1990-01-01

    The incidence and several characteristic features of fused and geminated teeth were studied radiographically, with full mouth periapical radiogram and pantomogram, in 4201 patients of mixed dentition and 5358 patients of permanent dentition. The obtained results were as follows: 1. The prevalence was revealed to 2.86%, 0.32%, 0.33%, and 0.06% in deciduous fused tooth, permanent fused tooth, deciduous geminated tooth and permanent geminated tooth respectively, and these anomalies were occurred in female more than male. 2. Fused teeth were observed predominantly in lower anterior teeth area, especially in lateral incisor and canine region, and many cases of deciduous geminated tooth were observed in upper central incisor region. 3. Congenital missing rates of succedaneous tooth in deciduous fused teeth were 57.1%, 85.7%, 71.0%, 69.0% in upper right and left central-lateral incisor regions, lower right and left lateral incisor-canine regions, respectively. 4. Prevalence of dental caries was 42.3%, 18.8% and 5.6% in deciduous fused, deciduous geminated and permanent fused tooth, respectively. 5. In classifying of fused and geminated teeth into 9 type, by following appearance such as number of crown, root, pulp chamber and pulp canal of those teeth, it was more favorable that Type I (2 crown, 2 root, 2 pulp chamber, 2 pulp canal) in deciduous fused tooth and Type IX (1 crown, 1 root, 1 pulp chamber, 1 pulp canal) in permanent used tooth, deciduous and permanent geminated tooth.

  12. Graph based techniques for tag cloud generation

    DEFF Research Database (Denmark)

    Leginus, Martin; Dolog, Peter; Lage, Ricardo Gomes

    2013-01-01

    Tag cloud is one of the navigation aids for exploring documents. Tag cloud also link documents through the user defined terms. We explore various graph based techniques to improve the tag cloud generation. Moreover, we introduce relevance measures based on underlying data such as ratings...... or citation counts for improved measurement of relevance of tag clouds. We show, that on the given data sets, our approach outperforms the state of the art baseline methods with respect to such relevance by 41 % on Movielens dataset and by 11 % on Bibsonomy data set....

  13. Sensor-based material tagging system

    International Nuclear Information System (INIS)

    Vercellotti, L.C.; Cox, R.W.; Ravas, R.J.; Schlotterer, J.C.

    1991-01-01

    Electronic identification tags are being developed for tracking material and personnel. In applying electronic identification tags to radioactive materials safeguards, it is important to measure attributes of the material to ensure that the tag remains with the material. The addition of a microcontroller with an on-board analog-to-digital converter to an electronic identification tag application-specific integrated-circuit has been demonstrated as means to provide the tag with sensor data. Each tag is assembled into a housing, which serves as a scale for measuring the weight of a paint-can-sized container and its contents. Temperature rise of the can above ambient is also measured, and a piezoelectric detector detects disturbances and immediately puts the tag into its alarm and beacon mode. Radiation measurement was also considered, but the background from nearby containers was found to be excessive. The sensor-based tagging system allows tracking of the material in cans as it is stored in vaults or is moved through the manufacturing process. The paper presents details of the sensor-based material tagging system and describes a demonstration system

  14. Using Interference to Block RFID Tags

    DEFF Research Database (Denmark)

    Krigslund, Rasmus; Popovski, Petar; Pedersen, Gert Frølund

    We propose a novel method to block RFID tags from responding, using intentional interference. We focus on the experimental evaluation, where we impose interference on the download and uplink, respectively. The results are positive, where modulated CCI shows most effective to block a tag.......We propose a novel method to block RFID tags from responding, using intentional interference. We focus on the experimental evaluation, where we impose interference on the download and uplink, respectively. The results are positive, where modulated CCI shows most effective to block a tag....

  15. Discharge residence of TLD tagged fish

    International Nuclear Information System (INIS)

    Romberg, G.P.; Prepejchal, W.

    1974-01-01

    Although visual observations suggested that fish remained in the discharge for considerable periods, temperature-sensitive tags indicated the majority of fish spend less than 50 hr or 10 percent of the time at discharge temperatures. During 1974 a second fish tagging study was conducted, using temperature-sensitive tags to yield discharge residence times of Lake Michigan salmonids at Point Beach thermal discharge. Preliminary results revealed that many fish tag values were close to Unit I line indicating that calculated maximum discharge residence times for these fish will be nearly 100 percent of the elapsed time

  16. Hemi-fused structure mediates and controls fusion and fission in live cells.

    Science.gov (United States)

    Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J; Krystofiak, Evan S; Villarreal, Seth A; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

    2016-06-23

    Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

  17. Chlorotoxin Fused to IgG-Fc Inhibits Glioblastoma Cell Motility via Receptor-Mediated Endocytosis

    Directory of Open Access Journals (Sweden)

    Tomonari Kasai

    2012-01-01

    Full Text Available Chlorotoxin is a 36-amino acid peptide derived from Leiurus quinquestriatus (scorpion venom, which has been shown to inhibit low-conductance chloride channels in colonic epithelial cells. Chlorotoxin also binds to matrix metalloproteinase-2 and other proteins on glioma cell surfaces. Glioma cells are considered to require the activation of matrix metalloproteinase-2 during invasion and migration. In this study, for targeting glioma, we designed two types of recombinant chlorotoxin fused to human IgG-Fcs with/without a hinge region. Chlorotoxin fused to IgG-Fcs was designed as a dimer of 60 kDa with a hinge region and a monomer of 30 kDa without a hinge region. The monomeric and dimeric forms of chlorotoxin inhibited cell proliferation at 300 nM and induced internalization in human glioma A172 cells. The monomer had a greater inhibitory effect than the dimer; therefore, monomeric chlorotoxin fused to IgG-Fc was multivalently displayed on the surface of bionanocapsules to develop a drug delivery system that targeted matrix metalloproteinase-2. The target-dependent internalization of bionanocapsules in A172 cells was observed when chlorotoxin was displayed on the bionanocapsules. This study indicates that chlorotoxin fused to IgG-Fcs could be useful for the active targeting of glioblastoma cells.

  18. Understanding error generation in fused deposition modeling

    International Nuclear Information System (INIS)

    Bochmann, Lennart; Transchel, Robert; Wegener, Konrad; Bayley, Cindy; Helu, Moneer; Dornfeld, David

    2015-01-01

    Additive manufacturing offers completely new possibilities for the manufacturing of parts. The advantages of flexibility and convenience of additive manufacturing have had a significant impact on many industries, and optimizing part quality is crucial for expanding its utilization. This research aims to determine the sources of imprecision in fused deposition modeling (FDM). Process errors in terms of surface quality, accuracy and precision are identified and quantified, and an error-budget approach is used to characterize errors of the machine tool. It was determined that accuracy and precision in the y direction (0.08–0.30 mm) are generally greater than in the x direction (0.12–0.62 mm) and the z direction (0.21–0.57 mm). Furthermore, accuracy and precision tend to decrease at increasing axis positions. The results of this work can be used to identify possible process improvements in the design and control of FDM technology. (paper)

  19. Understanding error generation in fused deposition modeling

    Science.gov (United States)

    Bochmann, Lennart; Bayley, Cindy; Helu, Moneer; Transchel, Robert; Wegener, Konrad; Dornfeld, David

    2015-03-01

    Additive manufacturing offers completely new possibilities for the manufacturing of parts. The advantages of flexibility and convenience of additive manufacturing have had a significant impact on many industries, and optimizing part quality is crucial for expanding its utilization. This research aims to determine the sources of imprecision in fused deposition modeling (FDM). Process errors in terms of surface quality, accuracy and precision are identified and quantified, and an error-budget approach is used to characterize errors of the machine tool. It was determined that accuracy and precision in the y direction (0.08-0.30 mm) are generally greater than in the x direction (0.12-0.62 mm) and the z direction (0.21-0.57 mm). Furthermore, accuracy and precision tend to decrease at increasing axis positions. The results of this work can be used to identify possible process improvements in the design and control of FDM technology.

  20. Mechanical losses in thin fused silica fibres

    International Nuclear Information System (INIS)

    Bilenko, I A; Braginsky, V B; Lourie, S L

    2004-01-01

    Intracavity topology of the readout system for LIGO III project and table-top QND mechanical measurements under development require the use of small probe masses and suspensions with a very low level of internal losses. A good choice is to use thin fused silica fibres similar to LIGO II mirrors suspensions. Mechanical losses of silica fibres are investigated in this work through the study of quality factor dependence on diameter for pendulum and violin modes of oscillations with diameters ranging from 1.5 to 40 μm. The estimated values of effective mechanical loss angle show noticeably greater growth with lower diameters than might be expected while extrapolating known results of research done for thicker fibres

  1. Mechanical losses in thin fused silica fibres

    Energy Technology Data Exchange (ETDEWEB)

    Bilenko, I A; Braginsky, V B; Lourie, S L [Department of Oscillatory Physics, Physics Faculty, Moscow State University (Russian Federation)

    2004-03-07

    Intracavity topology of the readout system for LIGO III project and table-top QND mechanical measurements under development require the use of small probe masses and suspensions with a very low level of internal losses. A good choice is to use thin fused silica fibres similar to LIGO II mirrors suspensions. Mechanical losses of silica fibres are investigated in this work through the study of quality factor dependence on diameter for pendulum and violin modes of oscillations with diameters ranging from 1.5 to 40 {mu}m. The estimated values of effective mechanical loss angle show noticeably greater growth with lower diameters than might be expected while extrapolating known results of research done for thicker fibres.

  2. Fused Entropy Algorithm in Optical Computed Tomography

    Directory of Open Access Journals (Sweden)

    Xiong Wan

    2014-02-01

    Full Text Available In most applications of optical computed tomography (OpCT, limited-view problems are often encountered, which can be solved to a certain extent with typical OpCT reconstructive algorithms. The concept of entropy first emerged in information theory has been introduced into OpCT algorithms, such as maximum entropy (ME algorithms and cross entropy (CE algorithms, which have demonstrated their superiority over traditional OpCT algorithms, yet have their own limitations. A fused entropy (FE algorithm, which follows an optimized criterion combining self-adaptively ME with CE, is proposed and investigated by comparisons with ME, CE and some traditional OpCT algorithms. Reconstructed results of several physical models show this FE algorithm has a good convergence and can achieve better precision than other algorithms, which verifies the feasibility of FE as an approach of optimizing computation, not only for OpCT, but also for other image processing applications.

  3. Highly selective end-tagged antimicrobial peptides derived from PRELP.

    Directory of Open Access Journals (Sweden)

    Martin Malmsten

    Full Text Available BACKGROUND: Antimicrobial peptides (AMPs are receiving increasing attention due to resistance development against conventional antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in an array of infections such as ocular infections, cystic fibrosis, wound and post-surgery infections, and sepsis. The goal of the study was to design novel AMPs against these pathogens. METHODOLOGY AND PRINCIPAL FINDINGS: Antibacterial activity was determined by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was evaluated by hemolysis and effects on human epithelial cells. Liposome and fluorescence studies provided mechanistic information. Protease sensitivity was evaluated after subjection to human leukocyte elastase, staphylococcal aureolysin and V8 proteinase, as well as P. aeruginosa elastase. Highly active peptides were evaluated in ex vivo skin infection models. C-terminal end-tagging by W and F amino acid residues increased antimicrobial potency of the peptide sequences GRRPRPRPRP and RRPRPRPRP, derived from proline arginine-rich and leucine-rich repeat protein (PRELP. The optimized peptides were antimicrobial against a range of gram-positive S. aureus and gram-negative P. aeruginosa clinical isolates, also in the presence of human plasma and blood. Simultaneously, they showed low toxicity against mammalian cells. Particularly W-tagged peptides displayed stability against P. aeruginosa elastase, and S. aureus V8 proteinase and aureolysin, and the peptide RRPRPRPRPWWWW-NH(2 was effective against various "superbugs" including vancomycin-resistant enterococci, multi-drug resistant P. aeruginosa, and methicillin-resistant S. aureus, as well as demonstrated efficiency in an ex vivo skin wound model of S. aureus and P. aeruginosa infection. CONCLUSIONS/SIGNIFICANCE: Hydrophobic C-terminal end-tagging of the cationic sequence RRPRPRPRP generates highly selective AMPs with potent

  4. Identification of human basic fetoprotein as glucose-6-phosphate isomerase by using N- and C-terminal sequence tags and terminal tag database.

    Science.gov (United States)

    Kuyama, Hiroki; Yoshizawa, Akiyasu C; Nakajima, Chihiro; Hosako, Mutsumi; Tanaka, Koichi

    2015-08-10

    Human basic fetoprotein (BFP), found in fetal serum and tissue extracts as well as in extracts of various cancer tissues, has long been known as a marker protein for cancers; however, the primary sequence has not yet been reported. This paper describes the identification of BFP using the N- and C-terminal amino acid sequence tags (Ac-AALTRDPQFQ and QQREARVQ, respectively) clarified by mass spectrometry-based methods, and a terminal tag database (ProteinCarta). In this study, BFP was identified as glucose-6-phosphate isomerase (G6PI_HUMAN). Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Engineering the ATLAS TAG Browser

    International Nuclear Information System (INIS)

    Zhang Qizhi

    2011-01-01

    ELSSI is a web-based event metadata (TAG) browser and event-level selection service for ATLAS. In this paper, we describe some of the challenges encountered in the process of developing ELSSI, and the software engineering strategies adopted to address those challenges. Approaches to management of access to data, browsing, data rendering, query building, query validation, execution, connection management, and communication with auxiliary services are discussed. We also describe strategies for dealing with data that may vary over time, such as run-dependent trigger decision decoding. Along with examples, we illustrate how programming techniques in multiple languages (PHP, JAVASCRIPT, XML, AJAX, and PL/SQL) have been blended to achieve the required results. Finally, we evaluate features of the ELSSI service in terms of functionality, scalability, and performance.

  6. To tag or not to tag: animal welfare, conservation and stakeholder considerations in fish tracking studies that use electronic tags

    Energy Technology Data Exchange (ETDEWEB)

    Cooke, Steven J.; Nguyen, Vivian M.; Murchie, Karen J.; Thiem, Jason D.; Donaldson, Michael R.; Hinch, Scott G.; Brown, Richard S.; Fisk, Aaron

    2013-11-01

    The advent and widespread adoption of electronic tags (including biotelemetry and biologging devices) for tracking animals has provided unprecedented information on the biology, management, and conservation of fish in the world’s oceans and inland waters. However, use of these tools is not without controversy. Even when scientific and management objectives may best be achieved using electronic tags, it is increasingly important to further consider other factors such as the welfare of tagged animals (i.e., the role of training and science-based surgical guidelines, anesthetic use, inability to maintain sterile conditions in field environments), the ethics of tagging threatened species vs. using surrogates, stakeholder perspectives on tagging (including aboriginals), as well as use of data emanating from such studies (e.g., by fishers to facilitate exploitation). Failure to do so will have the potential to create conflict and undermine scientific, management and public confidence in the use of this powerful tool. Indeed, there are already a number of examples of where tracking studies using electronic tags have been halted based on concerns raised by researchers, authorities, or stakeholders. Here we present a candid evaluation of several factors that should be considered when determining when to tag or not to tag fish with electronic devices. It is not our objective to judge the merit of previous studies. Rather, we hope to stimulate debate and discussion regarding the use of electronic tags to study fish. Relatedly, there is a need for more research to address these questions (e.g., what level of cleanliness is needed when conducting surgeries, what type of training should be required for fish surgery) including human dimensions studies to understand perspectives of different actors including society as a whole with respect to tagging and tracking studies.

  7. Teaching old NCATs new tricks: using non-canonical amino acid tagging to study neuronal plasticity

    Science.gov (United States)

    Hinz, FI; Dieterich, DC; Schuman, EM

    2016-01-01

    The non-canonical amino acid labeling techniques BONCAT (bioorthogonal non-canonical amino acid tagging) and FUNCAT (fluorescent non-canonical amino acid tagging) enable the specific identification and visualization of newly synthesized proteins. Recently, these techniques have been applied to neuronal systems to elucidate protein synthesis dynamics during plasticity, identify stimulation-induced proteomes and subproteomes and to investigate local protein synthesis in specific subcellular compartments. The next generation of tools and applications, reviewed here, includes the development of new tags, the quantitative identification of newly synthesized proteins, the application of NCAT to whole animals, and the ability to genetically restrict NCAT labeling. These techniques will enable not only improved detection but also allow new scientific questions to be tackled. PMID:23938204

  8. Comparing different types of patagial tags for use on vultures ...

    African Journals Online (AJOL)

    Raptor research often requires identifying individuals. Researchers place patagial tags on raptors to facilitate such identification. Researchers in southern African use two main types of patagial tags: hard plastic ear tags originally designed for cattle and soft vinyl tags. We deployed both types of tags on vultures in Botswana.

  9. Comparing different types of patagial tags for use on vultures ...

    African Journals Online (AJOL)

    Researchers in southern African use two main types of patagial tags: hard plastic ear tags originally designed for cattle and soft vinyl tags. We deployed both types of tags on vultures in Botswana. Based on our observations, we recommend using soft vinyl tags as they appear to be more aerodynamic and can be read from ...

  10. Evaluation of PIT-tagging in cyprinids

    DEFF Research Database (Denmark)

    Skov, Christian; Brodersen, J.; Brönmark, C.

    2005-01-01

    Laboratory and field experiments were used to investigate how different marking procedures, with 23 mm PIT (passive integrated transponders) - tags. affected mortality, body condition and tag expulsion in small roach Rutilus rutilus and rudd Scardinus erythrophthalmus (117 to 163 mm total length...

  11. In vivo two-photon fluorescence imaging with Cr:forsterite lasers using transgenic lines tagged by HcRed

    Science.gov (United States)

    Tsai, Tsung-Han; Chen, Szu-Yu; Tai, Shih-Peng; Lin, Cheng-Yung; Tsai, Huai-Jen; Sun, Chi-Kuang

    2005-03-01

    Transgenic lines carrying a specific tissue tagged by green-fluorescence-protein (GFP) have been a powerful tool to developmental biology because they encapsulate the expression of endogenous genes. Traditionally with two-photon fluorescence microscopy based on a femtosecond Ti:sapphire laser (with a wavelength between 700-980nm), green fluorescence can be excited by simultaneous absorption of two photons for high-resolution three-dimensional (3D) optical imaging. However for in vivo biological applications, Ti:sapphire-laser based optical technology presents several limitations including finite penetration depth, strong on-focus cell damage, and phototoxicity. For high optical penetration and minimized photodamages, two-photon imaging based on light sources with an optical wavelength located around the biological penetration window (~1300nm) is desired, where unwanted light-tissue interactions including scattering, absorption, and photodamages can all be minimized. Previous experiments around the optical penetration window indicated inefficient green fluorescence excitation of GFP through three-photon absorption. Red fluorescence protein is thus highly desired for future non-invasive in vivo two-photon imaging. Screening from embryos injected with DNA fragment containing a heart-specific regulatory element of zebrafish cardiac myosin light chain 2 gene (cmlc2) fused with HcRed gene, we generate a zebrafish line that has strong two-photon red fluorescence expressed in cardiac cells based on a 1230nm femtosecond light source working in the biological penetration window. Combined with its nonlinearity, high penetration depth, and minimized photodamages, this method provides superb imaging capability compared with the traditional GFP based two-photon microscopy, offering deep insight into the noninvasive in vivo studies of gene expression in vertebrate embryos.

  12. Sentiment topic mining based on comment tags

    Science.gov (United States)

    Zhang, Daohai; Liu, Xue; Li, Juan; Fan, Mingyue

    2018-03-01

    With the development of e-commerce, various comments based on tags are generated, how to extract valuable information from these comment tags has become an important content of business management decisions. This study takes HUAWEI mobile phone tags as an example using the sentiment analysis and topic LDA mining method. The first step is data preprocessing and classification of comment tag topic mining. And then make the sentiment classification for comment tags. Finally, mine the comments again and analyze the emotional theme distribution under different sentiment classification. The results show that HUAWEI mobile phone has a good user experience in terms of fluency, cost performance, appearance, etc. Meanwhile, it should pay more attention to independent research and development, product design and development. In addition, battery and speed performance should be enhanced.

  13. Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies

    Science.gov (United States)

    Setterquist, R. A.; Smith, G. K.; Oakley, T. H.; Lee, Y. H.; Fox, G. E.

    1996-01-01

    A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

  14. Tagged at first listen: an examination of social tagging practices in a music recommender system

    Directory of Open Access Journals (Sweden)

    Audrey Laplante

    2015-01-01

    Full Text Available http://dx.doi.org/10.5007/1518-2924.2015v20nesp1p33 Social tagging has become a very common way to index different types of resources on the web. Less prevalent in music than in other domains, social tagging is nevertheless used in a popular recommender system, Last.fm. Although the number of publications on tagging and folksonomies has exploded in the last few years, music tagging is still not well studied. In this paper, we present a study of tagging practices of Last.fm users. We examine the social tagging of songs during the first three months after their release. Our analysis shows that the release of a song triggers a burst in tagging activity that lasts two weeks, after what it decreases sharply and then remains fairly constant for the next ten weeks. We also find that a majority of songs do not get tagged during the first week and that tagging was positively related to popularity. Finally, we find that tags that have been frequently applied to a given song are more likely to be genre related, shorter in length, and relatively objective than tags that have been applied only once.

  15. Expression and affinity purification of recombinant proteins from plants

    Science.gov (United States)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  16. Pop-up Archival Transmitting (PAT) fish tag data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The cooperative tagging center (CTC) began deploying electronic tags in 2002. To date over 300 tags have been deployed. The following species have been monitored:...

  17. Fusing website usability and search engine optimisation

    Directory of Open Access Journals (Sweden)

    Eugene B. Visser

    2014-03-01

    Objectives: This research intended to establish a relationship between search engine optimisation and website usability in order to guide the industry. The authors found a discrepancy between the perceived roles of search engines and website usability. Method: The authors designed three test websites. Each had different combinations of usability, visibility and other attributes. They recorded and analysed the conversions and financial spending on these experimental websites. Finally, they designed a model that fuses search engine optimisation and website usability. Results: Initially, it seemed that website usability and search engine optimisation complemented each other. However, some contradictions between the two, based on content, keywords and their presentation, emerged. Industry experts do not acknowledge these contradictions, although they agree on the existence of the individual elements. The new model highlights the complementary and contradictory aspects. Conclusion: The authors found no evidence of any previous empirical experimental results that could confirm or refute the role of the model. In the fast-paced world of competition between commercial websites, this adds value and originality to the websites of organisations whose websites play important roles.

  18. Thermogravimetry analysis on fused borosilicate syntactic foams

    Science.gov (United States)

    Salleh, Zulzamri; Islam, Md Mainul; Epaarachchi, Jayantha Ananda

    2017-12-01

    Fused borosilicate/vinyl ester syntactic foams mostly used for thermal stability in many applications are considered to be characterised for their properties in higher temperature condition. Therefore, higher temperature characteristics need to be explored with respect to physical parameters such as porosity and void content. The filler, also known as glass microballoons, was incorporated in vinyl ester resin matrix in 2wt%, 4wt%, 6wt.%, 8wt.% and 10wt.%. These composites are characterised and degraded to examine the onset temperature, Tonset, peak temperature, Tpeak and end temperature, Tend using thermogravimetric analysis (TGA) method. The results for Tonset showed an increment while Tend and Tpeak showed decrement when glass microballoons were added in syntactic foams. Based on TGA data, they exhibited excellent thermal stability, showing 5% weight loss at 380-450 °C. Glass transition temperature (Tg) decreased when more glass microballoons were added, and also related to the effect on the physical properties of syntactic foams. Therefore, understanding of the relationship between thermal properties of syntactic foams and their physical properties such as porosity and void content will help in developing syntactic foams to optimise their thermal characteristics, which will be beneficial for engineering applications.

  19. Ultralightweight low-temperature fused Zerodur mirror

    Science.gov (United States)

    Miller, Jennifer; Crowe, David A.; Clark, Patrick; Marker, Alexander J.

    1995-09-01

    The performance and utility of optical systems can be significantly improved by using lightweight stable mirror components. Such components have been incorporated into a mirror assembly through a process that utilizes low temperature fusion to bond low CTE faceplates onto a lightweight core structure. The core structure is fabricated using an abrasive waterjet cutting technique that enables the designer to optimize core geometry to enhance the structural performance of the blank. The faceplates are bonded to the lightweight core resulting in a stiff structural mirror. A 15.125 inch Zerodur mirror was fabricated using this unique process and subjected to a test program to measure the optical stability of the mirror in support of either space-based applications. Strength testing was performed to verify the integrity of the fused joints and determine appropriate design allowable stress. A thermal test program was designed to assess performance at various temperature extremes and a mechanical load test was run to verify the capability of the blank to withstand operational loads without degrading the optical surface. The surface figure of the part was measured before, during, and after a 200 degree F temperature cycle with no change in figure quality. In addition, the mirror was subjected to a 10 g load for ten minutes with no change in the figure quality.

  20. Enhanced UHF RFID tags for drug tracing.

    Science.gov (United States)

    Catarinucci, Luca; Colella, Riccardo; De Blasi, Mario; Patrono, Luigi; Tarricone, Luciano

    2012-12-01

    Radio Frequency Identification (RFID) technology is playing a crucial role for item-level tracing systems in healthcare scenarios. The pharmaceutical supply chain is a fascinating application context, where RFID can guarantee transparency in the drug flow, supporting both suppliers and consumers against the growing counterfeiting problem. In such a context, the choice of the most adequate RFID tag, in terms of shape, frequency, size and reading range, is crucial. The potential presence of items containing materials hostile to the electromagnetic propagation exasperates the problem. In addition, the peculiarities of the different RFID-based checkpoints make even more stringent the requirements for the tag. In this work, the performance of several commercial UHF RFID tags in each step of the pharmaceutical supply chain has been evaluated, confirming the expected criticality. On such basis, a guideline for the electromagnetic design of new high-performance tags capable to overcome such criticalities has been defined. Finally, driven by such guidelines, a new enhanced tag has been designed, realized and tested. Due to patent pending issues, the antenna shape is not shown. Nevertheless, the optimal obtained results do not lose their validity. Indeed, on the one hand they demonstrate that high performance item level tracing systems can actually be implemented also in critical operating conditions. On the other hand, they encourage the tag designer to follow the identified guidelines so to realize enhanced UHF tags.

  1. DICOM involving XML path-tag

    Science.gov (United States)

    Zeng, Qiang; Yao, Zhihong; Liu, Lei

    2011-03-01

    Digital Imaging and Communications in Medicine (DICOM) is a standard for handling, storing, printing, and transmitting information in medical imaging. XML (Extensible Markup Language) is a set of rules for encoding documents in machine-readable form which has become more and more popular. The combination of these two is very necessary and promising. Using XML tags instead of numeric labels in DICOM files will effectively increase the readability and enhance the clear hierarchical structure of DICOM files. However, due to the fact that the XML tags rely heavily on the orders of the tags, the strong data dependency has a lot of influence on the flexibility of inserting and exchanging data. In order to improve the extensibility and sharing of DICOM files, this paper introduces XML Path-Tag to DICOM. When a DICOM file is converted to XML format, adding simple Path-Tag into the DICOM file in place of complex tags will keep the flexibility of a DICOM file while inserting data elements and give full play to the advantages of the structure and readability of an XML file. Our method can solve the weak readability problem of DICOM files and the tedious work of inserting data into an XML file. In addition, we set up a conversion engine that can transform among traditional DICOM files, XML-DCM and XML-DCM files involving XML Path-Tag efficiently.

  2. A Novel Inducible Protein Production System and Neomycin Resistance as Selection Marker for Methanosarcina mazei

    Directory of Open Access Journals (Sweden)

    Sebastian Mondorf

    2012-01-01

    Full Text Available Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei.

  3. Communication methods, systems, apparatus, and devices involving RF tag registration

    Science.gov (United States)

    Burghard, Brion J [W. Richland, WA; Skorpik, James R [Kennewick, WA

    2008-04-22

    One technique of the present invention includes a number of Radio Frequency (RF) tags that each have a different identifier. Information is broadcast to the tags from an RF tag interrogator. This information corresponds to a maximum quantity of tag response time slots that are available. This maximum quantity may be less than the total number of tags. The tags each select one of the time slots as a function of the information and a random number provided by each respective tag. The different identifiers are transmitted to the interrogator from at least a subset of the RF tags.

  4. Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins.

    Science.gov (United States)

    Hoffmann, Daniel; Ebrahimi, Mehrdad; Gerlach, Doreen; Salzig, Denise; Czermak, Peter

    2017-11-10

    The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.

  5. W/Top/Higgs-tagging in ATLAS

    CERN Document Server

    Norjoharuddeen, Nurfikri; The ATLAS collaboration

    2017-01-01

    We present updates of W, Top and Higgs tagging studies with the ATLAS detector. The performance of 2 variable taggers, HEPTopTagger and shower deconstruction are compared in Monte Carlo simulations. To asses the modelling of the taggers’ performance, the tagging efficiencies are measured, with the full 2015+2016 dataset, in semi-leptonic top quark pair events and the background rejections are measured in dijet and photon+jet topologies. Recent developments in subjet reconstruction techniques for high transverse momentum Higgs->bb tagging are also presented.

  6. Tagging Water Sources in Atmospheric Models

    Science.gov (United States)

    Bosilovich, M.

    2003-01-01

    Tagging of water sources in atmospheric models allows for quantitative diagnostics of how water is transported from its source region to its sink region. In this presentation, we review how this methodology is applied to global atmospheric models. We will present several applications of the methodology. In one example, the regional sources of water for the North American Monsoon system are evaluated by tagging the surface evaporation. In another example, the tagged water is used to quantify the global water cycling rate and residence time. We will also discuss the need for more research and the importance of these diagnostics in water cycle studies.

  7. Fused upper central incisors: management of two clinical cases.

    Science.gov (United States)

    Sfasciotti, Gian Luca; Marini, Roberta; Bossù, Maurizio; Ierardo, Gaetano; Annibali, Susanna

    2011-03-01

    This paper reports the management of two clinical cases, in which the upper right central incisor was fused with a supernumerary tooth and the upper left central incisor was macrodontic. A radiographic examination revealed that the fused teeth had two separate roots. Hemisectioning of the fused teeth was performed, the supernumerary portion was extracted and the remaining part was reshaped to remove any sharp margins and to achieve a normal morphology. The macrodontic central incisors were not treated. At 12-months post-surgery there were no periodontal problems and no hypersensitivity. Orthodontic treatment was performed to appropriately align the maxillary teeth and to correct the malocclusion.

  8. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

    Directory of Open Access Journals (Sweden)

    Bugli F

    2014-05-01

    Full Text Available Francesca Bugli,1 Valeria Caprettini,2 Margherita Cacaci,1 Cecilia Martini,1 Francesco Paroni Sterbini,1 Riccardo Torelli,1 Stefano Della Longa,3 Massimiliano Papi,4 Valentina Palmieri,4 Bruno Giardina,5 Brunella Posteraro,1 Maurizio Sanguinetti,1 Alessandro Arcovito5 1Istituto di Microbiologia, Università Cattolica del Sacro Cuore, 2Dipartimento di Fisica, Sapienza Università di Roma, Rome, 3Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita e dell’Ambiente, Università dell’Aquila, L’Aquila, 4Istituto di Fisica, 5Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy Abstract: In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few micron long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native

  9. Comparative Performance of Acoustic-tagged and PIT-tagged Juvenile Salmonids

    Energy Technology Data Exchange (ETDEWEB)

    Hockersmith, Eric E.; Brown, Richard S.; Liedtke, Theresa L.

    2008-02-01

    Numerous research tools and technologies are currently being used to evaluate fish passage and survival to determine the impacts of the Federal Columbia River Power System (FCRPS) on endangered and threatened juvenile salmonids, including PIT tags, balloon tags, hydroacoustic evaluations, radio telemetry, and acoustic telemetry. Each has advantages and disadvantages, but options are restricted in some situations because of limited capabilities of a specific technology, lack of detection capability downstream, or availability of adequate numbers of fish. However, there remains concern about the comparative effects of the tag or the tagging procedure on fish performance. The recently developed Juvenile Salmonid Acoustic Telemetry System (JSATS) acoustic transmitter is the smallest active acoustic tag currently available. The goal of this study was to determine whether fish tagged with the JSATS acoustic-telemetry tag can provide unbiased estimates of passage behavior and survival within the performance life of the tag. We conducted both field and laboratory studies to assess tag effects. For the field evaluation we released a total of 996 acoustic-tagged fish in conjunction with 21,026 PIT-tagged fish into the tailrace of Lower Granite Dam on 6 and 13 May. Travel times between release and downstream dams were not significantly different for the majority of the reaches between acoustic-tagged and PIT-tagged fish. In addition to the field evaluation, a series of laboratory experiments were conducted to determine if growth and survival of juvenile Chinook salmon surgically implanted with acoustic transmitters is different than untagged or PIT tagged juvenile Chinook salmon. Only yearling fish with integrated and non-integrated transmitters experienced mortalities, and these were low (<4.5%). Mortality among sub-yearling control and PIT-tag treatments ranged up to 7.7% while integrated and non-integrated treatments had slightly higher rates (up to 8.3% and 7

  10. Comparing the hierarchy of author given tags and repository given tags in a large document archive

    Science.gov (United States)

    Tibély, Gergely; Pollner, Péter; Palla, Gergely

    2016-10-01

    Folksonomies - large databases arising from collaborative tagging of items by independent users - are becoming an increasingly important way of categorizing information. In these systems users can tag items with free words, resulting in a tripartite item-tag-user network. Although there are no prescribed relations between tags, the way users think about the different categories presumably has some built in hierarchy, in which more special concepts are descendants of some more general categories. Several applications would benefit from the knowledge of this hierarchy. Here we apply a recent method to check the differences and similarities of hierarchies resulting from tags given by independent individuals and from tags given by a centrally managed repository system. The results from our method showed substantial differences between the lower part of the hierarchies, and in contrast, a relatively high similarity at the top of the hierarchies.

  11. User Interface Program for secure electronic tags

    International Nuclear Information System (INIS)

    Cai, Y.; Koehl, E.R.; Carlson, R.D.; Raptis, A.C.

    1995-05-01

    This report summarizes and documents the efforts of Argonne National Laboratory (ANL) in developing a secure tag communication user interface program comprising a tag monitor and a communication tool. This program can perform the same functions as the software that was developed at the Lawrence Livermore National Laboratory (LLNL), but it is enhanced with a user-friendly screen. It represents the first step in updating the TRANSCOM Tracking System (TRANSCOM) by incorporating a tag communication screen menu into the main menu of the TRANSCOM user program. A working version of TRANSCOM, enhanced with ANL secure-tag graphics, will strongly support the Department of Energy Warhead Dismantlement/Special Nuclear Materials Control initiatives. It will allow commercial satellite tracking of the movements and operational activities of treaty-limited items and transportation vehicles throughout Europe and the former USSR, as well as the continental US

  12. In vitro tagging of embryos with nanoparticles

    OpenAIRE

    Fynewever, Tricia L.; Agcaoili, Evelyn S.; Jacobson, John D.; Patton, William C.; Chan, Philip J.

    2006-01-01

    Purpose: To develop an in vitro method for tagging embryos and to compare the development of the embryos after nanoparticles injection versus externally-applied nanoparticles derived from either polystyrene or polyacrylonitrile.

  13. Flavour Tagging with the LHCb experiment

    CERN Multimedia

    Birnkraut, Alex

    2015-01-01

    Measurements of flavour oscillations and time-dependent CP asymmetries in neutral B meson systems require knowledge of the b quark production flavour. This identification is performed by the Flavour Tagging.

  14. 50 CFR 635.33 - Archival tags.

    Science.gov (United States)

    2010-10-01

    ... landing; furnish all requested information regarding the location and method of capture; and, as... recovery of the tag by a NMFS scientist, enforcement agent, or other person designated in writing by NMFS...

  15. User Interface Program for secure electronic tags

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Y.; Koehl, E.R.; Carlson, R.D.; Raptis, A.C.

    1995-05-01

    This report summarizes and documents the efforts of Argonne National Laboratory (ANL) in developing a secure tag communication user interface program comprising a tag monitor and a communication tool. This program can perform the same functions as the software that was developed at the Lawrence Livermore National Laboratory (LLNL), but it is enhanced with a user-friendly screen. It represents the first step in updating the TRANSCOM Tracking System (TRANSCOM) by incorporating a tag communication screen menu into the main menu of the TRANSCOM user program. A working version of TRANSCOM, enhanced with ANL secure-tag graphics, will strongly support the Department of Energy Warhead Dismantlement/Special Nuclear Materials Control initiatives. It will allow commercial satellite tracking of the movements and operational activities of treaty-limited items and transportation vehicles throughout Europe and the former USSR, as well as the continental US.

  16. b-flavour tagging in pp collisions

    CERN Multimedia

    Birnkraut, Alex

    2015-01-01

    An essential ingredient of all time-dependent CP violation studies of B mesons is the ability to tag the initial flavour of the B meson. The harsh environment of 7 and 8 TeV pp collisions makes this a particularly difficult enterprise. We report progresses in the flavour tagging of B0 and Bs mesons, including developments of novel techniques like the use of an opposite side charm tagger.

  17. W and top tagging scale factors

    CERN Document Server

    CMS Collaboration

    2017-01-01

    This note presents an improved determination of the efficiency and data/MC scale factors for the identification of hadronically decaying top quarks using the full 2016 CMS dataset. Also shown is an improved measurement of the W boson tagging performance using the full 2016 CMS dataset. Finally methods for the extraction of W tagging scale factors at high transverse momentum using fully merged top quarks are shown.

  18. A double built-in containment strategy for production of recombinant proteins in transgenic rice.

    Science.gov (United States)

    Zhang, Xianwen; Wang, Dongfang; Zhao, Sinan; Shen, Zhicheng

    2014-01-01

    Using transgenic rice as a bioreactor for mass production of pharmaceutical proteins could potentially reduce the cost of production significantly. However, a major concern over the bioreactor transgenic rice is the risk of its unintended spreading into environment and into food or feed supplies. Here we report a mitigating method to prevent unwanted transgenic rice spreading by a double built-in containment strategy, which sets a selectively termination method and a visual tag technology in the T-DNA for transformation. We created transgenic rice with an inserted T-DNA that harbors a human proinsulin gene fused with the far-red fluorescent protein gene mKate_S158A, an RNAi cassette suppressing the expression of the rice bentazon detoxification enzyme CYP81A6, and an EPSPS gene as the selection marker for transformation. Herbi