Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP).

PubMed

Guan, Dongli; Chen, Zhilei

2017-01-01

Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively. The N-intein/affinity tag is used to functionalize an affinity resin. The high affinity between the N- and C-fragment of DnaE intein enables the POI to be purified from the lysate via affinity to the resin, and the intein-mediated C-terminal cleavage reaction causes tagless POI to be released into the flow-through. The intein cleavage reaction is strongly inhibited by divalent ions (e.g., Zn 2+ ) under non-reducing conditions and is significantly enhanced by reducing conditions. The POI is cleaved efficiently regardless of the identity of the N-terminal amino acid except in the cases of threonine and proline, and the N-intein-functionalized affinity resin can be regenerated for multiple cycles of use.

Investigation of differences in the binding affinities of two analogous ligands for untagged and tagged p38 kinase using thermodynamic integration MD simulation.

PubMed

Sun, Ying-Chieh; Hsu, Wen-Chi; Hsu, Chia-Jen; Chang, Chia-Ming; Cheng, Kai-Hsiang

2015-11-01

Thermodynamic integration (TI) molecular dynamics (MD) simulations for the binding of a pair of a reference ("ref") ligand and an analogous ("analog") ligand to either tagged (with six extra residues at the N-terminus) or untagged p38 kinase proteins were carried out in order to probe how the binding affinity is influenced by the presence or absence of the peptide tag in p38 kinase. This possible effect of protein length on the binding affinity of a ligand-which is seldom addressed in the literature-is important because, even when two labs claim to have performed experiments with the same protein, they may actually have studied variants of the same protein with different lengths because they applied different protein expression conditions/procedures. Thus, if we wanted to compare ligand binding affinities measured in the two labs, it would be necessary to account for any variation in ligand binding affinity with protein length. The pair of ligand-p38 kinase complexes examined in this work (pdb codes: 3d7z and 3lhj, respectively) were ideal for investigating this effect. The experimentally determined binding energy for the ref ligand with the untagged p38 kinase was -10.9 kcal mol(-1), while that for the analog ligand with the tagged p38 kinase was -11.9 kcal mol(-1). The present TI-MD simulation of the mutation of the ref ligand into the analog ligand while the ligand is bound to the untagged p38 kinase predicted that the binding affinity of the analog ligand is 2.0 kcal mol(-1) greater than that of the ref ligand. A similar simulation also indicated that the same was true for ligand binding to the tagged protein, but in this case the binding affinity for the analog ligand is 2.5 kcal mol(-1) larger than that for the ref ligand. These results therefore suggest that the presence of the peptide tag on p38 kinase increased the difference in the binding energies of the ligands by a small amount of 0.5 kcal mol(-1). This result supports the assumption that the presence of a peptide tag has only a minor effect on ΔG values. The error bars in the computed ΔG values were then estimated via confidence interval analysis and a time autocorrelation function for the quantity dV/dλ. The estimated correlation time was ~0.5 ps and the error bar in the ΔG values estimated using nanosecond-scale simulations was ±0.3 kcal mol(-1) at a confidence level of 95%. These predicted results can be verified in future experiments and should prove useful in subsequent similar studies. Graphical Abstract Thermodynamic cycles for binding of two analogous ligands with untagged and tagged p38 kinases and associated Gibbs free energy.

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

PubMed

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.

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.

PubMed

Dean, Richard A; Butler, Georgina S; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R; Courty, José; Overall, Christopher M

2007-12-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.

Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis▿ †

PubMed Central

Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R.; Courty, José; Overall, Christopher M.

2007-01-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

Quantitative proteomic analysis of human breast epithelial cells with differential telomere length

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Li-Rong; Chan, King C.; Tahara, Hidetoshi

Telomeres play important functional roles in cell proliferation, cell cycle regulation, and genetic stability, in which telomere length is critical. In this study, quantitative proteome comparisons for the human breast epithelial cells with short and long telomeres (184-hTERT{sub L} vs. 184-hTERT{sub S} and 90P-hTERT{sub L} vs. 90P-hTERT{sub S}), resulting from transfection of the human telomerase reverse transcriptase (hTERT) gene, were performed using cleavable isotope-coded affinity tags. More than 2000 proteins were quantified in each comparative experiment, with approximately 77% of the proteins identified in both analyses. In the cells with long telomeres, significant and consistent alterations were observed in metabolismmore » (amino acid, nucleotide, and lipid metabolism), genetic information transmission (transcription and translation regulation, spliceosome and ribosome complexes), and cell signaling. Interestingly, the DNA excision repair pathway is enhanced, while integrin and its ligands are downregulated in the cells with long telomeres. These results may provide valuable information related to telomere functions.« less

High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

PubMed Central

Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

2016-01-01

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

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

PubMed

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 cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts. Copyright © 2016 Elsevier Inc. All rights reserved.

Metal Stable Isotope Tagging: Renaissance of Radioimmunoassay for Multiplex and Absolute Quantification of Biomolecules.

PubMed

Liu, Rui; Zhang, Shixi; Wei, Chao; Xing, Zhi; Zhang, Sichun; Zhang, Xinrong

2016-05-17

The unambiguous quantification of biomolecules is of great significance in fundamental biological research as well as practical clinical diagnosis. Due to the lack of a detectable moiety, the direct and highly sensitive quantification of biomolecules is often a "mission impossible". Consequently, tagging strategies to introduce detectable moieties for labeling target biomolecules were invented, which had a long and significant impact on studies of biomolecules in the past decades. For instance, immunoassays have been developed with radioisotope tagging by Yalow and Berson in the late 1950s. The later languishment of this technology can be almost exclusively ascribed to the use of radioactive isotopes, which led to the development of nonradioactive tagging strategy-based assays such as enzyme-linked immunosorbent assay, fluorescent immunoassay, and chemiluminescent and electrochemiluminescent immunoassay. Despite great success, these strategies suffered from drawbacks such as limited spectral window capacity for multiplex detection and inability to provide absolute quantification of biomolecules. After recalling the sequences of tagging strategies, an apparent question is why not use stable isotopes from the start? A reasonable explanation is the lack of reliable means for accurate and precise quantification of stable isotopes at that time. The situation has changed greatly at present, since several atomic mass spectrometric measures for metal stable isotopes have been developed. Among the newly developed techniques, inductively coupled plasma mass spectrometry is an ideal technique to determine metal stable isotope-tagged biomolecules, for its high sensitivity, wide dynamic linear range, and more importantly multiplex and absolute quantification ability. Since the first published report by our group, metal stable isotope tagging has become a revolutionary technique and gained great success in biomolecule quantification. An exciting research highlight in this area is the development and application of the mass cytometer, which fully exploited the multiplexing potential of metal stable isotope tagging. It realized the simultaneous detection of dozens of parameters in single cells, accurate immunophenotyping in cell populations, through modeling of intracellular signaling network and undoubted discrimination of function and connection of cell subsets. Metal stable isotope tagging has great potential applications in hematopoiesis, immunology, stem cells, cancer, and drug screening related research and opened a post-fluorescence era of cytometry. Herein, we review the development of biomolecule quantification using metal stable isotope tagging. Particularly, the power of multiplex and absolute quantification is demonstrated. We address the advantages, applicable situations, and limitations of metal stable isotope tagging strategies and propose suggestions for future developments. The transfer of enzymatic or fluorescent tagging to metal stable isotope tagging may occur in many aspects of biological and clinical practices in the near future, just as the revolution from radioactive isotope tagging to fluorescent tagging happened in the past.

Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

PubMed Central

Hagen, Sven; Baumann, Tobias; Wagner, Hanna J.; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M.; Müller, Kristian M.

2014-01-01

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats.

PubMed

Pershad, Kritika; Sullivan, Mark A; Kay, Brian K

2011-05-15

Affinity reagents that are generated by phage display are typically subcloned into an expression vector for further biochemical characterization. This insert transfer process is time consuming and laborious especially if many inserts are to be subcloned. To simplify the transfer process, we have constructed a "drop-out" phagemid vector that can be rapidly converted to an expression vector by a simple restriction enzyme digestion with MfeI (to "drop-out" the gene III coding sequence), which generates alkaline phosphatase (AP) fusions of the affinity reagents on religation. Subsequently, restriction digestion with AscI drops out the AP coding region and religation generates affinity reagents with a C-terminal six-histidine tag. To validate the usefulness of this vector, four different human single chain Fragments of variable regions (scFv) were tested, three of which show specific binding to three zebrafish (Danio rerio) proteins, namely suppression of tumorigenicity 13, recoverin, and Ppib and the fourth binds to human Lactoferrin protein. For each of the constructs tested, the gene III and AP drop-out efficiency was between 90% and 100%. This vector is especially useful in speeding up the downstream screening of affinity reagents and bypassing the time-consuming subcloning experiments. Copyright © 2011 Elsevier Inc. All rights reserved.

Multiple tag labeling method for DNA sequencing

DOEpatents

Mathies, Richard A.; Huang, Xiaohua C.; Quesada, Mark A.

1995-01-01

A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

A Dual Protease Approach for Expression and Affinity Purification of Recombinant Proteins

PubMed Central

Raran-Kurussi, Sreejith; Waugh, David S.

2016-01-01

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. PMID:27105777

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

PubMed

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.

An Overview of Enzymatic Reagents for the Removal of Affinity Tags

PubMed Central

Waugh, David S.

2011-01-01

Although they are often exploited to facilitate the expression and purification of recombinant proteins, every affinity tag, whether large or small, has the potential to interfere with the structure and function of its fusion partner. For this reason, reliable methods for removing affinity tags are needed. Only enzymes have the requisite specificity to be generally useful reagents for this purpose. In this review, the advantages and disadvantages of some commonly used endo- and exoproteases are discussed in light of the latest information. PMID:21871965

Identification of midgut proteins that are differentially expressed in trypanosome-susceptible and normal tsetse flies (Glossina morsitans morsitans).

PubMed

Haddow, J D; Haines, L R; Gooding, R H; Olafson, R W; Pearson, T W

2005-05-01

Molecules in the midgut of tsetse flies (Diptera: Glossinidiae) are thought to play important roles in the life cycle of African trypanosomes by influencing initial parasite establishment and subsequent differentiation events that ultimately lead to maturation of mammal-infective trypanosomes. The molecular composition of the tsetse midgut is, therefore, of critical importance to disease transmission by these medically important vectors. In this study we compared protein expression profiles of midguts of the salmon mutant and wild type Glossina morsitans morsitans Westwood that display marked differences in their susceptibility to infection by African trypanosomes. Isotope coded affinity tag (ICAT) technology was used to identify 207 proteins including 17 that were up regulated and nine that were down regulated in the salmon mutants. Several of the up regulated molecules were previously described as tsetse midgut or salivary gland proteins. Of particular interest was the up regulation in the salmon flies of tsetse midgut EP protein, a recently described molecule with lectin-like activity that was also found to be induced in tsetse by bacterial challenge. The up regulation of the EP protein in midguts of salmon mutants was confirmed by two-dimensional gel electrophoresis and tandem mass spectrometry.

Design of self-coded combinatorial libraries to facilitate direct analysis of ligands by mass spectrometry.

PubMed

Hughes, I

1998-09-24

The direct analysis of selected components from combinatorial libraries by sensitive methods such as mass spectrometry is potentially more efficient than deconvolution and tagging strategies since additional steps of resynthesis or introduction of molecular tags are avoided. A substituent selection procedure is described that eliminates the mass degeneracy commonly observed in libraries prepared by "split-and-mix" methods, without recourse to high-resolution mass measurements. A set of simple rules guides the choice of substituents such that all components of the library have unique nominal masses. Additional rules extend the scope by ensuring that characteristic isotopic mass patterns distinguish isobaric components. The method is applicable to libraries having from two to four varying substituent groups and can encode from a few hundred to several thousand components. No restrictions are imposed on the manner in which the "self-coded" library is synthesized or screened.

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

PubMed

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.

Redox regulation of mitochondrial proteins and proteomes by cysteine thiol switches.

PubMed

Nietzel, Thomas; Mostertz, Jörg; Hochgräfe, Falko; Schwarzländer, Markus

2017-03-01

Mitochondria are hotspots of cellular redox biochemistry. Respiration as a defining mitochondrial function is made up of a series of electron transfers that are ultimately coupled to maintaining the proton motive force, ATP production and cellular energy supply. The individual reaction steps involved require tight control and flexible regulation to maintain energy and redox balance in the cell under fluctuating demands. Redox regulation by thiol switching has been a long-standing candidate mechanism to support rapid adjustment of mitochondrial protein function at the posttranslational level. Here we review recent advances in our understanding of cysteine thiol switches in the mitochondrial proteome with a focus on their operation in vivo. We assess the conceptual basis for thiol switching in mitochondria and discuss to what extent insights gained from in vitro studies may be valid in vivo, considering thermodynamic, kinetic and structural constraints. We compare functional proteomic approaches that have been used to assess mitochondrial protein thiol switches, including thioredoxin trapping, redox difference gel electrophoresis (redoxDIGE), isotope-coded affinity tag (OxICAT) and iodoacetyl tandem mass tag (iodoTMT) labelling strategies. We discuss conditions that may favour active thiol switching in mitochondrial proteomes in vivo, and appraise recent advances in dissecting their impact using combinations of in vivo redox sensing and quantitative redox proteomics. Finally we focus on four central facets of mitochondrial biology, aging, carbon metabolism, energy coupling and electron transport, exemplifying the current emergence of a mechanistic understanding of mitochondrial regulation by thiol switching in living plants and animals. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag.

PubMed

Abdelhamid, Mohamed A A; Ikeda, Takeshi; Motomura, Kei; Tanaka, Tatsuya; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio

2016-11-01

We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica biomineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric l-arginine, at concentrations of 0.3-0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of ∼70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75-90% recovery with ∼90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of l-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

PubMed

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.

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

PubMed

Kökpinar, Öznur; Walter, Johanna-Gabriela; Shoham, Yuval; Stahl, Frank; Scheper, Thomas

2011-10-01

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months. Copyright © 2011 Wiley Periodicals, Inc.

The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

NASA Astrophysics Data System (ADS)

Giannone, Richard J.; Liu, Yie; Wang, Yisong

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands

PubMed Central

Encell, Lance P; Friedman Ohana, Rachel; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Wood, Monika G; Los, Georgyi V; McDougall, Mark G; Zimprich, Chad; Karassina, Natasha; Learish, Randall D; Hurst, Robin; Hartnett, James; Wheeler, Sarah; Stecha, Pete; English, Jami; Zhao, Kate; Mendez, Jacqui; Benink, Hélène A; Murphy, Nancy; Daniels, Danette L; Slater, Michael R; Urh, Marjeta; Darzins, Aldis; Klaubert, Dieter H; Bulleit, Robert F; Wood, Keith V

2012-01-01

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins. PMID:23248739

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

PubMed

Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

2015-07-01

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin

PubMed Central

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 acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond. PMID:28670287

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

PubMed

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.

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

PubMed Central

Petrescu, Anca D.; Huang, Huan; Hostetler, Heather A.; Schroeder, Friedhelm; Kier, Ann B.

2008-01-01

Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (Kd=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (Kd=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP. PMID:18178100

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli.

PubMed

Verdon, Julien; Girardin, Nicolas; Marchand, Adrienne; Héchard, Yann; Berjeaud, Jean-Marc

2013-06-01

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for (15)N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification.

PubMed

Mayer, Daniel; Baginsky, Sacha; Schwemmle, Martin

2005-11-01

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.

Method and apparatus for manufacturing gas tags

DOEpatents

Gross, K.C.; Laug, M.T.

1996-12-17

For use in the manufacture of gas tags employed in a gas tagging failure detection system for a nuclear reactor, a plurality of commercial feed gases each having a respective noble gas isotopic composition are blended under computer control to provide various tag gas mixtures having selected isotopic ratios which are optimized for specified defined conditions such as cost. Using a new approach employing a discrete variable structure rather than the known continuous-variable optimization problem, the computer controlled gas tag manufacturing process employs an analytical formalism from condensed matter physics known as stochastic relaxation, which is a special case of simulated annealing, for input feed gas selection. For a tag blending process involving M tag isotopes with N distinct feed gas mixtures commercially available from an enriched gas supplier, the manufacturing process calculates the cost difference between multiple combinations and specifies gas mixtures which approach the optimum defined conditions. The manufacturing process is then used to control tag blending apparatus incorporating tag gas canisters connected by stainless-steel tubing with computer controlled valves, with the canisters automatically filled with metered quantities of the required feed gases. 4 figs.

Method and apparatus for manufacturing gas tags

DOEpatents

Gross, Kenny C.; Laug, Matthew T.

1996-01-01

For use in the manufacture of gas tags employed in a gas tagging failure detection system for a nuclear reactor, a plurality of commercial feed gases each having a respective noble gas isotopic composition are blended under computer control to provide various tag gas mixtures having selected isotopic ratios which are optimized for specified defined conditions such as cost. Using a new approach employing a discrete variable structure rather than the known continuous-variable optimization problem, the computer controlled gas tag manufacturing process employs an analytical formalism from condensed matter physics known as stochastic relaxation, which is a special case of simulated annealing, for input feed gas selection. For a tag blending process involving M tag isotopes with N distinct feed gas mixtures commercially available from an enriched gas supplier, the manufacturing process calculates the cost difference between multiple combinations and specifies gas mixtures which approach the optimum defined conditions. The manufacturing process is then used to control tag blending apparatus incorporating tag gas canisters connected by stainless-steel tubing with computer controlled valves, with the canisters automatically filled with metered quantities of the required feed gases.

The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal

PubMed Central

Vergis, James M.; Wiener, Michael C.

2011-01-01

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (Enterokinase, Factor Xa, Human Rhinovirus 3C Protease, SUMOstar, Tobacco Etch Virus Protease, and Thrombin) by use of a panel of ninety-four individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and Factor Xa were only affected by a small number of detergents, making them good choices as well. PMID:21539919

The Means: Cytometry and Mass Spectrometry Converge in a Single Cell Deep Profiling Platform

PubMed Central

Weis-Garcia, Frances; Bandura, Dmitry; Baranov, Vladimir; Ornatsky, Olga; Tanner, Scott

2013-01-01

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a distinct flavor of mass spectrometry that has had little association with cell biology: it remains the state of the art for the determination of the atomic composition of materials. Unrelatedly, flow cytometry is the superior method for distinguishing the heterogeneity of cells through the determination of antigen signatures using tagged antibodies. Simply replacing fluorophore tags with stable isotopes of the heavy metals, and measuring these cell-by-cell with ICP-MS, dramatically increases the number of probes that can be simultaneously measured in cytometry and enables a transformative increase in the resolution of rare cell populations in complex biological samples. While this can be thought of as a novel incarnation of single-cell targeted proteomics, the metal-labeling reagents, ICP-MS of single cells, and accompanying informatics comprise a new field of technology termed Mass Cytometry. While the conception of mass cytometry is simple the embodiment to address the issues of multi-parameter flow cytometry has been far more challenging. There are many elements, and many more stable isotopes of those elements, that might be used as distinct reporter tags. Still, there are many approaches to conjugating metals to antibodies (or other affinity reagents) and work in this area along with developing new applications is ongoing. The mass resolution and linear (quantitative) dynamic range of ICP-MS allows those many stable isotopes to be measured simultaneously and without the spectral overlap issues that limit fluorescence assay. However, the adaptation of ICP-MS to allow high-speed simultaneous measurement with single cell distinction at high throughput required innovation of the cell introduction system, ion optics (sampling, transmission and beam-shaping), mass analysis, and signal handling and processing. An overview of “the nuts and bolts” of Mass Cytometry is presented.

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments.

PubMed

Cannataro, Mario; Cuda, Giovanni; Gaspari, Marco; Greco, Sergio; Tradigo, Giuseppe; Veltri, Pierangelo

2007-07-15

Isotope-coded affinity tags (ICAT) is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS). The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties but different masses). Then, after whole sample digestion, the labeled peptides are captured selectively using the biotin tag contained in both ICAT reagents. Finally, the simplified peptide mixture is analyzed by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nevertheless, the ICAT LC-MS/MS method still suffers from insufficient sample-to-sample reproducibility on peptide identification. In particular, the number and the type of peptides identified in different experiments can vary considerably and, thus, the statistical (comparative) analysis of sample sets is very challenging. Low information overlap at the peptide and, consequently, at the protein level, is very detrimental in situations where the number of samples to be analyzed is high. We designed a method for improving the data processing and peptide identification in sample sets subjected to ICAT labeling and LC-MS/MS analysis, based on cross validating MS/MS results. Such a method has been implemented in a tool, called EIPeptiDi, which boosts the ICAT data analysis software improving peptide identification throughout the input data set. Heavy/Light (H/L) pairs quantified but not identified by the MS/MS routine, are assigned to peptide sequences identified in other samples, by using similarity criteria based on chromatographic retention time and Heavy/Light mass attributes. EIPeptiDi significantly improves the number of identified peptides per sample, proving that the proposed method has a considerable impact on the protein identification process and, consequently, on the amount of potentially critical information in clinical studies. The EIPeptiDi tool is available at http://bioingegneria.unicz.it/~veltri/projects/eipeptidi/ with a demo data set. EIPeptiDi significantly increases the number of peptides identified and quantified in analyzed samples, thus reducing the number of unassigned H/L pairs and allowing a better comparative analysis of sample data sets.

Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

PubMed

Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

2016-03-01

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Reversible chemoselective tagging and functionalization of methionine containing peptides.

PubMed

Kramer, Jessica R; Deming, Timothy J

2013-06-07

Reagents were developed to allow chemoselective tagging of methionine residues in peptides and polypeptides, subsequent bioorthogonal functionalization of the tags, and cleavage of the tags when desired. This methodology can be used for triggered release of therapeutic peptides, or release of tagged protein digests from affinity columns.

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative ProteomicsW⃞

PubMed Central

Majeran, Wojciech; Cai, Yang; Sun, Qi; van Wijk, Klaas J.

2005-01-01

Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway. PMID:16243905

Analysis of the STAT3 interactome using in-situ biotinylation and SILAC.

PubMed

Blumert, Conny; Kalkhof, Stefan; Brocke-Heidrich, Katja; Kohajda, Tibor; von Bergen, Martin; Horn, Friedemann

2013-12-06

Signal transducer and activator of transcription 3 (STAT3) is activated by a variety of cytokines and growth factors. To generate a comprehensive data set of proteins interacting specifically with STAT3, we applied stable isotope labeling with amino acids in cell culture (SILAC). For high-affinity pull-down using streptavidin, we fused STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag), which did not affect STAT3 functions. By this approach, 3642 coprecipitated proteins were detected in human embryonic kidney-293 cells. Filtering using statistical and functional criteria finally extracted 136 proteins as putative interaction partners of STAT3. Both, a physical interaction network analysis and the enrichment of known and predicted interaction partners suggested that our filtering criteria successfully enriched true STAT3 interactors. Our approach identified numerous novel interactors, including ones previously predicted to associate with STAT3. By reciprocal coprecipitation, we were able to verify the physical association between STAT3 and selected interactors, including the novel interaction with TOX4, a member of the TOX high mobility group box family. Applying the same method, we next investigated the activation-dependency of the STAT3 interactome. Again, we identified both known and novel interactions. Thus, our approach allows to study protein-protein interaction effectively and comprehensively. The location, activity, function, degradation, and synthesis of proteins are significantly regulated by interactions of proteins with other proteins, biopolymers and small molecules. Thus, the comprehensive characterization of interactions of proteins in a given proteome is the next milestone on the path to understanding the biochemistry of the cell. In order to generate a comprehensive interactome dataset of proteins specifically interacting with a selected bait protein, we fused our bait protein STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag). This bio-tag allows an affinity pull-down using streptavidin but affected neither the activation of STAT3 by tyrosine phosphorylation nor its transactivating potential. We combined SILAC for accurate relative protein quantification, subcellular fractionation to increase the coverage of interacting proteins, high-affinity pull-down and a stringent filtering method to successfully analyze the interactome of STAT3. With our approach we confirmed several already known and identified numerous novel STAT3 interactors. The approach applied provides a rapid and effective method, which is broadly applicable for studying protein-protein interactions and their dependency on post-translational modifications. © 2013. Published by Elsevier B.V. All rights reserved.

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a smallmore » change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.« less

Tags to Track Illicit Uranium and Plutonium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haire, M. Jonathan; Forsberg, Charles W.

2007-07-01

With the expansion of nuclear power, it is essential to avoid nuclear materials from falling into the hands of rogue nations, terrorists, and other opportunists. This paper examines the idea of detection and attribution tags for nuclear materials. For a detection tag, it is proposed to add small amounts [about one part per billion (ppb)] of {sup 232}U to enriched uranium to brighten its radioactive signature. Enriched uranium would then be as detectable as plutonium and thus increase the likelihood of intercepting illicit enriched uranium. The use of rare earth oxide elements is proposed as a new type of 'attribution'more » tag for uranium and thorium from mills, uranium and plutonium fuels, and other nuclear materials. Rare earth oxides are chosen because they are chemically compatible with the fuel cycle, can survive high-temperature processing operations in fuel fabrication, and can be chosen to have minimal neutronic impact within the nuclear reactor core. The mixture of rare earths and/or rare earth isotopes provides a unique 'bar code' for each tag. If illicit nuclear materials are recovered, the attribution tag can identify the source and lot of nuclear material, and thus help police reduce the possible number of suspects in the diversion of nuclear materials based on who had access. (authors)« less

Improved gas tagging and cover gas combination for nuclear reactor

DOEpatents

Gross, K.C.; Laug, M.T.

1983-09-26

The invention discloses the use of stable isotopes of neon and argon, sealed as tags in different cladding nuclear fuel elements to be used in a liquid metal fast breeder reactor. Cladding failure allows fission gases and these tag isotopes to escape and to combine with the cover gas. The isotopes are Ne/sup 20/, Ne/sup 21/ and Ne/sup 22/ and Ar/sup 36/, Ar/sup 38/ and Ar/sup 40/, and the cover gas is He. Serially connected cryogenically operated charcoal beds are used to clean the cover gas and to separate out the tags. The first or cover gas cleanup bed is held between 0 and -25/sup 0/C to remove the fission gases from the cover gas and tags, and the second or tag recovery system bed between -170 and -185/sup 0/C to isolate the tags from the cover gas. Spectrometric analysis is used to identify the specific tags that are recovered, and thus the specific leaking fuel element. By cataloging the fuel element tags to the location of the fuel elements in the reactor, the location of the leaking fuel element can then be determined.

Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

PubMed

Hua, Weiwei; Lou, Yimin; Xu, Weiyuan; Cheng, Zhixian; Gong, Xingwen; Huang, Jianying

2016-01-01

Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

Optimal use of tandem biotin and V5 tags in ChIP assays

PubMed Central

Kolodziej, Katarzyna E; Pourfarzad, Farzin; de Boer, Ernie; Krpic, Sanja; Grosveld, Frank; Strouboulis, John

2009-01-01

Background Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes. PMID:19196479

Solid tags for identifying failed reactor components

DOEpatents

Bunch, Wilbur L.; Schenter, Robert E.

1987-01-01

A solid tag material which generates stable detectable, identifiable, and measurable isotopic gases on exposure to a neutron flux to be placed in a nuclear reactor component, particularly a fuel element, in order to identify the reactor component in event of its failure. Several tag materials consisting of salts which generate a multiplicity of gaseous isotopes in predetermined ratios are used to identify different reactor components.

Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications.

PubMed

Young, Carissa L; Britton, Zachary T; Robinson, Anne S

2012-05-01

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Endoprotease profiling with double-tagged peptide substrates: a new diagnostic approach in oncology.

PubMed

Peccerella, Teresa; Lukan, Nadine; Hofheinz, Ralf; Schadendorf, Dirk; Kostrezewa, Markus; Neumaier, Michael; Findeisen, Peter

2010-02-01

The measurement of disease-related proteolytic activity in complex biological matrices like serum is of emerging interest to improve the diagnosis of malignant diseases. We developed a mass spectrometry (MS)-based functional proteomic profiling approach that tracks degradation of artificial endoprotease substrates in serum specimens. The synthetic reporter peptides that are cleaved by tumor-associated endopeptidases were systematically optimized with regard to flanking affinity tags, linkers, and stabilizing elements. Serum specimens were incubated with reporter peptides under standardized conditions and the peptides subsequently extracted with affinity chromatography before MS. In a pilot study an optimized reporter peptide with the cleavage motif WKPYDAADL was added to serum specimens from colorectal tumor patients (n = 50) and healthy controls (n = 50). This reporter peptide comprised a known cleavage site for the cysteine-endopeptidase "cancer procoagulant." Serial affinity chromatography using biotin- and 6xHis tags was superior to the single affinity enrichment using only 6xHis tags. Furthermore, protease-resistant stop elements ensured signal accumulation after prolonged incubation. In contrast, signals from reporter peptides without stop elements vanished completely after prolonged incubation owing to their total degradation. Reporter-peptide spiking showed good reproducibility, and the difference in proteolytic activity between serum specimens from cancer patients and controls was highly significant (P < 0.001). The introduction of a few structural key elements (affinity tags, linkers, d-amino acids) into synthetic reporter peptides increases the diagnostic sensitivity for MS-based protease profiling of serum specimens. This new approach might lead to functional MS-based protease profiling for improved disease classification.

pH-regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments.

PubMed

Levi Mortera, Stefano; Dioni, Ilaria; Greco, Viviana; Neri, Cristina; Rovero, Paolo; Urbani, Andrea

2014-05-01

Among the most common stable-isotope labeling strategies, the reaction of formaldehyde with peptides in the presence of NaCNBH₃ features many attractive aspects that are conducive to its employment in quantitation experiments in proteomics. Reductive amination, with formaldehyde and d(2)-formaldehyde, is reported to be a fast, easy, and specific reaction, undoubtedly inexpensive if compared with commercially available kits for differential isotope coding. Acetaldehyde and d(4)-acetaldehyde could be employed as well without a substantial increase in terms of cost, and should provide a wider spacing between the differentially tagged peptides in the mass spectrum. Nevertheless, only a single paper reports about a diethylation approach for quantitation. We undertook a systematic analytical investigation on the reductive amination of some standard peptides pointing out the occasional occurrence of side reactions in dependence of pH or reagents order of addition, particularly observing the formation of cyclic adducts ascribable to rearrangements involving the generated Schiff-base and all the nucleophilic sites of its chemical environment. We also tried to evaluate how much this side-products amount may impair isotope coded relative quantitation.

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

PubMed

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.

GSyellow, a Multifaceted Tag for Functional Protein Analysis in Monocot and Dicot Plants.

PubMed

Besbrugge, Nienke; Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; Kulkarni, Shubhada R; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Bontinck, Michiel; Aesaert, Stijn; Impens, Francis; Gevaert, Kris; Van Damme, Daniel; Van Lijsebettens, Mieke; Inzé, Dirk; Vandepoele, Klaas; Nelissen, Hilde; De Jaeger, Geert

2018-06-01

The ability to tag proteins has boosted the emergence of generic molecular methods for protein functional analysis. Fluorescent protein tags are used to visualize protein localization, and affinity tags enable the mapping of molecular interactions by, for example, tandem affinity purification or chromatin immunoprecipitation. To apply these widely used molecular techniques on a single transgenic plant line, we developed a multifunctional tandem affinity purification tag, named GS yellow , which combines the streptavidin-binding peptide tag with citrine yellow fluorescent protein. We demonstrated the versatility of the GS yellow tag in the dicot Arabidopsis ( Arabidopsis thaliana ) using a set of benchmark proteins. For proof of concept in monocots, we assessed the localization and dynamic interaction profile of the leaf growth regulator ANGUSTIFOLIA3 (AN3), fused to the GS yellow tag, along the growth zone of the maize ( Zea mays ) leaf. To further explore the function of ZmAN3, we mapped its DNA-binding landscape in the growth zone of the maize leaf through chromatin immunoprecipitation sequencing. Comparison with AN3 target genes mapped in the developing maize tassel or in Arabidopsis cell cultures revealed strong conservation of AN3 target genes between different maize tissues and across monocots and dicots, respectively. In conclusion, the GS yellow tag offers a powerful molecular tool for distinct types of protein functional analyses in dicots and monocots. As this approach involves transforming a single construct, it is likely to accelerate both basic and translational plant research. © 2018 American Society of Plant Biologists. All rights reserved.

Pseudo-orthogonal frequency coded wireless SAW RFID temperature sensor tags.

PubMed

Saldanha, Nancy; Malocha, Donald C

2012-08-01

SAW sensors are ideal for various wireless, passive multi-sensor applications because they are small, rugged, radiation hard, and offer a wide range of material choices for operation over broad temperature ranges. The readable distance of a tag in a multi-sensor environment is dependent on the insertion loss of the device and the processing gain of the system. Single-frequency code division multiple access (CDMA) tags that are used in high-volume commercial applications must have universal coding schemes and large numbers of codes. The use of a large number of bits at the common center frequency to achieve sufficient code diversity in CDMA tags necessitates reflector banks with >30 dB loss. Orthogonal frequency coding is a spread-spectrum approach that employs frequency and time diversity to achieve enhanced tag properties. The use of orthogonal frequency coded (OFC) SAW tags reduces adjacent reflector interactions for low insertion loss, increased range, complex coding, and system processing gain. This work describes a SAW tag-sensor platform that reduces device loss by implementing long reflector banks with optimized spectral coding. This new pseudo-OFC (POFC) coding is defined and contrasted with the previously defined OFC coding scheme. Auto- and cross-correlation properties of the chips and their relation to reflectivity per strip and reflector length are discussed. Results at 250 MHz of 8-chip OFC and POFC SAW tags will be compared. The key parameters of insertion loss, cross-correlation, and autocorrelation of the two types of frequency-coded tags will be analyzed, contrasted, and discussed. It is shown that coded reflector banks can be achieved with near-zero loss and still maintain good coding properties. Experimental results and results predicted by the coupling of modes model are presented for varying reflector designs and codes. A prototype 915-MHz POFC sensor tag is used as a wireless temperature sensor and the results are shown.

The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A: Insights into their use as tools for the removal of affinity tags

PubMed Central

Austin, Brian P.; Tözsér, József; Bagossi, Péter; Tropea, Joseph E.; Waugh, David S.

2012-01-01

Carboxypeptidases may serve as tools for removal for C-terminal affinity tags. In the present study, we describe the expression and purification of an A-type carboxypeptidase from the fungal pathogen Metarhizium anisopliae (MeCPA) that has been genetically engineered to facilitate the removal of polyhistidine tags from the C-termini of recombinant proteins. A complete, systematic analysis of the specificity of MeCPA in comparison with that of bovine carboxypeptidase A (BoCPA) was carried out. Our results indicate that the specificity of the two enzymes is similar but not identical. Histidine residues are removed more efficiently by MeCPA. The very inefficient digestion of peptides with C-terminal lysine or arginine residues, along with the complete inability of the enzyme to remove a C-terminal proline suggests a strategy for designing C-terminal affinity tags that can be trimmed by MeCPA (or BoCPA) to produce a digestion product with a homogeneous endpoint. PMID:21073956

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

PubMed

Crusius, Kerstin; Finster, Silke; McClary, John; Xia, Wei; Larsen, Brent; Schneider, Douglas; Lu, Hong-Tao; Biancalana, Sara; Xuan, Jian-Ai; Newton, Alicia; Allen, Debbie; Bringmann, Peter; Cobb, Ronald R

2006-10-01

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

HaloTag Technology: A Versatile Platform for Biomedical Applications

PubMed Central

2015-01-01

Exploration of protein function and interaction is critical for discovering links among genomics, proteomics, and disease state; yet, the immense complexity of proteomics found in biological systems currently limits our investigational capacity. Although affinity and autofluorescent tags are widely employed for protein analysis, these methods have been met with limited success because they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows protein function and interaction to be comprehensively analyzed using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review, we examine all current applications of the HaloTag technology platform for biomedical applications, such as the study of protein isolation and purification, protein function, protein–protein and protein–DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed along with potential future applications. PMID:25974629

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tykvart, J.; Sacha, P.; Barinka, C.

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.more » 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.« less

Quantitative proteomic analysis of CMS-related changes in Honglian CMS rice anther.

PubMed

Sun, Qingping; Hu, Chaofeng; Hu, Jun; Li, Shaoqing; Zhu, Yingguo

2009-10-01

Honglian (HL) cytoplasmic male sterility (CMS) is one of the rice CMS types and has been widely used in hybrid rice production in China. The CMS line (Yuetai A, YTA) has a Yuetai B (maintainer line, YTB) nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of Yuetai B. The fertility of hybrid (HL-6) was restored by restorer gene in nuclear genome of restorer line (9311). We used isotope-code affinity tag (ICAT) technology to perform the protein profiling of uninucleate stage rice anther and identify the CMS-HL related proteins. Two separate ICAT analyses were performed in this study: (1) anthers from YTA versus anthers from YTB, and (2) anthers from YTA versus anthers from HL-6. Based on the two analyses, a total of 97 unique proteins were identified and quantified in uninucleate stage rice anther under the error rate of less than 10%, of which eight proteins showed abundance changes of at least twofold between YTA and YTB. Triosephosphate isomerase, fructokinase II, DNA-binding protein GBP16 and ribosomal protein L3B were over-expressed in YTB, while oligopeptide transporter, floral organ regulator 1, kinase and S-adenosyl-L: -methionine synthetase were over-expressed in YTA. Reduction of the proteins associated with energy production and lesser ATP equivalents detected in CMS anther indicated that the low level of energy production played an important role in inducing CMS-HL.

Thiol-based Redox Proteins in Brassica napus Guard Cell Abscisic Acid and Methyl Jasmonate Signaling

PubMed Central

Zhu, Mengmeng; Zhu, Ning; Song, Wen-yuan; Harmon, Alice C.; Assmann, Sarah M.; Chen, Sixue

2014-01-01

SUMMARY Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in different physiological processes. Little is known, however, about redox sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in-gel electrophoresis (DIGE) and isotope-coded affinity tag (ICAT). In total, 65 and 118 potential redox responsive proteins were identified in ABA and MeJA treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra-molecular disulfide bonds. Most of the proteins fall into the functional groups of energy, stress and defense, and metabolism. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA and MeJA treated samples. A total of 44 cysteines was mapped in all the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a SNRK2 kinase and an isopropylmalate dehydrogenase were confirmed to be redox regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in guard cell ABA and MeJA signal transduction. PMID:24580573

Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag.

PubMed

Moldes, Cristina; García, José L; García, Pedro

2004-08-01

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.

Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

PubMed

Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

2015-01-01

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

Individually Identifiable Surface Acoustic Wave Sensors, Tags and Systems

NASA Technical Reports Server (NTRS)

Hines, Jacqueline H. (Inventor); Solie, Leland P. (Inventor); Tucker, Dana Y. G. (Inventor); Hines, Andrew T. (Inventor)

2017-01-01

A surface-launched acoustic wave sensor tag system for remotely sensing and/or providing identification information using sets of surface acoustic wave (SAW) sensor tag devices is characterized by acoustic wave device embodiments that include coding and other diversity techniques to produce groups of sensors that interact minimally, reducing or alleviating code collision problems typical of prior art coded SAW sensors and tags, and specific device embodiments of said coded SAW sensor tags and systems. These sensor/tag devices operate in a system which consists of one or more uniquely identifiable sensor/tag devices and a wireless interrogator. The sensor device incorporates an antenna for receiving incident RF energy and re-radiating the tag identification information and the sensor measured parameter(s). Since there is no power source in or connected to the sensor, it is a passive sensor. The device is wirelessly interrogated by the interrogator.

Scaffold design of trivalent chelator heads dictates high-affinity and stable His-tagged protein labeling in vitro and in cellulo.

PubMed

Gatterdam, Karl; Joest, Eike F; Gatterdam, Volker; Tampé, Robert

2018-05-29

Small chemical/biological interaction pairs are at the forefront in tracing proteins' function and interaction at high signal-to-background ratio in cellular pathways. Pharma ventures have eager plans to develop trisNTA probes for in vitro and in vivo screening of His-tagged protein targets. However, the optimal design of scaffold, linker, and chelator head yet deserves systematic investigations to achieve highest affinity and kinetic stability for in vitro and especially cell applications. In this study, we report on a library of N-nitrilotriacetic acid (NTA) based multivalent chelator heads (MCHs) built up on linear, cyclic, and dendritic scaffolds and contrast these with regard to their binding affinity and stability for labeling of cellular His-tagged proteins. Furthermore, we assign a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we describe fundamental differences between the MCH scaffold and define a cyclic trisNTA chelator, which displays the highest affinity and kinetic stability of all reversible, low-molecular weight interaction pairs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

PubMed Central

Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

2016-01-01

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

Impact of an N-terminal Poly Histidine Tag on Protein Thermal Stability

USDA-ARS?s Scientific Manuscript database

For years, the use of polyhistidine tags (His-tags) have been a staple in the isolation of recombinant proteins in immobilized metal affinity chromatography experiments. Their usage has been widely beneficial in increasing protein purity from crude cell lysates. For some recombinant proteins, a cons...

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

PubMed Central

2013-01-01

Background 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. Results 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. Conclusion 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 new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient. PMID:23687945

The Use of Affinity Tags to Overcome Obstacles in Recombinant Protein Expression and Purification.

PubMed

Amarasinghe, Chinthaka; Jin, Jian-Ping

2015-01-01

Research and industrial demands for recombinant proteins continue to increase over time for their broad applications in structural and functional studies and as therapeutic agents. These applications often require large quantities of recombinant protein at desirable purity, which highlights the importance of developing and improving production approaches that provide high level expression and readily achievable purity of recombinant protein. E. coli is the most widely used host for the expression of a diverse range of proteins at low cost. However, there are common pitfalls that can severely limit the expression of exogenous proteins, such as stability, low solubility and toxicity to the host cell. To overcome these obstacles, one strategy that has found to be promising is the use of affinity tags or carrier peptide to aid in the folding of the target protein, increase solubility, lower toxicity and increase the level of expression. In the meantime, the tags and fusion proteins can be designed to facilitate affinity purification. Since the fusion protein may not exhibit the native conformation of the target protein, various strategies have been developed to remove the tag during or after purification to avoid potential complications in structural and functional studies and to obtain native biological activities. Despite extensive research and rapid development along these lines, there are unsolved problems and imperfect applications. This focused review compares and contrasts various strategies that employ affinity tags to improve bacterial expression and to facilitate purification of recombinant proteins. The pros and cons of the approaches are discussed for more effective applications and new directions of future improvement.

Retention of coded wire tags, and their effect on maturation and survival of yellow mealworms (Coleoptera: Tenebrionidae)

USGS Publications Warehouse

Schaffler, James J.; Isely, J.J.

2001-01-01

This study demonstrates that coded wire tags can be used to mark certain insect larvae without adverse effects on maturation, and that tags are retained through the adult phase in high enough proportion for practical application. Coded wire tags also offer the benefit that marked organisms can be identified to the batch or individual level.

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

PubMed Central

Schotte, Lise; Rombaut, Bart; Thys, Bert

2012-01-01

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads. PMID:22688388

Purification of phage display-modified bacteriophage T4 by affinity chromatography

PubMed Central

2011-01-01

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development. PMID:21627821

Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins.

PubMed

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

PubMed Central

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible. PMID:23874971

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

PubMed

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.

Evaluation and implementation of QR Code Identity Tag system for Healthcare in Turkey.

PubMed

Uzun, Vassilya; Bilgin, Sami

2016-01-01

For this study, we designed a QR Code Identity Tag system to integrate into the Turkish healthcare system. This system provides QR code-based medical identification alerts and an in-hospital patient identification system. Every member of the medical system is assigned a unique QR Code Tag; to facilitate medical identification alerts, the QR Code Identity Tag can be worn as a bracelet or necklace or carried as an ID card. Patients must always possess the QR Code Identity bracelets within hospital grounds. These QR code bracelets link to the QR Code Identity website, where detailed information is stored; a smartphone or standalone QR code scanner can be used to scan the code. The design of this system allows authorized personnel (e.g., paramedics, firefighters, or police) to access more detailed patient information than the average smartphone user: emergency service professionals are authorized to access patient medical histories to improve the accuracy of medical treatment. In Istanbul, we tested the self-designed system with 174 participants. To analyze the QR Code Identity Tag system's usability, the participants completed the System Usability Scale questionnaire after using the system.

Self-Powered Wireless Affinity-Based Biosensor Based on Integration of Paper-Based Microfluidics and Self-Assembled RFID Antennas.

PubMed

Yuan, Mingquan; Alocilja, Evangelyn C; Chakrabartty, Shantanu

2016-08-01

This paper presents a wireless, self-powered, affinity-based biosensor based on the integration of paper-based microfluidics with our previously reported method for self-assembling radio-frequency (RF) antennas. At the core of the proposed approach is a silver-enhancement technique that grows portions of a RF antenna in regions where target antigens hybridize with target specific affinity probes. The hybridization regions are defined by a network of nitrocellulose based microfluidic channels which implement a self-powered approach to sample the reagent and control its flow and mixing. The integration substrate for the biosensor has been constructed using polyethylene and the patterning of the antenna on the substrate has been achieved using a low-cost ink-jet printing technique. The substrate has been integrated with passive radio-frequency identification (RFID) tags to demonstrate that the resulting sensor-tag can be used for continuous monitoring in a food supply-chain where direct measurement of analytes is typically considered to be impractical. We validate the proof-of-concept operation of the proposed sensor-tag using IgG as a model analyte and using a 915 MHz Ultra-high-frequency (UHF) RFID tagging technology.

Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe.

PubMed

Zilio, Nicola; Boddy, Michael N

2017-03-01

The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins. © 2017 Cold Spring Harbor Laboratory Press.

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

PubMed

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.

The use of a proteinaceous "cushion" with a polystyrene-binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface.

PubMed

Imanaka, Hiroyuki; Yamadzumi, Daisuke; Yanagita, Keisuke; Ishida, Naoyuki; Nakanishi, Kazuhiro; Imamura, Koreyoshi

2016-03-01

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a "cushion" between the PS-affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS-affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527-534, 2016. © 2016 American Institute of Chemical Engineers.

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag.

PubMed

Li, Junhua; Zhang, Yang; Yang, Yanjun

2013-03-01

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.

A novel expression vector for the improved solubility of recombinant scorpion venom in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Tianqing; Ming, Hongyan; Deng, Lili

Recombinant scorpion anti-excitation peptide (rANEP) has previously been expressed using the pET32a system and purified via affinity chromatography. However, rANEP is expressed in BL21(DE3) cells as an inclusion body, and the affinity tag can not be removed. To overcome this problem, we used a variety of protein, DsbA, MBP, TrxA, intein, and affinity tags in fusion and co-expression to achieve soluble and functional rANEP without any affinity tag. In the pCIT-ANEP expression vector, the highest soluble expression level was approximately 90% of the total cellular proteins in E. coli, and the rANEP was cleaved by the intein protein and subsequently purifiedmore » to obtain rANEP, which had the same activity as the natural ANEP. The purity of rANEP obtained using this method was over 95%, with a quantity of 5.1 mg from of purified rANEP from 1 L of culture. This method could expand the application of the soluble expression of disulfide-rich peptides in E. coli.« less

Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

USDA-ARS?s Scientific Manuscript database

Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of the current study was to evaluate the His-tag pr...

Overview of the Purification of Recombinant Proteins

PubMed Central

Wingfield, Paul T.

2015-01-01

When the first version of this unit was written in 1995 protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E.coli, protein expression and purification from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302

Overview of the purification of recombinant proteins.

PubMed

Wingfield, Paul T

2015-04-01

When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.

Tracking animals in freshwater with electronic tags: past, present and future

USGS Publications Warehouse

Cooke, Steven J.; Midwood, Jonathan D.; Thiem, Jason D.; Klimley, Peter; Lucas, Martyn C.; Thorstad, Eva B.; Eiler, John; Holbrook, Chris; Ebner, Brendan C.

2013-01-01

Considerable technical developments over the past half century have enabled widespread application of electronic tags to the study of animals in the wild, including in freshwater environments. We review the constraints associated with freshwater telemetry and biologging and the technical developments relevant to their use. Technical constraints for tracking animals are often influenced by the characteristics of the animals being studied and the environment they inhabit. Collectively, they influence which and how technologies can be used and their relative effectiveness. Although radio telemetry has historically been the most commonly used technology in freshwater, passive integrated transponder (PIT) technology, acoustic telemetry and biologgers are becoming more popular. Most telemetry studies have focused on fish, although an increasing number have focused on other taxa, such as turtles, crustaceans and molluscs. Key technical developments for freshwater systems include: miniaturization of tags for tracking small-size life stages and species, fixed stations and coded tags for tracking large samples of animals over long distances and large temporal scales, inexpensive PIT systems that enable mass tagging to yield population- and community-level relevant sample sizes, incorporation of sensors into electronic tags, validation of tag attachment procedures with a focus on maintaining animal welfare, incorporation of different techniques (for example, genetics, stable isotopes) and peripheral technologies (for example, geographic information systems, hydroacoustics), development of novel analytical techniques, and extensive international collaboration. Innovations are still needed in tag miniaturization, data analysis and visualization, and in tracking animals over larger spatial scales (for example, pelagic areas of lakes) and in challenging environments (for example, large dynamic floodplain systems, under ice). There seems to be a particular need for adapting various global positioning system and satellite tagging approaches to freshwater. Electronic tagging provides a mechanism to collect detailed information from imperilled animals and species that have no direct economic value. Current and future advances will continue to improve our knowledge of the natural history of aquatic animals and ecological processes in freshwater ecosystems while facilitating evidence-based resource management and conservation.

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag.

PubMed

Biancucci, Marco; Dolores, Jazel S; Wong, Jennifer; Grimshaw, Sarah; Anderson, Wayne F; Satchell, Karla J F; Kwon, Keehwan

2017-01-05

Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag". The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.

Bidirectional immobilization of affinity-tagged cytochrome c on electrode surfaces.

PubMed

Schröper, Florian; Baumann, Arnd; Offenhäusser, Andreas; Mayer, Dirk

2010-08-07

Here, we report a new strategy for the directed bivalent immobilization of cyt c on or between gold electrodes. C-terminal modification with cys- or his-tag did not affect the functional integrity of the protein. In combination with electrostatic protein binding, these tags enable a bifunctional immobilization between two electrodes or alternatively one electrode and interacting enzymes.

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides.

PubMed

Winton, Alexander J; Baptiste, Janae L; Allen, Mark A

2018-09-01

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. Copyright © 2018. Published by Elsevier Inc.

Functional study of miR-27a in human hepatic stellate cells by proteomic analysis: comprehensive view and a role in myogenic tans-differentiation.

PubMed

Ji, Yuhua; Zhang, Jinsheng; Wang, Wenwen; Ji, Juling

2014-01-01

We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validated. The results not only delineated the underlying mechanism of miR-27a in modulating fat metabolism and cell proliferation, but also revealed a novel role of miR-27a in promoting myogenic tans-differentiation during HSCs activation. This study also exemplified proteomics strategy as a powerful tool for the functional study of miRNA.

Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.

PubMed

Zhu, Mengmeng; Zhu, Ning; Song, Wen-yuan; Harmon, Alice C; Assmann, Sarah M; Chen, Sixue

2014-05-01

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox-sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard-cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in-gel electrophoresis and isotope-coded affinity tagging. In total, 65 and 118 potential redox-responsive proteins were identified in ABA- and MeJA-treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra-molecular disulfide bonds. Most of the proteins fall into the functional groups of 'energy', 'stress and defense' and 'metabolism'. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA- and MeJA-treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non-fermenting 1-related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox-regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies

PubMed Central

Anthony, Kelsey C.; You, Changjiang; Piehler, Jacob; Pomeranz Krummel, Daniel A.

2014-01-01

SUMMARY There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle (AuNPtris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of AuNPtris-NTA labeled proteins by electron microscopy is further ensured by the reagent’s short conformationally restricted linker. We have employed AuNPtris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. AuNPtris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our new labeling reagent should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. PMID:24560806

A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana

PubMed Central

Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

2014-01-01

Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes. PMID:25482810

Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

PubMed Central

Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

2012-01-01

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

Plant proteome analysis: a 2006 update.

PubMed

Jorrín, Jesús V; Maldonado, Ana M; Castillejo, Ma Angeles

2007-08-01

This 2006 'Plant Proteomics Update' is a continuation of the two previously published in 'Proteomics' by 2004 (Canovas et al., Proteomics 2004, 4, 285-298) and 2006 (Rossignol et al., Proteomics 2006, 6, 5529-5548) and it aims to bring up-to-date the contribution of proteomics to plant biology on the basis of the original research papers published throughout 2006, with references to those appearing last year. According to the published papers and topics addressed, we can conclude that, as observed for the three previous years, there has been a quantitative, but not qualitative leap in plant proteomics. The full potential of proteomics is far from being exploited in plant biology research, especially if compared to other organisms, mainly yeast and humans, and a number of challenges, mainly technological, remain to be tackled. The original papers published last year numbered nearly 100 and deal with the proteome of at least 26 plant species, with a high percentage for Arabidopsis thaliana (28) and rice (11). Scientific objectives ranged from proteomic analysis of organs/tissues/cell suspensions (57) or subcellular fractions (29), to the study of plant development (12), the effect of hormones and signalling molecules (8) and response to symbionts (4) and stresses (27). A small number of contributions have covered PTMs (8) and protein interactions (4). 2-DE (specifically IEF-SDS-PAGE) coupled to MS still constitutes the almost unique platform utilized in plant proteome analysis. The application of gel-free protein separation methods and 'second generation' proteomic techniques such as multidimensional protein identification technology (MudPIT), and those for quantitative proteomics including DIGE, isotope-coded affinity tags (ICAT), iTRAQ and stable isotope labelling by amino acids in cell culture (SILAC) still remains anecdotal. This review is divided into seven sections: Introduction, Methodology, Subcellular proteomes, Development, Responses to biotic and abiotic stresses, PTMs and Protein interactions. Section 8 summarizes the major pitfalls and challenges of plant proteomics.

Combination of Mass Signal Amplification and Isotope-Labeled Alkanethiols for the Multiplexed Detection of miRNAs.

PubMed

Kang, Hyunook; Hong, Seol-Hye; Sung, Jiha; Yeo, Woon-Seok

2017-08-04

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope-labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am-tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am-tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am-tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO 4 concentrations. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein.

PubMed

Galvão, Carolina W; Pedrosa, Fábio O; Souza, Emanuel M; Yates, M Geoffrey; Chubatsu, Leda S; Steffens, Maria Berenice R

2004-06-01

The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.

Review on SAW RFID tags.

PubMed

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.

Expression and purification of mouse peptide ESP4 in Escherichia coli.

PubMed

Hirakane, Makoto; Taniguchi, Masahiro; Yoshinaga, Sosuke; Misumi, Shogo; Terasawa, Hiroaki

2014-04-01

Pheromones are species-specific chemical signals that regulate a wide range of social and sexual behaviors in many animals. In mice, the male-specific peptide ESP1 (exocrine gland-secreting peptide 1) is secreted into tear fluids and enhances female sexual receptive behavior. ESP1 belongs to the ESP family, a multigene family with 38 genes in mice. ESP1 shares the highest homology with ESP4. ESP1 is expressed in the extraorbital lacrimal gland, whereas ESP4 is expressed in some exocrine glands. Thus, ESP4 is expected to have a function that has not been elucidated yet. Large amounts of the purified ESP4 protein are required for structural and biochemical studies. Here we present an expression and purification scheme for the recombinant ESP4 protein. The N-terminally histidine-tagged ESP4 fusion protein was expressed in Escherichia coli as inclusion bodies, which were solubilized and purified by nickel affinity chromatography. The histidine tag was cleaved with thrombin and removed by a second nickel affinity chromatography step. The ESP4 protein was isolated with high purity by reversed-phase chromatography. For NMR analyses, we prepared a stable isotope-labeled ESP4 protein. Three repeated freeze-drying steps after the reversed-phase chromatography were required, to remove a volatile contaminating compound and to obtain an NMR spectrum with a homogeneous line shape. AMS-modification and far-UV CD spectroscopic analyses suggested that ESP4 has an intramolecular disulfide bridge and a helical structure, respectively. The present study provides a powerful tool for structural and biochemical studies of ESP4, leading toward the elucidation of the roles of the ESP family members. Copyright © 2014 Elsevier Inc. All rights reserved.

Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.

PubMed

Schilling, Birgit; Rardin, Matthew J; MacLean, Brendan X; Zawadzka, Anna M; Frewen, Barbara E; Cusack, Michael P; Sorensen, Dylan J; Bereman, Michael S; Jing, Enxuan; Wu, Christine C; Verdin, Eric; Kahn, C Ronald; Maccoss, Michael J; Gibson, Bradford W

2012-05-01

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.

Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline

PubMed Central

Schilling, Birgit; Rardin, Matthew J.; MacLean, Brendan X.; Zawadzka, Anna M.; Frewen, Barbara E.; Cusack, Michael P.; Sorensen, Dylan J.; Bereman, Michael S.; Jing, Enxuan; Wu, Christine C.; Verdin, Eric; Kahn, C. Ronald; MacCoss, Michael J.; Gibson, Bradford W.

2012-01-01

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models. PMID:22454539

Method of remote powering and detecting multiple UWB passive tags in an RFID system

DOEpatents

Dowla, Farid U [Castro Valley, CA; Nekoogar, Faranak [San Ramon, CA; Benzel, David M [Livermore, CA; Dallum, Gregory E [Livermore, CA; Spiridon, Alex [Palo Alto, CA

2012-05-29

A new Radio Frequency Identification (RFID), tracking, powering apparatus/system and method using coded Ultra-wideband (UWB) signaling is introduced. The proposed hardware and techniques disclosed herein utilize a plurality of passive UWB transponders in a field of an RFID-radar system. The radar system itself enables multiple passive tags to be remotely powered (activated) at about the same time frame via predetermined frequency UWB pulsed formats. Once such tags are in an activated state, an UWB radar transmits specific "interrogating codes" to put predetermined tags in an awakened status. Such predetermined tags can then communicate by a unique "response code" so as to be detected by an UWB system using radar methods.

New trends and affinity tag designs for recombinant protein purification.

PubMed

Wood, David W

2014-06-01

Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different purification tags have been developed, including short peptides, epitopes, folded protein domains, non-chromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research. Copyright © 2014 Elsevier Ltd. All rights reserved.

Barley as a green factory for the production of functional Flt3 ligand.

PubMed

Erlendsson, Lýdur S; Muench, Marcus O; Hellman, Ulf; Hrafnkelsdóttir, Soffía M; Jonsson, Anders; Balmer, Yves; Mäntylä, Einar; Orvar, Björn L

2010-02-01

Biologically active recombinant human Flt3 ligand was expressed and isolated from transgenic barley seeds. Its expression is controlled by a tissue specific promoter that confines accumulation of the recombinant protein to the endosperm tissue of the seed. The recombinant Flt3 ligand variant expressed in the seeds contains an HQ-tag for affinity purification on immobilized metal ion affinity chromatography (IMAC) resin. The tagged protein was purified from seed extracts to near homogeneity using sequential chromatography on IMAC affinity resin and cation exchange resin. We also show that the recombinant Flt3 ligand protein undergoes posttranslational modifications: it is a glycoprotein containing alpha-1,3-fucose and alpha-1,2-xylose. The HQ-tagged Flt3 ligand variant exhibits comparable biological activity to commercial Flt3 ligand. This is the first report showing expression and accumulation of recombinant human growth factor in barley seeds with a yield of active protein similar to a bacterial expression system. The present results demonstrate that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors.

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

PubMed

Finzi, Andrés; Cloutier, Jonathan; Cohen, Eric A

2003-07-01

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

PubMed

Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

2015-09-01

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular Targets of Dietary Polyphenol Resveratrol

DTIC Science & Technology

2005-03-01

attempts to generate affinity columns tagged with other polyphenols, e.g., epigallocatechin gallate ( EGCG ). Conceivably such columns, if generated, would...Similar affinity chromatography with the related polyphenol Epigallocatechin gallate does not produce similar results.” Answer: We did not make...addition, the PI does not provid expression. If there is “increased ex many bind the resveratrol affinity co related polyphenol Epigallocatechin Response

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

PubMed

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.

Tag retention, growth, and survival of red swamp crayfish Procambarus clarkii marked with coded wire tags

USGS Publications Warehouse

Isely, J.J.; Eversole, A.G.

1998-01-01

Juvenile red swamp crayfish (or crawfish), Procambarus clarkii (20-41 mm in total length) were collected from a crayfish culture pond by dipnetting and tagged with sequentially numbered, standard length, binary-coded wire tags. Four replicates of 50 crayfish were impaled perpendicular to the long axis of the abdomen with a fixed needle. Tags were injected transversely into the ventral surface of the first or second abdominal segment and were imbedded in the musculature just beneath the abdominal sternum. Tags were visible upon inspection. Additionally, two replicates of 50 crayfish were not tagged and were used as controls. Growth, survival, and tag retention were evaluated after 7 d in individual containers, after 100 d in aquaria, and after 200 d in field cages. Tag retention during each sample period was 100%, and average mortality of tagged crayfish within 7 d of tagging was 1%. Mortality during the remainder of the study was high (75-91%) but was similar between treatment and control samples. Most of the deaths were probably due to cannibalism. Average total length increased threefold during the course of the study, and crayfish reached maturity. Because crayfish were mature by the end of the study, we concluded that the coded wire tag was retained through the life history of the crayfish.

A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

PubMed

Chang, Tsung-Che; Adak, Avijit K; Lin, Ting-Wei; Li, Pei-Jhen; Chen, Yi-Ju; Lai, Chain-Hui; Liang, Chien-Fu; Chen, Yu-Ju; Lin, Chun-Cheng

2016-03-15

The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Application of coupled affinity-sizing chromatography for the detection of proteolyzed HSA-tagged proteins.

PubMed

London, Anne Serdakowski; Patel, Kunal; Quinn, Lisa; Lemmerer, Martin

2015-04-01

Coupled affinity liquid chromatography and size exclusion chromatography (ALC-SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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.

Z-path SAW RFID tag.

PubMed

Härmä, Sanna; Plessky, Victor P; Hartmann, Clinton S; Steichen, William

2008-01-01

Surface acoustic wave (SAW) radio-frequency identification (RFID) tags are soon expected to be produced in very high volumes. The size and cost of a SAW RFID tag will be key parameters for many applications. Therefore, it is of primary importance to reduce the chip size. In this work, we describe the design principles of a 2.4-GHz SAW RFID tag that is significantly smaller than earlier reported tags. We also present simulated and experimental results. The coded signal should arrive at the reader with a certain delay (typically about 1 micros), i.e., after the reception of environmental echoes. If the tag uses a bidirectional interdigital transducer (IDT), space for the initial delay is needed on both sides of the IDT. In this work, we replace the bidirectional IDT by a unidirectional one. This halves the space required by the initial delay because all the code reflectors must now be placed on the same side of the IDT. We reduce tag size even further by using a Z-path geometry in which the same space in x-direction is used for both the initial delay and the code reflectors. Chip length is thus determined only by the space required by the code reflectors.

Comparative analysis of viral RNA signatures on different RIG-I-like receptors

PubMed Central

Sanchez David, Raul Y; Combredet, Chantal; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Coppée, Jean-Yves; Mura, Marie; Guerbois Galla, Mathilde; Despres, Philippe; Tangy, Frédéric; Komarova, Anastassia V

2016-01-01

The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection. DOI: http://dx.doi.org/10.7554/eLife.11275.001 PMID:27011352

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

PubMed Central

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

Purification of bacteriophage lambda repressor

PubMed Central

Gao, Ning; Shearwin, Keith; Mack, John; Finzi, Laura; Dunlap, David

2013-01-01

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy “footprinting” assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops. PMID:23831434

[Identification of the interacting proteins with S100A8 or S100A9 by affinity purification and mass spectrometry].

PubMed

Wang, Jing; Zhang, Xuemei; Li, Zheng; Li, Xiayu; Ma, Jian; Shen, Shourong

2017-04-28

To identify the interacting proteins with S100A8 or S100A9 in HEK293 cell line by flag-tag affinity purification and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS).
 Methods: The p3×Flag-CMV-S100A8 and p3×Flag-CMV-S100A9 expression vectors were constructed by inserting S100A8 or S100A9 coding sequence. The recombinant plasmids were then transfected into HEK293 cells. Affinity purification and LC-MS/MS were applied to identify the proteins interacting with S100A8 or S100A9. Bioinformatics analysis was used to seek the gene ontology of the interacting proteins. Co-immunoprecipitation (Co-IP) was applied to confirm the proteins interacted with S100A8 or S100A9.
 Results: Fourteen proteins including pyruvate kinase, muscle (PKM), nucleophosmin (NPM1) and eukaryotic translation initiation factor 5A (EIF5A), which potentially interacted with S100A8, were successfully identified by Flag-tag affinity purification followed by LC-MS/MS analysis. Six proteins, such as tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (14-3-3ε) and PKM, which potentially interacted with S100A9, were successfully identified. Gene ontology analysis of the identified proteins suggested that proteins interacted with S100A8 or S100A9 were involved in several biological pathways, including canonical glycolysis, positive regulation of NF-κB transcription factor activity, negative regulation of apoptotic process, cell-cell adhesion, etc. Co-IP experiment confirmed that PKM2 can interact with both S100A8 and S100A9, and 14-3-3ε can interact with S100A8.
 Conclusion: PKM2 is identified to interact with both S100A8 and S100A9, while 14-3-3ε can interact with S100A9. These results may provide a new clue for the role of S100A8 or S100A9 in the progression of colitis-associated colorectal cancer.

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

PubMed Central

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita

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. PMID:29360877

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

PubMed

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.

Synthesis of a Polyhistidine-bearing Amphipol and its Use for Immobilizing Membrane Proteins.

PubMed

Giusti, Fabrice; Kessler, Pascal; Hansen, Randi Westh; Della Pia, Eduardo A; Le Bon, Christel; Mourier, Gilles; Popot, Jean-Luc; Martinez, Karen L; Zoonens, Manuela

2015-12-14

Amphipols (APols) are short amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions. In the present study, A8-35, a polyacrylate-based APol, was grafted with hexahistidine tags (His6-tags). The synthesis and characterization of this novel functionalized APol, named HistAPol, 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 regeneration of the supports and dispenses with any genetic modification of the target protein, provides a novel, promising tool for attaching MPs onto solid supports while stabilizing them.

Recovery of coded wire tags at a caspian tern colony in San Francisco Bay: A technique to evaluate impacts of avian predation on juvenile salmonids

USGS Publications Warehouse

Evans, A.F.; Roby, D.D.; Collis, K.; Cramer, B.M.; Sheggeby, J.A.; Adrean, L.J.; Battaglia, D.S.; Lyons, Donald E.

2011-01-01

We recovered coded wire tags (CWTs) from a colony of Caspian terns Hydroprogne caspia on Brooks Island in San Francisco Bay, California, to evaluate predation on juvenile salmonids originating from the Sacramento and San Joaquin rivers. Subsamples of colony substrate representing 11.7% of the nesting habitat used by the terns yielded 2,079 salmonid CWTs from fish released and subsequently consumed by terns in 2008. The estimated number of CWTs deposited on the entire tern colony was 40,143 (ranging from 26,763 to 80,288), once adjustments were made to account for tag loss and the total amount of nesting habitat used by terns. Tags ingested by terns and then egested on the colony were undamaged, and the tags' complete numeric codes were still identifiable. The CWTs found on the tern colony indicated that hatchery Chinook salmon Oncorhynchus tshawytscha trucked to and released in San Pablo Bay were significantly more likely to be consumed by Caspian terns than Chinook salmon that migrated in-river to the bay; 99.7% of all tags recovered were from bay-released Chinook salmon. Of the CWTs recovered on the tern colony, 98.0% were from fall-run Chinook salmon, indicating a higher susceptibility to tern predation than for the spring run type. None of the approximately 518,000 wild Chinook salmon that were coded-wire-tagged and released in the basin were recovered on the tern colony, suggesting that the impacts on wild, U.S. Endangered Species Act-listed Chinook salmon populations were minimal in 2008. Overall, we estimate that 0.3% of the approximately 12.3 million coded-wire-tagged Chinook salmon released in the basin in 2008 were subsequently consumed by Caspian terns from the Brooks Island colony. These results indicate that CWTs implanted in juvenile salmon can be recovered from a piscivorous waterbird colony and used to evaluate smolt losses for runs that are tagged. Abstract We recovered coded wire tags (CWTs) from a colony of Caspian terns Hydroprogne caspia on Brooks Island in San Francisco Bay, California, to evaluate predation on juvenile salmonids originating from the Sacramento and San Joaquin rivers. Subsamples of colony substrate representing 11.7% of the nesting habitat used by the terns yielded 2,079 salmonid CWTs from fish released and subsequently consumed by terns in 2008. The estimated number of CWTs deposited on the entire tern colony was 40,143 (ranging from 26,763 to 80,288), once adjustments were made to account for tag loss and the total amount of nesting habitat used by terns. Tags ingested by terns and then egested on the colony were undamaged, and the tags' complete numeric codes were still identifiable. The CWTs found on the tern colony indicated that hatchery Chinook salmon Oncorhynchus tshawytscha trucked to and released in San Pablo Bay were significantly more likely to be consumed by Caspian terns than Chinook salmon that migrated in-river to the bay; 99.7% of all tags recovered were from bay-released Chinook salmon. Of the CWTs recovered on the tern colony, 98.0% were from fall-run Chinook salmon, indicating a higher susceptibility to tern predation than for the spring run type. None of the approximately 518,000 wild Chinook salmon that were coded-wire-tagged and released in the basin were recovered on the tern colony, suggesting that the impacts on wild, U.S. Endangered Species Act-listed Chinook salmon populations were minimal in 2008. Overall, we estimate that 0.3% of the approximately 12.3 million coded-wire-tagged Chinook salmon released in the basin in 2008 were subsequently consumed by Caspian terns from the Brooks Island colony. These results indicate that CWTs implanted in juvenile salmon can be recovered from a piscivorous waterbird colony and used to evaluate smolt losses for runs that are tagged ?? American Fisheries Society 2011.

Effects of coded-wire-tagging on stream-dwelling Sea Lamprey larvae

USGS Publications Warehouse

Johnson, Nicholas; Swink, William D.; Dawson, Heather A.; Jones, Michael L.

2016-01-01

The effects of coded wire tagging Sea Lamprey Petromyzon marinus larvae from a known-aged stream-dwelling population were assessed. Tagged larvae were significantly shorter on average than untagged larvae from 3 to 18 months after tagging. However, 30 months after tagging, the length distribution of tagged and untagged larvae did not differ and tagged Sea Lampreys were in better condition (i.e., higher condition factor) and more likely to have undergone metamorphosis than the untagged population. The reason why tagged larvae were more likely to metamorphose is not clear, but the increased likelihood of metamorphosis could have been a compensatory response to the period of slower growth after tagging. Slower growth after tagging was consistent across larval size-classes, so handling and displacement from quality habitat during the early part of the growing season was likely the cause rather than the tag burden. The tag effects observed in this study, if caused by displacement and handling, may be minimized in future studies if tagging is conducted during autumn after growth has concluded for the year.

Immunodiagnostic Value of Echinococcus Granulosus Recombinant B8/1 Subunit of Antigen B.

PubMed

Savardashtaki, Amir; Sarkari, Bahador; Arianfar, Farzane; Mostafavi-Pour, Zohreh

2017-06-01

Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized. The sequence was synthesized and inserted into pGEX-4T-1 vector. Purification was performed with GST tag affinity column. Diagnostic performance of the produced recombinant antigen, native antigen B and a commercial ELISA kit were further evaluated in an ELISA system, using a panel of sera from CE patients and controls. SDS-PAGE demonstrated that the protein of interest had a high expression level and purity after GST tag affinity purification. Western blotting verified the immunoreactivity of the produced recombinant antigen with the sera of CE patients. In an ELISA system, the sensitivity and specificity (for human CE diagnosis) of the recombinant antigen, native antigen B and commercial kit were respectively 93% and 92%, 87% and 90% and 97% and 95%. The produced recombinant antigen showed a high diagnostic value which can be recommended for serodiagnosis of CE in Iran and other CE-endemic areas. Utilizing the combination of other subunits of AgB8 would improve the performance value of the introduced ELISA system.

The Design and Analysis of Salmonid Tagging Studies in the Columbia Basin; Volume XII; A Multinomial Model for Estimating Ocean Survival from Salmonid Coded Wire-Tag Data.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryding, Kristen E.; Skalski, John R.

1999-06-01

The purpose of this report is to illustrate the development of a stochastic model using coded wire-tag (CWT) release and age-at-return data, in order to regress first year ocean survival probabilities against coastal ocean conditions and climate covariates.

Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification, and structural analysis.

PubMed

Baldwin, M A; Medzihradszky, K F; Lock, C M; Fisher, B; Settineri, T A; Burlingame, A L

2001-04-15

The design and operation of a novel UV-MALDI ionization source on a commercial QqoaTOF mass spectrometer (Applied Biosystem/MDS Sciex QSTAR Pulsar) is described. Samples are loaded on a 96-well target plate, the movement of which is under software control and can be readily automated. Unlike conventional high-energy MALDI-TOF, the ions are produced with low energies (5-10 eV) in a region of relatively low vacuum (8 mTorr). Thus, they are cooled by extensive low-energy collisions before selection in the quadrupole mass analyzer (Q1), potentially giving a quasi-continuous ion beam ideally suited to the oaTOF used for mass analysis of the fragment ions, although ion yields from individual laser shots may vary widely. Ion dissociation is induced by collisions with argon in an rf-only quadrupole cell, giving typical low-energy CID spectra for protonated peptide ions. Ions separated in the oaTOF are registered by a four-anode detector and time-to-digital converter and accumulated in "bins" that are 625 ps wide. Peak shapes depend upon the number of ion counts in adjacent bins. As expected, the accuracy of mass measurement is shown to be dependent upon the number of ions recorded for a particular peak. With internal calibration, mass accuracy better than 10 ppm is attainable for peaks that contain sufficient ions to give well-defined Gaussian profiles. By virtue of its high resolution, capability for accurate mass measurements, and sensitivity in the low-femotomole range, this instrument is ideally suited to protein identification for proteomic applications by generation of peptide tags, manual sequence interpretation, identification of modifications such as phosphorylation, and protein structural elucidation. Unlike the multiply charged ions typical of electrospray ionization, the singly charged MALDI-generated peptide ions show a linear dependence of optimal collision energy upon molecular mass, which is advantageous for automated operation. It is shown that the novel pulsing technique of this instrument that increases the sensitivity for precursor ions scans is applicable to the identification of peptides labeled with isotope-coded affinity tags.

Weighted SAW reflector gratings for orthogonal frequency coded SAW tags and sensors

NASA Technical Reports Server (NTRS)

Puccio, Derek (Inventor); Malocha, Donald (Inventor)

2011-01-01

Weighted surface acoustic wave reflector gratings for coding identification tags and sensors to enable unique sensor operation and identification for a multi-sensor environment. In an embodiment, the weighted reflectors are variable while in another embodiment the reflector gratings are apodized. The weighting technique allows the designer to decrease reflectively and allows for more chips to be implemented in a device and, consequently, more coding diversity. As a result, more tags and sensors can be implemented using a given bandwidth when compared with uniform reflectors. Use of weighted reflector gratings with OFC makes various phase shifting schemes possible, such as in-phase and quadrature implementations of coded waveforms resulting in reduced device size and increased coding.

Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

PubMed Central

Berger, Christian; Ho, Jenny T.C.; Kimura, Tomohiro; Hess, Sonja; Gawrisch, Klaus; Yeliseev, Alexei

2010-01-01

We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and 15N2-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2 per liter of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two 15N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of 15N chemical shifts strongly suggests that the 15N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation. PMID:20044006

Summary of Findings from Coded Wire Tag Analysis from Spring Chinook Salmon Spawning Surveys in the Clearwater Basin, Technical Report 2001.

DOE Office of Scientific and Technical Information (OSTI.GOV)

S.P. Cramer & Associates, Inc.

2002-05-31

We recently received data on the decoded coded wire tags (CWT's) recovered from spring chinook snouts we collected during spawning surveys in the Clearwater Basin last fall (2001). We were curious about what could be learned from the tags recovered (even though our project is over), so we did some cursory analyses and have described our findings in the attached memo. Snouts were processed and codes determined by Idaho Department of Fish and Game. Most snouts did not contain CWTs, because most ad-clipped fish were not given a CWT. Further, because adults were outplanted live, we do not know whatmore » codes they contained. Each of the hatcheries from which outplanted adults were obtained had several CWT code groups returning. That means that the best we can do with the codes recovered is compare the hatchery of origin for the tag with the hatchery from which outplants were taken. The results are interesting and not exactly as we would have predicted.« less

Spatial and Temporal Trends in Stable Carbon and Oxygen Isotope Ratios of Juvenile Winter Flounder

EPA Science Inventory

Isotopic ratios of fish otoliths have been used in numerous studies as natural tags or markers to aid the study of connectivity among fish populations. We investigated the use of spatial and temporal changes in the stable carbon and oxygen isotope ratios of otoliths to different...

Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging.

PubMed

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 dihydrofolate reductase and the antibiotic trimethoprim and was 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. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. © 2013 by John Wiley & Sons, Inc.

Error probability for RFID SAW tags with pulse position coding and peak-pulse detection.

PubMed

Shmaliy, Yuriy S; Plessky, Victor; Cerda-Villafaña, Gustavo; Ibarra-Manzano, Oscar

2012-11-01

This paper addresses the code reading error probability (EP) in radio-frequency identification (RFID) SAW tags with pulse position coding (PPC) and peak-pulse detection. EP is found in a most general form, assuming M groups of codes with N slots each and allowing individual SNRs in each slot. The basic case of zero signal in all off-pulses and equal signals in all on-pulses is investigated in detail. We show that if a SAW-tag with PPC is designed such that the spurious responses are attenuated by more than 20 dB below on-pulses, then EP can be achieved at the level of 10(-8) (one false read per 108 readings) with SNR >17 dB for any reasonable M and N. The tag reader range is estimated as a function of the transmitted power and EP.

Complete mitochondrial genome of Bactrocera arecae (Insecta: Tephritidae) by next-generation sequencing and molecular phylogeny of Dacini tribe

PubMed Central

Yong, Hoi-Sen; Song, Sze-Looi; Lim, Phaik-Eem; Chan, Kok-Gan; Chow, Wan-Loo; Eamsobhana, Praphathip

2015-01-01

The whole mitochondrial genome of the pest fruit fly Bactrocera arecae was obtained from next-generation sequencing of genomic DNA. It had a total length of 15,900 bp, consisting of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region (A + T-rich control region). The control region (952 bp) was flanked by rrnS and trnI genes. The start codons included 6 ATG, 3 ATT and 1 each of ATA, ATC, GTG and TCG. Eight TAA, two TAG, one incomplete TA and two incomplete T stop codons were represented in the protein-coding genes. The cloverleaf structure for trnS1 lacked the D-loop, and that of trnN and trnF lacked the TΨC-loop. Molecular phylogeny based on 13 protein-coding genes was concordant with 37 mitochondrial genes, with B. arecae having closest genetic affinity to B. tryoni. The subgenus Bactrocera of Dacini tribe and the Dacinae subfamily (Dacini and Ceratitidini tribes) were monophyletic. The whole mitogenome of B. arecae will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general. PMID:26472633

78 FR 5797 - Availability of e-Tag Information to Commission Staff; Notice Specifying webRegistry Code

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-28

... e-Tag Information to Commission Staff; Notice Specifying webRegistry Code In Order No. 771,\\1\\ the... stated that, ``following issuance of this Final Rule and the Commission's registration in the OATI web... in the Purchasing-Seller Entity section of OATI webRegistry. This code should be used to designate...

Evidence for lack of homing by sea lampreys

USGS Publications Warehouse

Bergstedt, Roger A.; Seelye, James G.

1995-01-01

Recently metamorphosed sea lampreys Petromyzon marinus were captured in the Devil River, a tributary to Lake Huron, during summer and autumn 1990. They were tagged with a coded wire tag and returned to the river to continue their migration to Lake Huron to begin the parasitic (juvenile) phase of their life. During the spawning run in spring 1992 when the tagged animals were expected to mature and return to spawn, sea lampreys were trapped in nine tributaries to Lake Huron, including the Devil River; 47,946 animals were examined for coded wire tags, and 41 tagged animals were recovered. None of the 45 mature sea lampreys captured in the Devil River in 1992 were tagged, a proportion (0%) significantly lower than the proportion of the recently metamorphosed sea lampreys tagged in 1990. The distribution of tag recoveries among streams lakewide, however, was proportional to catch. Tagged sea lampreys did not appear to home, but instead seemed to select spawning streams through innate attraction to other sensory cues.

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

PubMed

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.

Method for nonlinear optimization for gas tagging and other systems

DOEpatents

Chen, Ting; Gross, Kenny C.; Wegerich, Stephan

1998-01-01

A method and system for providing nuclear fuel rods with a configuration of isotopic gas tags. The method includes selecting a true location of a first gas tag node, selecting initial locations for the remaining n-1 nodes using target gas tag compositions, generating a set of random gene pools with L nodes, applying a Hopfield network for computing on energy, or cost, for each of the L gene pools and using selected constraints to establish minimum energy states to identify optimal gas tag nodes with each energy compared to a convergence threshold and then upon identifying the gas tag node continuing this procedure until establishing the next gas tag node until all remaining n nodes have been established.

Method for nonlinear optimization for gas tagging and other systems

DOEpatents

Chen, T.; Gross, K.C.; Wegerich, S.

1998-01-06

A method and system are disclosed for providing nuclear fuel rods with a configuration of isotopic gas tags. The method includes selecting a true location of a first gas tag node, selecting initial locations for the remaining n-1 nodes using target gas tag compositions, generating a set of random gene pools with L nodes, applying a Hopfield network for computing on energy, or cost, for each of the L gene pools and using selected constraints to establish minimum energy states to identify optimal gas tag nodes with each energy compared to a convergence threshold and then upon identifying the gas tag node continuing this procedure until establishing the next gas tag node until all remaining n nodes have been established. 6 figs.

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

PubMed

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

2014-12-01

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule

PubMed Central

Nakanishi, Tsuyoshi; Ando, Eiji; Furuta, Masaru; Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru; Tsunasawa, Susumu; Nishimura, Osamu

2007-01-01

We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3. PMID:18166671

RNase One Gene Isolation, Expression, and Affinity Purification Models Research Experimental Progression and Culminates with Guided Inquiry-Based Experiments

ERIC Educational Resources Information Center

Bailey, Cheryl P.

2009-01-01

This new biochemistry laboratory course moves through a progression of experiments that generates a platform for guided inquiry-based experiments. RNase One gene is isolated from prokaryotic genomic DNA, expressed as a tagged protein, affinity purified, and tested for activity and substrate specificity. Student pairs present detailed explanations…

High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems.

PubMed

Tremblay, Reynald; Feng, Mary; Menassa, Rima; Huner, Norman P A; Jevnikar, Anthony M; Ma, Shengwu

2011-04-01

Soybean agglutinin (SBA) is a specific N-acetylgalactosamine-binding plant lectin that can agglutinate a wide variety of cells. SBA has great potential for medical and biotechnology-focused applications, including screening and treatment of breast cancer, isolation of fetal cells from maternal blood for genetic screening, the possibility as a carrier system for oral drug delivery, and utilization as an affinity tag for high-quality purification of tagged proteins. The success of these applications, to a large degree, critically depends on the development of a highly efficient expression system for a source of recombinant SBA (rSBA). Here, we demonstrate the utility of transient and stable expression systems in Nicotiana benthamiana and potato, respectively, for the production of rSBA, with the transgenic protein accumulated to 4% of total soluble protein (TSP) in Nicotiana benthamiana leaves and 0.3% of TSP in potato tubers. Furthermore, we show that both plant-derived rSBAs retain their ability to induce the agglutination of red blood cells, are similarly glycosylated when compared with native SBA, retained their binding specificity for N-acetylgalactosamine, and were highly resistant to degradation in simulated gastric and intestinal fluids. Affinity column purification using N-acetylgalactosamine as a specific ligand resulted in high recovery and purity of rSBA. This work is the first step toward use of rSBA for various new applications, including the development of rSBA as a novel affinity tag for simplified purification of tagged proteins and as a new carrier molecule for delivery of oral drugs.

A dual affinity-tag strategy for the expression and purification of human linker histone H1.4 in Escherichia coli.

PubMed

Ryan, Daniel P; Tremethick, David J

2016-04-01

Linker histones are an abundant and critical component of the eukaryotic chromatin landscape. They play key roles in regulating the higher order structure of chromatin and many genetic processes. Higher eukaryotes possess a number of different linker histone subtypes and new data are consistently emerging that indicate these subtypes are functionally distinct. We were interested in studying one of the most abundant human linker histone subtypes, H1.4. We have produced recombinant full-length H1.4 in Escherichia coli. An N-terminal Glutathione-S-Transferase tag was used to promote soluble expression and was combined with a C-terminal hexahistidine tag to facilitate a simple non-denaturing two-step affinity chromatography procedure that results in highly pure full-length H1.4. The purified H1.4 was shown to be functional via in vitro chromatin assembly experiments and remains active after extended storage at -80 °C. Copyright © 2015 Elsevier Inc. All rights reserved.

An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

PubMed

Ferguson, A D; Breed, J; Diederichs, K; Welte, W; Coulton, J W

1998-07-01

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

Annual Coded Wire Tag Program; Missing Production Groups, 1996 Annual Report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pastor, Stephen M.

1997-01-01

In 1989 the Bonneville Power Administration (BPA) began funding the evaluation of production groups of juvenile anadromous fish not being coded-wire tagged for other programs. These groups were the ''Missing Production Groups''. Production fish released by the U.S. Fish and Wildlife Service (USFWS) without representative coded-wire tags during the 1980's are indicated as blank spaces on the survival graphs in this report. The objectives of the ''Missing Production Groups'' program are: (1) to estimate the total survival of each production group, (2) to estimate the contribution of each production group to various fisheries, and (3) to prepare an annual reportmore » for all USFWS hatcheries in the Columbia River basin. Coded-wire tag recovery information will be used to evaluate the relative success of individual brood stocks. This information can also be used by salmon harvest managers to develop plans to allow the harvest of excess hatchery fish while protecting threatened, endangered, or other stocks of concern. In order to meet these objectives, a minimum of one marked group of fish is necessary for each production release. The level of marking varies according to location, species, and age at release. In general, 50,000 fish are marked with a coded-wire tag (CWT) to represent each production release group at hatcheries below John Day Dam. More than 100,000 fish per group are usually marked at hatcheries above John Day Dam. All fish release information, including marked/unmarked ratios, is reported to the Pacific States Marine Fisheries Commission (PSMFC). Fish recovered in the various fisheries or at the hatcheries are sampled to recover coded-wire tags. This recovery information is also reported to PSMFC.« less

The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barrette-Ng, Isabelle H.; Wu, Sau-Ching; Tjia, Wai-Mui

2013-05-01

The structure of the SBP-Tag–streptavidin complex reveals a novel mode of peptide recognition in which a single peptide binds simultaneously to biotin-binding pockets from adjacent subunits of streptavidin. The molecular details of peptide recognition suggest how the SBP-Tag can be further modified to become an even more useful tag for a wider range of biotechnological applications. The 38-residue SBP-Tag binds to streptavidin more tightly (K{sub d} ≃ 2.5–4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-bindingmore » pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12–14) and a C-terminal HPQ sequence (residues 31–33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17–28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1–10 and 35–38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11–34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag–streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.« less

Application of a New Dual Localization-Affinity Purification Tag Reveals Novel Aspects of Protein Kinase Biology in Aspergillus nidulans

PubMed Central

De Souza, Colin P.; Hashmi, Shahr B.; Osmani, Aysha H.; Osmani, Stephen A.

2014-01-01

Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation. PMID:24599037

Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

PubMed

De Souza, Colin P; Hashmi, Shahr B; Osmani, Aysha H; Osmani, Stephen A

2014-01-01

Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation.

Ethanol Attenuates Histiotrophic Nutrition Pathways and Alters the Intracellular Redox Environment and Thiol Proteome during Rat Organogenesis

PubMed Central

Jilek, Joseph L.; Sant, Karilyn E.; Cho, Katherine H.; Reed, Matthew S.; Pohl, Jan; Hansen, Jason M.; Harris, Craig

2015-01-01

Ethanol (EtOH) is a reactive oxygen-generating teratogen involved in the etiology of structural and functional developmental defects. Embryonic nutrition, redox environment, and changes in the thiol proteome following EtOH exposures (1.56.0 mg/ml) were studied in rat whole embryo culture. Glutathione (GSH) and cysteine (Cys) concentrations with their respective intracellular redox potentials (Eh) were determined using high-performance liquid chromatography. EtOH reduced GSH and Cys concentrations in embryo (EMB) and visceral yolk sac (VYS) tissues, and also in yolk sac and amniotic fluids. These changes produced greater oxidation as indicated by increasingly positive Eh values. EtOH reduced histiotrophic nutrition pathway activities as measured by the clearance of fluorescin isothiocyanate (FITC)-albumin from culture media. A significant decrease in total FITC clearance was observed at all concentrations, reaching approximately 50% at the highest dose. EtOH-induced changes to the thiol proteome were measured in EMBs and VYSs using isotope-coded affinity tags. Decreased concentrations for specific proteins from cytoskeletal dynamics and endocytosis pathways (α-actinin, α-tubulin, cubilin, and actin-related protein 2); nuclear translocation (Ran and RanBP1); and maintenance of receptor-mediated endocytosis (cubilin) were observed. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis also identified a decrease in ribosomal proteins in both EMB and VYS. Results show that EtOH interferes with nutrient uptake to reduce availability of amino acids and micronutrients required by the conceptus. Intracellular antioxidants such as GSH and Cys are depleted following EtOH and Eh values increase. Thiol proteome analysis in the EMB and VYS show selectively altered actin/cytoskeleton, endocytosis, ribosome biogenesis and function, nuclear transport, and stress-related responses. PMID:26185205

Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of cd toxicity.

PubMed

Go, Young-Mi; Roede, James R; Orr, Michael; Liang, Yongliang; Jones, Dean P

2014-05-01

Cadmium (Cd) exposure contributes to human diseases affecting liver, kidney, lung, and other organ systems, but mechanisms underlying the pleotropic nature of these toxicities are poorly understood. Cd accumulates in humans from dietary, environmental (including cigarette smoke), and occupational sources, and has a twenty-year biologic half-life. Our previous mouse and cell studies showed that environmental low-dose Cd exposure altered protein redox states resulting in stimulation of inflammatory signaling and disruption of the actin cytoskeleton system, suggesting that Cd could impact multiple mechanisms of disease. In the current study, we investigated the effects of acute Cd exposure on the redox proteome and metabolome of mouse liver mitochondria to gain insight into associated toxicological mechanisms and functions. We analyzed redox states of liver mitochondrial proteins by redox proteomics using isotope coded affinity tag (ICAT) combined mass spectrometry. Redox ICAT identified 2687 cysteine-containing peptides (peptidyl Cys) of which 1667 peptidyl Cys (657 proteins) were detected in both control and Cd-exposed samples. Of these, 46% (1247 peptidyl Cys, 547 proteins) were oxidized by Cd more than 1.5-fold relative to controls. Bioinformatics analysis using MetaCore software showed that Cd affected 86 pathways, including 24 Cys in proteins functioning in branched chain amino acid (BCAA) and 14 Cys in proteins functioning in fatty acid (acylcarnitine/carnitine) metabolism. Consistently, high-resolution metabolomics data showed that Cd treatment altered levels of BCAA and carnitine metabolites. Together, these results show that mitochondrial protein redox and metabolites are targets in Cd-induced hepatotoxicity. The results further indicate that redox proteomics and metabolomics can be used in an integrated systems approach to investigate complex disease mechanisms.

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

PubMed

Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

2006-01-01

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

76 FR 71315 - Endangered and Threatened Species; Take of Anadromous Fish

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-17

... the fish and would then sample them for biological information (fin tissue and scale samples). They..., measured, weighed, tissue-sampled, and checked for external marks and coded-wire tags depending on the.... Then the researchers would remove and preserve fish body tissues, otoliths, and coded wire tags (from...

Preparation of Protein Samples for NMR Structure, Function, and Small Molecule Screening Studies

PubMed Central

Acton, Thomas B.; Xiao, Rong; Anderson, Stephen; Aramini, James; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Kornhaber, Gregory; Lau, Jessica; Lee, Dong Yup; Liu, Gaohua; Maglaqui, Melissa; Ma, Lichung; Mao, Lei; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Shastry, Ritu; Swapna, G.V.T.; Tang, Yeufeng; Tong, Saichiu; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.

2014-01-01

In this chapter, we concentrate on the production of high quality protein samples for NMR studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium, and outline our high-throughput strategies for producing high quality protein samples for nuclear magnetic resonance (NMR) studies. Our strategy is based on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6X-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (> 97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5,000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this paper describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening and structural genomics research. PMID:21371586

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco.

PubMed

Nagaki, Kiyotaka; Shibata, Fukashi; Kanatani, Asaka; Kashihara, Kazunari; Murata, Minoru

2012-04-01

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres. © Springer-Verlag 2011

Nickel and zinc isotope fractionation in hyperaccumulating and nonaccumulating plants.

PubMed

Deng, Teng-Hao-Bo; Cloquet, Christophe; Tang, Ye-Tao; Sterckeman, Thibault; Echevarria, Guillaume; Estrade, Nicolas; Morel, Jean-Louis; Qiu, Rong-Liang

2014-10-21

Until now, there has been little data on the isotope fractionation of nickel (Ni) in higher plants and how this can be affected by plant Ni and zinc (Zn) homeostasis. A hydroponic cultivation was conducted to investigate the isotope fractionation of Ni and Zn during plant uptake and translocation processes. The nonaccumulator Thlaspi arvense, the Ni hyperaccumulator Alyssum murale and the Ni and Zn hyperaccumulator Noccaea caerulescens were grown in low (2 μM) and high (50 μM) Ni and Zn solutions. Results showed that plants were inclined to absorb light Ni isotopes, presumably due to the functioning of low-affinity transport systems across root cell membrane. The Ni isotope fractionation between plant and solution was greater in the hyperaccumulators grown in low Zn treatments (Δ(60)Ni(plant-solution) = -0.90 to -0.63‰) than that in the nonaccumulator T. arvense (Δ(60)Ni(plant-solution) = -0.21‰), thus indicating a greater permeability of the low-affinity transport system in hyperaccumulators. Light isotope enrichment of Zn was observed in most of the plants (Δ(66)Zn(plant-solution) = -0.23 to -0.10‰), but to a lesser extent than for Ni. The rapid uptake of Zn on the root surfaces caused concentration gradients, which induced ion diffusion in the rhizosphere and could result in light Zn isotope enrichment in the hyperaccumulator N. caerulescens. In high Zn treatment, Zn could compete with Ni during the uptake process, which reduced Ni concentration in plants and decreased the extent of Ni isotope fractionation (Δ(60)Ni(plant-solution) = -0.11 to -0.07‰), indicating that plants might take up Ni through a low-affinity transport system of Zn. We propose that isotope composition analysis for transition elements could become an empirical tool to study plant physiological processes.

Stable isotope, site-specific mass tagging for protein identification

DOEpatents

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.

Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing*

PubMed Central

Welle, Kevin A.; Zhang, Tian; Hryhorenko, Jennifer R.; Shen, Shichen; Qu, Jun; Ghaemmaghami, Sina

2016-01-01

Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. The results indicate that upon reaching quiescence, fibroblasts compensate for lack of cellular growth by globally downregulating protein synthesis and upregulating protein degradation. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data. PMID:27765818

Host Immune Selection of Rumen Bacteria through Salivary Secretory IgA

PubMed Central

Fouhse, Janelle M.; Smiegielski, Luke; Tuplin, Melanie; Guan, Le Luo; Willing, Benjamin P.

2017-01-01

The rumen microbiome is integral to efficient production in cattle and shows strong host specificity, yet little is known about what host factors shape rumen microbial composition. Secretory immunoglobulin A (SIgA) is produced in large amounts in the saliva, can coat both commensal and pathogenic microbes within the gut, and presents a plausible mechanism of host specificity. However, the role salivary SIgA plays in commensal bacteria selection in ruminants remains elusive. The main objectives of this study were to develop an immuno-affinity benchtop method to isolate SIgA-tagged microbiota and to determine if salivary SIgA preferentially binds selected bacteria. We hypothesized that SIgA-tagged bacteria would differ from total bacteria, thus supporting a potential host-derived mechanism in commensal bacterial selection. Whole rumen (n = 9) and oral secretion samples (n = 10) were incubated with magnetic beads conjugated with anti-secretory IgA antibodies to enrich SIgA-tagged microbiota. Microbial DNA from the oral secretion, whole rumen, SIgA-tagged oral secretion, and SIgA-tagged rumen was isolated for amplicon sequencing of V1–V3 region of 16S rDNA genes. Whole rumen and oral secretion had distinctive (P < 0.05) bacterial compositions indicated by the non-parametric multidimensional scaling plot using Euclidean distance metrics. The SIgA-tagged microbiota from rumen and oral secretion had similar abundance of Bacteroidetes, Actinobacteria, Fibrobacter, candidate phyla TM7, and Tenericutes and are clustered tightly. Composition of SIgA-tagged oral secretion microbiota was more similar to whole rumen microbiota than whole oral secretion due to enrichment of rumen bacteria (Lachnospiraceae) and depletion of oral taxa (Streptococcus, Rothia, Neisseriaceae, and Lactobacillales). In conclusion, SIgA-tagged oral secretion microbiota had an increased resemblance to whole rumen microbiota, suggesting salivary SIgA-coating may be one host-derived mechanism impacting commensal colonization. Further studies, to explore the variations in antibody affinity between different animals as a driver of microbial composition are warranted. PMID:28553275

Preparation of His-tagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus.

PubMed

Cheng, Yangjian; Niu, Jianjun; Zhang, Yongyou; Huang, Jianwei; Li, Qingge

2006-10-01

Armored RNA has been increasingly used as both an external and internal positive control in nucleic acid-based assays for RNA virus. In order to facilitate armored RNA purification, a His6 tag was introduced into the loop region of the MS2 coat protein, which allows the exposure of multiple His tags on the surface during armored RNA assembly. The His-tagged armored RNA particles were purified to homogeneity and verified to be free of DNA contamination in a single run of affinity chromatography. A fragment of severe acute respiratory syndrome coronavirus (SARS-CoV) genome targeted for SARS-CoV detection was chosen for an external positive control preparation. A plant-specific gene sequence was chosen for a universal noncompetitive internal positive control preparation. Both controls were purified by Co2+ affinity chromatography and were included in a real-time reverse transcription-PCR assay for SARS-CoV. The noncompetitive internal positive control can be added to clinical samples before RNA extraction and enables the identification of potential inhibitive effects without interfering with target amplification. The external control could be used for the quantification of viral loads in clinical samples.

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

PubMed

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.

Strep-Tagged Protein Purification.

PubMed

Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schäfer, Frank

2015-01-01

The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given. Depending on the amount of Strep-tagged protein in the lysate, a protocol for batch binding and subsequent washing and eluting by gravity flow can be used. Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable. In addition, Strep-tagged protein purification can also be automated using prepacked columns for FPLC or other liquid-handling chromatography instrumentation, but automated purification is not discussed in this protocol. The protocols described here can be regarded as an update of the Strep-Tag Protein Handbook (Qiagen, 2009). © 2015 Elsevier Inc. All rights reserved.

Global combined precursor isotopic labeling and isobaric tagging (cPILOT) approach with selective MS(3) acquisition.

PubMed

Evans, Adam R; Robinson, Renã A S

2013-11-01

Recently, we reported a novel proteomics quantitation scheme termed "combined precursor isotopic labeling and isobaric tagging (cPILOT)" that allows for the identification and quantitation of nitrated peptides in as many as 12-16 samples in a single experiment. cPILOT offers enhanced multiplexing and posttranslational modification specificity, however excludes global quantitation for all peptides present in a mixture and underestimates reporter ion ratios similar to other isobaric tagging methods due to precursor co-isolation. Here, we present a novel chemical workflow for cPILOT that can be used for global tagging of all peptides in a mixture. Specifically, through low pH precursor dimethylation of tryptic or LysC peptides followed by high pH tandem mass tags, the same reporter ion can be used twice in a single experiment. Also, to improve triple-stage mass spectrometry (MS(3) ) data acquisition, a selective MS(3) method that focuses on product selection of the y1 fragment of lysine-terminated peptides is incorporated into the workflow. This novel cPILOT workflow has potential for global peptide quantitation that could lead to enhanced sample multiplexing and increase the number of quantifiable spectra obtained from MS(3) acquisition methods. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Annual Stock Assessment - CWT [Coded Wire Tag program] (USFWS), Annual Report 2007.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pastor, Stephen M.

2009-07-21

In 1989 the Bonneville Power Administration (BPA) began funding the evaluation of production groups of juvenile anadromous fish not being coded-wire tagged for other programs. These groups were the 'Missing Production Groups'. Production fish released by the U.S. Fish and Wildlife Service (FWS) without representative coded-wire tags during the 1980s are indicated as blank spaces on the survival graphs in this report. This program is now referred to as 'Annual Stock Assessment - CWT'. The objectives of the 'Annual Stock Assessment' program are to: (1) estimate the total survival of each production group, (2) estimate the contribution of each productionmore » group to fisheries, and (3) prepare an annual report for USFWS hatcheries in the Columbia River basin. Coded-wire tag recovery information will be used to evaluate the relative success of individual brood stocks. This information can also be used by salmon harvest managers to develop plans to allow the harvest of excess hatchery fish while protecting threatened, endangered, or other stocks of concern. All fish release information, including marked/unmarked ratios, is reported to the Pacific States Marine Fisheries Commission (PSMFC). Fish recovered in the various fisheries or at the hatcheries are sampled to recover coded-wire tags. This recovery information is also reported to PSMFC. This report has been prepared annually starting with the report labeled 'Annual Report 1994'. Although the current report has the title 'Annual Report 2007', it was written in fall of 2008 using data available from RMIS that same year, and submitted as final in January 2009. The main objective of the report is to evaluate survival of groups which have been tagged under this ongoing project.« less

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase

PubMed Central

Li, Yifeng; Franklin, Sarah; Zhang, Michael J; Vondriska, Thomas M

2011-01-01

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His6 purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems. PMID:21080425

Tracing the origins, migrations, and other movements of fishes using stable isotopes

EPA Science Inventory

Although stable isotope studies are increasingly used to trace fish origin, migration, and movement, the approach has not been as widely adopted as extrinsic marking and tagging. This is, at least in part, because many fishery scientists do not have a background in biogeochemistr...

A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules.

PubMed

Tsunehiro, Masaya; Meki, Yuma; Matsuoka, Kanako; Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru

2013-04-15

A simple and efficient method based on magnetic-bead technology has been developed for the separation of phosphorylated and nonphosphorylated low-molecular-weight biomolecules, such as nucleotides, phosphorylated amino acids, or phosphopeptides. The phosphate-binding site on the bead is an alkoxide-bridged dinuclear zinc(II) complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag), which is linked to a hydrophilic cross-linked agarose coating on a magnetic core particle. All steps for the phosphate-affinity separation are conducted in buffers of neutral pH with 50 μL of the magnetic beads in a 1.5-mL microtube. The entire separation protocol for phosphomonoester-type compounds, from addition to elution, requires less than 12 min per sample if the buffers and the zinc(II)-bound Phos-tag magnetic beads have been prepared in advance. The phosphate-affinity magnetic beads are reusable at least 15 times without a decrease in their phosphate-binding ability and they are stable for three months in propan-2-ol. Copyright © 2013 Elsevier B.V. All rights reserved.

System and method for a parallel immunoassay system

DOEpatents

Stevens, Fred J.

2002-01-01

A method and system for detecting a target antigen using massively parallel immunoassay technology. In this system, high affinity antibodies of the antigen are covalently linked to small beads or particles. The beads are exposed to a solution containing DNA-oligomer-mimics of the antigen. The mimics which are reactive with the covalently attached antibody or antibodies will bind to the appropriate antibody molecule on the bead. The particles or beads are then washed to remove any unbound DNA-oligomer-mimics and are then immobilized or trapped. The bead-antibody complexes are then exposed to a test solution which may contain the targeted antigens. If the antigen is present it will replace the mimic since it has a greater affinity for the respective antibody. The particles are then removed from the solution leaving a residual solution. This residual solution is applied a DNA chip containing many samples of complimentary DNA. If the DNA tag from a mimic binds with its complimentary DNA, it indicates the presence of the target antigen. A flourescent tag can be used to more easily identify the bound DNA tag.

TAGS 85/2N RTG Power for Viking Lander Capsule

DOE R&D Accomplishments Database

1969-08-01

Results of studies performed by Isotopes, Inc., Nuclear Systems Division, to optimize and baseline a TAGS 85/2N RTG for the Viking Lander Capsule prime electrical power source are presented. These studies generally encompassed identifying the Viking RTG mission profile and design requirements, and establishing a baseline RTG design consistent with these requirements.

Relative quantification of biomarkers using mixed-isotope labeling coupled with MS

PubMed Central

Chapman, Heidi M; Schutt, Katherine L; Dieter, Emily M; Lamos, Shane M

2013-01-01

The identification and quantification of important biomarkers is a critical first step in the elucidation of biological systems. Biomarkers take many forms as cellular responses to stimuli and can be manifested during transcription, translation, and/or metabolic processing. Increasingly, researchers have relied upon mixed-isotope labeling (MIL) coupled with MS to perform relative quantification of biomarkers between two or more biological samples. MIL effectively tags biomarkers of interest for ease of identification and quantification within the mass spectrometer by using isotopic labels that introduce a heavy and light form of the tag. In addition to MIL coupled with MS, a number of other approaches have been used to quantify biomarkers including protein gel staining, enzymatic labeling, metabolic labeling, and several label-free approaches that generate quantitative data from the MS signal response. This review focuses on MIL techniques coupled with MS for the quantification of protein and small-molecule biomarkers. PMID:23157360

ICC-CLASS: isotopically-coded cleavable crosslinking analysis software suite

PubMed Central

2010-01-01

Background Successful application of crosslinking combined with mass spectrometry for studying proteins and protein complexes requires specifically-designed crosslinking reagents, experimental techniques, and data analysis software. Using isotopically-coded ("heavy and light") versions of the crosslinker and cleavable crosslinking reagents is analytically advantageous for mass spectrometric applications and provides a "handle" that can be used to distinguish crosslinked peptides of different types, and to increase the confidence of the identification of the crosslinks. Results Here, we describe a program suite designed for the analysis of mass spectrometric data obtained with isotopically-coded cleavable crosslinkers. The suite contains three programs called: DX, DXDX, and DXMSMS. DX searches the mass spectra for the presence of ion signal doublets resulting from the light and heavy isotopic forms of the isotopically-coded crosslinking reagent used. DXDX searches for possible mass matches between cleaved and uncleaved isotopically-coded crosslinks based on the established chemistry of the cleavage reaction for a given crosslinking reagent. DXMSMS assigns the crosslinks to the known protein sequences, based on the isotopically-coded and un-coded MS/MS fragmentation data of uncleaved and cleaved peptide crosslinks. Conclusion The combination of these three programs, which are tailored to the analytical features of the specific isotopically-coded cleavable crosslinking reagents used, represents a powerful software tool for automated high-accuracy peptide crosslink identification. See: http://www.creativemolecules.com/CM_Software.htm PMID:20109223

Tagging as a Social Literacy Practice

ERIC Educational Resources Information Center

MacGillivray, Laurie; Curwen, Margaret Sauceda

2007-01-01

Tagging is not simply an act of vandalism or violence; it is a social practice with its own rules and codes--a literacy practice imbued with intent and meaning. Three aspects of tagging reflect its nature as a literate practice: (1) The purpose of tagging to achieve particular social goals and group affiliations; (2) The role of talent to be…

Pharmacoproteomics of a metalloproteinase hydroxamate inhibitor in breast cancer cells: dynamics of membrane type 1 matrix metalloproteinase-mediated membrane protein shedding.

PubMed

Butler, Georgina S; Dean, Richard A; Tam, Eric M; Overall, Christopher M

2008-08-01

Broad-spectrum matrix metalloproteinase (MMP) inhibitors (MMPI) were unsuccessful in cancer clinical trials, partly due to side effects resulting from limited knowledge of the full repertoire of MMP substrates, termed the substrate degradome, and hence the in vivo functions of MMPs. To gain further insight into the degradome of MMP-14 (membrane type 1 MMP) an MMPI, prinomastat (drug code AG3340), was used to reduce proteolytic processing and ectodomain shedding in human MDA-MB-231 breast cancer cells transfected with MMP-14. We report a quantitative proteomic evaluation of the targets and effects of the inhibitor in this cell-based system. Proteins in cell-conditioned medium (the secretome) and membrane fractions with levels that were modulated by the MMPI were identified by isotope-coded affinity tag (ICAT) labeling and tandem mass spectrometry. Comparisons of the expression of MMP-14 with that of a vector control resulted in increased MMP-14/vector ICAT ratios for many proteins in conditioned medium, indicating MMP-14-mediated ectodomain shedding. Following MMPI treatment, the MMPI/vehicle ICAT ratio was reversed, suggesting that MMP-14-mediated shedding of these proteins was blocked by the inhibitor. The reduction in shedding or the release of substrates from pericellular sites in the presence of the MMPI was frequently accompanied by the accumulation of the protein in the plasma membrane, as indicated by high MMPI/vehicle ICAT ratios. Considered together, this is a strong predictor of biologically relevant substrates cleaved in the cellular context that led to the identification of many undescribed MMP-14 substrates, 20 of which we validated biochemically, including DJ-1, galectin-1, Hsp90alpha, pentraxin 3, progranulin, Cyr61, peptidyl-prolyl cis-trans isomerase A, and dickkopf-1. Other proteins with altered levels, such as Kunitz-type protease inhibitor 1 and beta-2-microglobulin, were not substrates in biochemical assays, suggesting an indirect affect of the MMPI, which might be important in drug development as biomarkers or, in preclinical phases, to predict systemic drug actions and adverse side effects. Hence, this approach describes the dynamic pattern of cell membrane ectodomain shedding and its perturbation upon metalloproteinase drug treatment.

Identification coding schemes for modulated reflectance systems

DOEpatents

Coates, Don M [Santa Fe, NM; Briles, Scott D [Los Alamos, NM; Neagley, Daniel L [Albuquerque, NM; Platts, David [Santa Fe, NM; Clark, David D [Santa Fe, NM

2006-08-22

An identifying coding apparatus employing modulated reflectance technology involving a base station emitting a RF signal, with a tag, located remotely from the base station, and containing at least one antenna and predetermined other passive circuit components, receiving the RF signal and reflecting back to the base station a modulated signal indicative of characteristics related to the tag.

[Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it].

PubMed

Weng, Daihui; Lei, Yingfeng; Dong, Yangchao; Han, Peijun; Ye, Chuantao; Yang, Jing; Wang, Yuan; Yin, Wen

2015-12-01

To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay. Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay. The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system. TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.

Summary of evidence for an anticodonic basis for the origin of the genetic code

NASA Technical Reports Server (NTRS)

Lacey, J. C., Jr.; Mullins, D. W., Jr.

1981-01-01

This article summarizes data supporting the hypothesis that the genetic code origin was based on relationships (probably affinities) between amino acids and their anticodon nucleotides. Selective activation seems to follow from selective affinity and consequently, incorporation of amino acids into peptides can also be selective. It is suggested that these selectivities in affinity and activation, coupled with the base pairing specificities, allowed the origin of the code and the process of translation.

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

PubMed

Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

2018-05-22

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

Method of preparing gas tags for identification of single and multiple failures of nuclear reactor fuel assemblies

DOEpatents

McCormick, Norman J.

1976-01-01

For use in the identification of failed fuel assemblies in a nuclear reactor, the ratios of the tag gas isotopic concentrations are located on curved surfaces to enable the ratios corresponding to failure of a single fuel assembly to be distinguished from those formed from any combination of two or more failed assemblies.

Using Digital Acoustic Recording Tags to Detect Marine Mammals on Navy Ranges and Study their Responses to Naval Sonar

DTIC Science & Technology

2011-02-01

written in C and assembly languages. 2) executable code for the low-power wakeup controller in the tag. This software is responsible for the VHF...used in the tag software. The multi-rate processing in the new tag necessitated a more complex task- scheduling software architecture. The effort of

Short range spread-spectrum radiolocation system and method

DOEpatents

Smith, Stephen F.

2003-04-29

A short range radiolocation system and associated methods that allow the location of an item, such as equipment, containers, pallets, vehicles, or personnel, within a defined area. A small, battery powered, self-contained tag is provided to an item to be located. The tag includes a spread-spectrum transmitter that transmits a spread-spectrum code and identification information. A plurality of receivers positioned about the area receive signals from a transmitting tag. The position of the tag, and hence the item, is located by triangulation. The system employs three different ranging techniques for providing coarse, intermediate, and fine spatial position resolution. Coarse positioning information is provided by use of direct-sequence code phase transmitted as a spread-spectrum signal. Intermediate positioning information is provided by the use of a difference signal transmitted with the direct-sequence spread-spectrum code. Fine positioning information is provided by use of carrier phase measurements. An algorithm is employed to combine the three data sets to provide accurate location measurements.

An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.

PubMed

Pollier, Jacob; González-Guzmán, Miguel; Ardiles-Diaz, Wilson; Geelen, Danny; Goossens, Alain

2011-01-01

cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

PubMed

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

Targeted silver nanoparticles for ratiometric cell phenotyping

NASA Astrophysics Data System (ADS)

Willmore, Anne-Mari A.; Simón-Gracia, Lorena; Toome, Kadri; Paiste, Päärn; Kotamraju, Venkata Ramana; Mölder, Tarmo; Sugahara, Kazuki N.; Ruoslahti, Erkki; Braun, Gary B.; Teesalu, Tambet

2016-04-01

Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 +/- 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 +/- 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo.Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 +/- 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 +/- 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo. Electronic supplementary information (ESI) available: TEM images of isotopic AgNPs, cell antibody staining, coadministration ICP-MS data, and biotin control particle ICP-MS data. See DOI: 10.1039/C5NR07928D

Dual-isotope PET using positron-gamma emitters.

PubMed

Andreyev, A; Celler, A

2011-07-21

Positron emission tomography (PET) is widely recognized as a highly effective functional imaging modality. Unfortunately, standard PET cannot be used for dual-isotope imaging (which would allow for simultaneous investigation of two different biological processes), because positron-electron annihilation products from different tracers are indistinguishable in terms of energy. Methods that have been proposed for dual-isotope PET rely on differences in half-lives of the participating isotopes; these approaches, however, require making assumptions concerning kinetic behavior of the tracers and may not lead to optimal results. In this paper we propose a novel approach for dual-isotope PET and investigate its performance using GATE simulations. Our method requires one of the two radioactive isotopes to be a pure positron emitter and the second isotope to emit an additional high-energy gamma in a cascade simultaneously with positron emission. Detection of this auxiliary prompt gamma in coincidence with the annihilation event allows us to identify the corresponding 511 keV photon pair as originating from the same isotope. Two list-mode datasets are created: a primary dataset that contains all detected 511 keV photon pairs from both isotopes, and a second, tagged (much smaller) dataset that contains only those PET events for which a coincident prompt gamma has also been detected. An image reconstructed from the tagged dataset reflects the distribution of the second positron-gamma radiotracer and serves as a prior for the reconstruction of the primary dataset. Our preliminary simulation study with partially overlapping (18)F/(22)Na and (18)F/(60)Cu radiotracer distributions showed that in these two cases the dual-isotope PET method allowed for separation of the two activity distributions and recovered total activities with relative errors of about 5%.

Retention of internal anchor tags by juvenile striped bass

USGS Publications Warehouse

Van Den Avyle, M.J.; Wallin, J.E.

2001-01-01

We marked hatchery-reared striped bass Morone saxatilis (145-265 mm total length) with internal anchor tags and monitored retention for 28 months after stocking in the Savannah River, Georgia and South Carolina. Anchor tags (with an 18-mm, T-shaped anchor and 42-mm streamer) were surgically implanted ventrally, and coded wire tags (1 mm long and 0.25 mm in diameter) were placed into the cheek muscle to help identify subsequent recaptures. The estimated probability of retention (SD) of anchor tags was 0.94 (0.05) at 4 months, 0.64 (0.13) at 16 months, and 0.33 (0.19) at 28 months. Of 10 fish recaptured with only coded wire tags, 5 showed an externally visible wound or scar near the point of anchor tag insertion. The incidence of wounds or scars, which we interpreted as evidence of tag shedding, increased to 50% in recaptures taken at 28 months (three of six fish). Our estimates for retention of anchor tags were generally lower than those in other studies of striped bass, possibly because of differences in the style of anchor or sizes of fish used. Because of its low rate of retention, the type of anchor tag we used may not be suitable for long-term assessments of stock enhancement programs that use striped bass of the sizes we evaluated.

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

NASA Astrophysics Data System (ADS)

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

A General Strategy for Targeting Drugs to Bone.

PubMed

Jahnke, Wolfgang; Bold, Guido; Marzinzik, Andreas L; Ofner, Silvio; Pellé, Xavier; Cotesta, Simona; Bourgier, Emmanuelle; Lehmann, Sylvie; Henry, Chrystelle; Hemmig, René; Stauffer, Frédéric; Hartwieg, J Constanze D; Green, Jonathan R; Rondeau, Jean-Michel

2015-11-23

Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS). Furthermore, bisphosphonate bone affinity comes at the expense of very low and variable oral bioavailability. FPPS inhibitors were developed with a monophosphonate as a bone-affinity tag that confers moderate affinity to bone, which can furthermore be tuned to the desired level, and the relationship between structure and bone affinity was evaluated by using an NMR-based bone-binding assay. The concept of targeting drugs to bone with moderate affinity, while retaining oral bioavailability, has broad application to a variety of other bone-targeted drugs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface acoustic wave coding for orthogonal frequency coded devices

NASA Technical Reports Server (NTRS)

Malocha, Donald (Inventor); Kozlovski, Nikolai (Inventor)

2011-01-01

Methods and systems for coding SAW OFC devices to mitigate code collisions in a wireless multi-tag system. Each device producing plural stepped frequencies as an OFC signal with a chip offset delay to increase code diversity. A method for assigning a different OCF to each device includes using a matrix based on the number of OFCs needed and the number chips per code, populating each matrix cell with OFC chip, and assigning the codes from the matrix to the devices. The asynchronous passive multi-tag system includes plural surface acoustic wave devices each producing a different OFC signal having the same number of chips and including a chip offset time delay, an algorithm for assigning OFCs to each device, and a transceiver to transmit an interrogation signal and receive OFC signals in response with minimal code collisions during transmission.

Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

PubMed Central

Tan, Dan; Li, Qiang; Zhang, Mei-Jun; Liu, Chao; Ma, Chengying; Zhang, Pan; Ding, Yue-He; Fan, Sheng-Bo; Tao, Li; Yang, Bing; Li, Xiangke; Ma, Shoucai; Liu, Junjie; Feng, Boya; Liu, Xiaohui; Wang, Hong-Wei; He, Si-Min; Gao, Ning; Ye, Keqiong; Dong, Meng-Qiu; Lei, Xiaoguang

2016-01-01

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction. DOI: http://dx.doi.org/10.7554/eLife.12509.001 PMID:26952210

Neutron-Encoded Protein Quantification by Peptide Carbamylation

NASA Astrophysics Data System (ADS)

Ulbrich, Arne; Merrill, Anna E.; Hebert, Alexander S.; Westphall, Michael S.; Keller, Mark P.; Attie, Alan D.; Coon, Joshua J.

2014-01-01

We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.

Investigation of the Mechanism of Electron Capture and Electron Transfer Dissociation of Peptides with a Covalently Attached Free Radical Hydrogen Atom Scavenger.

PubMed

Sohn, Chang Ho; Yin, Sheng; Peng, Ivory; Loo, Joseph A; Beauchamp, J L

2015-11-15

The mechanisms of electron capture and electron transfer dissociation (ECD and ETD) are investigated by covalently attaching a free-radical hydrogen atom scavenger to a peptide. The 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) radical was chosen as the scavenger due to its high hydrogen atom affinity (ca. 280 kJ/mol) and low electron affinity (ca. 0.45 ev), and was derivatized to the model peptide, FQX TEMPO EEQQQTEDELQDK. The X TEMPO residue represents a cysteinyl residue derivatized with an acetamido-TEMPO group. The acetamide group without TEMPO was also examined as a control. The gas phase proton affinity (882 kJ/mol) of TEMPO is similar to backbone amide carbonyls (889 kJ/mol), minimizing perturbation to internal solvation and sites of protonation of the derivatized peptides. Collision induced dissociation (CID) of the TEMPO tagged peptide dication generated stable odd-electron b and y type ions without indication of any TEMPO radical induced fragmentation initiated by hydrogen abstraction. The type and abundance of fragment ions observed in the CID spectra of the TEMPO and acetamide tagged peptides are very similar. However, ECD of the TEMPO labeled peptide dication yielded no backbone cleavage. We propose that a labile hydrogen atom in the charge reduced radical ions is scavenged by the TEMPO radical moiety, resulting in inhibition of N-C α backbone cleavage processes. Supplemental activation after electron attachment (ETcaD) and CID of the charge-reduced precursor ion generated by electron transfer of the TEMPO tagged peptide dication produced a series of b + H (b H ) and y + H (y H ) ions along with some c ions having suppressed intensities, consistent with stable O-H bond formation at the TEMPO group. In summary, the results indicate that ECD and ETD backbone cleavage processes are inhibited by scavenging of a labile hydrogen atom by the localized TEMPO radical moiety. This observation supports the conjecture that ECD and ETD processes involve long-lived intermediates formed by electron capture/transfer in which a labile hydrogen atom is present and plays a key role with low energy processes leading to c and z ion formation. Ab initio and density functional calculations are performed to support our conclusion, which depends most importantly on the proton affinity, electron affinity and hydrogen atom affinity of the TEMPO moiety.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

PubMed

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-11-14

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

PubMed Central

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-01-01

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

Design of Distortion-Invariant Optical ID Tags for Remote Identification and Verification of Objects

NASA Astrophysics Data System (ADS)

Pérez-Cabré, Elisabet; Millán, María Sagrario; Javidi, Bahram

Optical identification (ID) tags [1] have a promising future in a number of applications such as the surveillance of vehicles in transportation, control of restricted areas for homeland security, item tracking on conveyor belts or other industrial environment, etc. More specifically, passive optical ID tag [1] was introduced as an optical code containing a signature (that is, a characteristic image or other relevant information of the object), which permits its real-time remote detection and identification. Since their introduction in the literature [1], some contributions have been proposed to increase their usefulness and robustness. To increase security and avoid counterfeiting, the signature was introduced in the optical code as an encrypted function [2-5] following the double-phase encryption technique [6]. Moreover, the design of the optical ID tag was done in such a way that tolerance to variations in scale and rotation was achieved [2-5]. To do that, the encrypted information was multiplexed and distributed in the optical code following an appropriate topology. Further studies were carried out to analyze the influence of different sources of noise. In some proposals [5, 7], the designed ID tag consists of two optical codes where the complex-valued encrypted signature was separately introduced in two real-valued functions according to its magnitude and phase distributions. This solution was introduced to overcome some difficulties in the readout of complex values in outdoors environments. Recently, the fully phase encryption technique [8] has been proposed to increase noise robustness of the authentication system.

99mTc-tagged chicken liver as a marker of solid food in the human stomach.

PubMed

Meyer, J H; MacGregor, I L; Gueller, R; Martin, P; Cavalieri, R

1976-04-01

Past measurement of gastric emptying of solid food in man has depended on external counting of surface-absorbed isotopes without verification that isotopic labels remain attached to solid food in the stomach. In this study chicken liver was isotopically labeled with 99mTc incorporated uniformly and intracellularly throughout the liver substance. In vitro studies showed less than 10% loss of 99mTc from liver incubated with pepsin HC1. By contrast, up to 90% of 51Cr absorbed to scrambled eggs became detached under similar conditions. In feeding experiments less than 10% of 99mTc was liberated from fed 99mTc liver, while significantly more 51Cr became detached from egg under identical intragastric conditions. We conclude that 99mTc-tagged chicken liver is an adequate marker of the rate of emptying of solid food and appears to be more reliable than 51Cr-labeled scrambled eggs from which 51Cr dissociates in the stomach.

In vivo phosphorylation of a peptide tag for protein purification.

PubMed

Goux, Marine; Fateh, Amina; Defontaine, Alain; Cinier, Mathieu; Tellier, Charles

2016-05-01

To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag. The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III). The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

NASA Technical Reports Server (NTRS)

Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

2000-01-01

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

A set of ligation-independent in vitro translation vectors for eukaryotic protein production.

PubMed

Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

2008-03-27

The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

Stable isotope analysis of vertebrae reveals ontogenetic changes in habitat in an endothermic pelagic shark

PubMed Central

Carlisle, Aaron B.; Goldman, Kenneth J.; Litvin, Steven Y.; Madigan, Daniel J.; Bigman, Jennifer S.; Swithenbank, Alan M.; Kline, Thomas C.; Block, Barbara A.

2015-01-01

Ontogenetic changes in habitat are driven by shifting life-history requirements and play an important role in population dynamics. However, large portions of the life history of many pelagic species are still poorly understood or unknown. We used a novel combination of stable isotope analysis of vertebral annuli, Bayesian mixing models, isoscapes and electronic tag data to reconstruct ontogenetic patterns of habitat and resource use in a pelagic apex predator, the salmon shark (Lamna ditropis). Results identified the North Pacific Transition Zone as the major nursery area for salmon sharks and revealed an ontogenetic shift around the age of maturity from oceanic to increased use of neritic habitats. The nursery habitat may reflect trade-offs between prey availability, predation pressure and thermal constraints on juvenile endothermic sharks. The ontogenetic shift in habitat coincided with a reduction of isotopic niche, possibly reflecting specialization upon particular prey or habitats. Using tagging data to inform Bayesian isotopic mixing models revealed that adult sharks primarily use neritic habitats of Alaska yet receive a trophic subsidy from oceanic habitats. Integrating the multiple methods used here provides a powerful approach to retrospectively study the ecology and life history of migratory species throughout their ontogeny. PMID:25621332

IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells

PubMed Central

Orlando, Ron; Lim, Jae-Min; Atwood, James A.; Angel, Peggi M.; Fang, Meng; Aoki, Kazuhiro; Alvarez-Manilla, Gerardo; Moremen, Kelley W.; York, William S.; Tiemeyer, Michael; Pierce, Michael; Dalton, Stephen; Wells, Lance

2012-01-01

Robust quantification is an essential component of comparative –omic strategies. In this regard, glycomics lags behind proteomics. Although various isotope-tagging and direct quantification methods have recently enhanced comparative glycan analysis, a cell culture labeling strategy, that could provide for glycomics the advantages that SILAC provides for proteomics, has not been described. Here we report the development of IDAWG, Isotopic Detection of Aminosugars With Glutamine, for the incorporation of differential mass tags into the glycans of cultured cells. In this method, culture media containing amide-15N-Gln is used to metabolically label cellular aminosugars with heavy nitrogen. Because the amide side chain of Gln is the sole source of nitrogen for the biosynthesis of GlcNAc, GalNAc, and sialic acid, we demonstrate that culturing mouse embryonic stems cells for 72 hours in the presence of amide-15N-Gln media results in nearly complete incorporation of 15N into N-linked and O-linked glycans. The isotopically heavy monosaccharide residues provide additional information for interpreting glycan fragmentation and also allow quantification in both full MS and MS/MS modes. Thus, IDAWG is a simple to implement, yet powerful quantitative tool for the glycomics toolbox. PMID:19449840

Rhenium-osmium and samarium-neodymium isotopic systematics of the Stillwater complex

NASA Technical Reports Server (NTRS)

Lambert, David D.; Shirey, Steven B.; Carlson, Richard W.; Morgan, John W.; Walker, Richard J.

1989-01-01

The role of magma mixing in the formation of strategic platinum-group element ore deposits is examined using isotopic data from the Stillwater Complex, Montana. Nd and Os isotopic data show that the intrusion formed from at least two distinct magmas: ultramafic (U-type) affinity magmas and anorthositic (A-type) affinity magmas. The U-type magmas formed from a lithospheric mantle source containing recycled crustal materials and the A-type magmas originated either by crustal contamination of basaltic magmas or by partial melting of basalt in the lower crust. The results also suggest that the platinum-group element ore deposits were derived from A-type magmas which were injected into the U-type magma chamber at several stages during the development of the ultramafic series.

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

PubMed

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.

SPECHT - single-stage phosphopeptide enrichment and stable-isotope chemical tagging: quantitative phosphoproteomics of insulin action in muscle.

PubMed

Kettenbach, Arminja N; Sano, Hiroyuki; Keller, Susanna R; Lienhard, Gustav E; Gerber, Scott A

2015-01-30

The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues. Through the use of a quantitatively reproducible, proteome-wide phosphopeptide enrichment strategy, we demonstrated the feasibility of post-phosphopeptide purification chemical labeling and tagging as an enabling approach for quantitative phosphoproteomics of primary tissues. Using reductive dimethyl labeling as a generalized chemical tagging strategy, we compared the performance of post-phosphopeptide purification chemical tagging to the well established community standard, SILAC, in insulin-stimulated tissue culture cells. We then extended our method to the analysis of low-dose insulin signaling in murine muscle tissue, and report on the analytical and biological significance of our results. Copyright © 2014 Elsevier B.V. All rights reserved.

Cloning and expression of antibacterial goat lactoferricin from Escherichia coli AD494(DE3)pLysS expression system.

PubMed

Chen, Gen-Hung; Yin, Li-Jung; Chiang, I-Hua; Jiang, Shann-Tzong

2008-12-01

Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His*Tag and GLfcin S-His*Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His*Tag and GLfcin S-His*Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His*Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His*Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100 degrees C.

Method of isotope separation by chemi-ionization

DOEpatents

Wexler, Sol; Young, Charles E.

1977-05-17

A method for separating specific isotopes present in an isotopic mixture by aerodynamically accelerating a gaseous compound to form a jet of molecules, and passing the jet through a stream of electron donor atoms whereby an electron transfer takes place, thus forming negative ions of the molecules. The molecular ions are then passed through a radiofrequency quadrupole mass filter to separate the specific isotopes. This method may be used for any compounds having a sufficiently high electron affinity to permit negative ion formation, and is especially useful for the separation of plutonium and uranium isotopes.

Generic tags for Mn(ii) and Gd(iii) spin labels for distance measurements in proteins.

PubMed

Yang, Yin; Gong, Yan-Jun; Litvinov, Aleksei; Liu, Hong-Kai; Yang, Feng; Su, Xun-Cheng; Goldfarb, Daniella

2017-10-11

High-affinity chelating tags for Gd(iii) and Mn(ii) ions that provide valuable high-resolution distance restraints for biomolecules were used as spin labels for double electron-electron resonance (DEER) measurements. The availability of a generic tag that can bind both metal ions and provide a narrow and predictable distance distribution for both ions is attractive owing to their different EPR-related characteristics. Herein we introduced two paramagnetic tags, 4PSPyMTA and 4PSPyNPDA, which are conjugated to cysteine residues through a stable thioether bond, forming a short and, depending on the metal ion coordination mode, a rigid tether with the protein. These tags exhibit high affinity for both Mn(ii) and Gd(iii) ions. The DEER performance of the 4PSPyMTA and 4PSPyNPDA tags, in complex with Gd(iii) or Mn(ii), was evaluated for three double cysteine mutants of ubiquitin, and the Gd(iii)-Gd(iii) and Mn(ii)-Mn(ii) distance distributions they generated were compared. All three Gd(iii) complexes of the ubiquitin-PyMTA and ubiquitin-PyNPDA conjugates produced similar and expected distance distributions. In contrast, significant variations in the maxima and widths of the distance distributions were observed for the Mn(ii) analogs. Furthermore, whereas PyNPDA-Gd(iii) and PyNPDA-Mn(ii) delivered similar distance distributions, appreciable differences were observed for two mutants with PyMTA, with the Mn(ii) analog exhibiting a broader distance distribution and shorter distances. ELDOR (electron-electron double resonance)-detected NMR measurements revealed some distribution in the Mn(ii) coordination environment for the protein conjugates of both tags but not for the free tags. The broader distance distributions generated by 4PSPyMTA-Mn(ii), as compared with Gd(iii), were attributed to the distributed location of the Mn(ii) ion within the PyMTA chelate owing to its smaller size and lower coordination number that leave the pyridine nitrogen uncoordinated. Accordingly, in terms of distance resolution, 4PSPyNPDA can serve as an effective generic tag for Gd(iii) and Mn(ii), whereas 4PSPyMTA is efficient for Gd(iii) only. This comparison between Gd(iii) and Mn(ii) suggests that PyMTA model compounds may not predict sufficiently well the performance of PyMTA-Mn(ii) as a tag for high-resolution distance measurements in proteins because the protein environment can influence its coordination mode.

Comprehensive quantification of triacylglycerols in soybean seeds by electrospray ionization mass spectrometry with multiple neutral loss scans

DOE PAGES

Li, Maoyin; Butka, Emily; Wang, Xuemin

2014-10-10

Soybean seeds are an important source of vegetable oil and biomaterials. The content of individual triacylglycerol species (TAG) in soybean seeds is difficult to quantify in an accurate and rapid way. The present study establishes an approach to quantify TAG species in soybean seeds utilizing an electrospray ionization tandem mass spectrometry with multiple neutral loss scans. Ten neutral loss scans were performed to detect the fatty acyl chains of TAG, including palmitic (P, 1650), linolenic (Ln, 1853), linoleic (L, 1852), oleic (O, 1851), stearic (S, 1850), eicosadienoic (2052), gadoleic (2051), arachidic (2050), erucic (2251), and behenic (2250). The abundance ofmore » ten fatty acyl chains at 46 TAG masses (mass-to-charge ratio, m/z) were determined after isotopic deconvolution and correction by adjustment factors at each TAG mass. The direct sample infusion and multiple internal standards correction allowed a rapid and accurate quantification of TAG species. Ninety-three TAG species were resolved and their levels were determined.The most abundant TAG species were LLL, OLL, LLLn, PLL, OLLn, OOL, POL, and SLL. Many new species were detected and quantified. As a result, this shotgun lipidomics approach should facilitate the study of TAG metabolism and genetic breeding of soybean seeds for desirable TAG content and composition.« less

A Synthesis of Tagging Studies Examining the Behaviour and Survival of Anadromous Salmonids in Marine Environments

PubMed Central

Drenner, S. Matthew; Clark, Timothy D.; Whitney, Charlotte K.; Martins, Eduardo G.; Cooke, Steven J.; Hinch, Scott G.

2012-01-01

This paper synthesizes tagging studies to highlight the current state of knowledge concerning the behaviour and survival of anadromous salmonids in the marine environment. Scientific literature was reviewed to quantify the number and type of studies that have investigated behaviour and survival of anadromous forms of Pacific salmon (Oncorhynchus spp.), Atlantic salmon (Salmo salar), brown trout (Salmo trutta), steelhead (Oncorhynchus mykiss), and cutthroat trout (Oncorhynchus clarkii). We examined three categories of tags including electronic (e.g. acoustic, radio, archival), passive (e.g. external marks, Carlin, coded wire, passive integrated transponder [PIT]), and biological (e.g. otolith, genetic, scale, parasites). Based on 207 papers, survival rates and behaviour in marine environments were found to be extremely variable spatially and temporally, with some of the most influential factors being temperature, population, physiological state, and fish size. Salmonids at all life stages were consistently found to swim at an average speed of approximately one body length per second, which likely corresponds with the speed at which transport costs are minimal. We found that there is relatively little research conducted on open-ocean migrating salmonids, and some species (e.g. masu [O. masou] and amago [O. rhodurus]) are underrepresented in the literature. The most common forms of tagging used across life stages were various forms of external tags, coded wire tags, and acoustic tags, however, the majority of studies did not measure tagging/handling effects on the fish, tag loss/failure, or tag detection probabilities when estimating survival. Through the interdisciplinary application of existing and novel technologies, future research examining the behaviour and survival of anadromous salmonids could incorporate important drivers such as oceanography, tagging/handling effects, predation, and physiology. PMID:22431962

A synthesis of tagging studies examining the behaviour and survival of anadromous salmonids in marine environments.

PubMed

Drenner, S Matthew; Clark, Timothy D; Whitney, Charlotte K; Martins, Eduardo G; Cooke, Steven J; Hinch, Scott G

2012-01-01

This paper synthesizes tagging studies to highlight the current state of knowledge concerning the behaviour and survival of anadromous salmonids in the marine environment. Scientific literature was reviewed to quantify the number and type of studies that have investigated behaviour and survival of anadromous forms of Pacific salmon (Oncorhynchus spp.), Atlantic salmon (Salmo salar), brown trout (Salmo trutta), steelhead (Oncorhynchus mykiss), and cutthroat trout (Oncorhynchus clarkii). We examined three categories of tags including electronic (e.g. acoustic, radio, archival), passive (e.g. external marks, Carlin, coded wire, passive integrated transponder [PIT]), and biological (e.g. otolith, genetic, scale, parasites). Based on 207 papers, survival rates and behaviour in marine environments were found to be extremely variable spatially and temporally, with some of the most influential factors being temperature, population, physiological state, and fish size. Salmonids at all life stages were consistently found to swim at an average speed of approximately one body length per second, which likely corresponds with the speed at which transport costs are minimal. We found that there is relatively little research conducted on open-ocean migrating salmonids, and some species (e.g. masu [O. masou] and amago [O. rhodurus]) are underrepresented in the literature. The most common forms of tagging used across life stages were various forms of external tags, coded wire tags, and acoustic tags, however, the majority of studies did not measure tagging/handling effects on the fish, tag loss/failure, or tag detection probabilities when estimating survival. Through the interdisciplinary application of existing and novel technologies, future research examining the behaviour and survival of anadromous salmonids could incorporate important drivers such as oceanography, tagging/handling effects, predation, and physiology.

Identifying organism involved in new and regenerated production using TAG-SIP

NASA Astrophysics Data System (ADS)

Morando, M.; Capone, D. G.

2016-02-01

The coupling of stable isotope probing (SIP) with high throughput sequencing (TAG-SIP), allows examination of DNA from individual taxa for the incorporation of a specific isotopically labeled substrate, facilitating an in-depth investigation of the activity and functional diversity of in situ microbial communities. This approach was applied to the monthly San Pedro Ocean Time-series (SPOT), during April of 2014 in order to characterize the organisms involved in new and regenerated production by investigating the assimilation of 15N-NO3-, 15N-NH4+, and 15N-urea at several light depths throughout the euphotic zone. Overall, very little variation was seen between the DNA banding patterns and density of each discrete OTU compared over multiple control treatments, i.e. unlabeled substrate was added to each control and so any disparity between the DNA banding of these OTU replicates reflects methodological variation. The lack of disparity found here further demonstrates TAG-SIP's high precision, accuracy, and more importantly validates the TAG-SIP's reproducibility in both gradient formation and DNA sedimentation with respect to density. The mean density of these discrete control OTU DNA bands (n=7) were then compared to those of their isotopically treated equivalent OTU after a 24h incubation in order to accurately assess and identify significant shifts in DNA density. Therefore we are confident that differences in density between control and treated sample DNA greater than the variation quantified among the controls themselves, is direct evidence of `heavy' isotope incorporation, i.e. metabolic activity and growth. Direct evidence of activity was found in a broad range of taxa, thought not every treatment yielded positive results. As expected the majority of the organisms identified as assimilators were found within the 15N-NH4+ treatments. Many taxa displayed evidence of uptake in one or more but not all treatments providing evidence on which taxa are metabolizing a broad range of substrates as opposed to those which are specializing. Overall, this dataset provides unique insight into which organisms contribute to new biomass formation and ultimately to carbon export and those that help maintain the population through the recycling of nutrients.

Cobalt carbonyl complexes as probes for alkyne-tagged lipids[S

PubMed Central

Tallman, Keri A.; Armstrong, Michelle D.; Milne, Stephen B.; Marnett, Lawrence J.; Brown, H. Alex; Porter, Ned A.

2013-01-01

Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a “tag” that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV349nm. The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity “pull-down” strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection. PMID:23307946

Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.

PubMed

White, Jim F; Grisshammer, Reinhard

2010-09-07

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.

Impact of Chronic Alcohol Ingestion on Cardiac Muscle Protein Expression

PubMed Central

Fogle, Rachel L.; Lynch, Christopher J.; Palopoli, Mary; Deiter, Gina; Stanley, Bruce A.; Vary, Thomas C.

2014-01-01

Background Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved. Methods The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT™). Following the reaction with the ICAT™ reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches. Results Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system. Conclusions Based on the changes in proteins, we speculate modulation of cardiac muscle protein expression represents a fundamental alteration induced by chronic alcohol consumption, consistent with changes in myocardial wall thickness measured under the same conditions. PMID:20477769

Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

PubMed

Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

2015-09-01

The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

Soluble expression and one-step purification of recombinant mouse interferon-λ3 in Escherichia coli.

PubMed

Wang, Y Q; Zhou, M; Zeng, L M; Gao, Q Y; Yuan, X L; Li, Y; Li, M C

2015-02-01

Interferon (IFN)-λ3, a member of the type III IFN family, is a pleiotropic cytokine that exhibits potent antiproliferative, antiviral, and immunoregulatory activities. For further functional study of IFN-λ3, we developed an efficient procedure that includes cloning, expression, and purification to obtain relatively large quantity of mouse IFN-λ3 fusion protein. The mature IFN-λ3 protein-coding region was cloned into the prokaryotic expression vector pET-44. IFN-λ3 contains a hexahistidine tag at its C-terminus. We used Ni(2+)-nitrilotriacetic acid agarose-affinity chromatography to purify the expressed soluble protein. The purified IFN-λ3 inhibited significantly IL-13 production in stimulated RAW264.7 macrophages. Our findings show that the production of soluble IFN-λ3 proteins by the pET-44 vector in Escherichia coli is a good alternative for the production of native IFN-λ3 and could be useful for the production of other IFN proteins.

Tracking the potyviral P1 protein in Nicotiana benthamiana plants during plum pox virus infection.

PubMed

Vozárová, Z; Glasa, M; Šubr, Z W

The P1 protein is derived from the N terminus of potyvirus-coded polyprotein. In addition to the proteolytic activity essential for its maturation, it probably participates in suppression of host defense and/or in virus replication. Clear validation of the P1 in vivo function(s), however, is not yet available. We applied an infectious cDNA clone of plum pox virus (PPV), where the P1 was N-fused with a hexahistidine tag, to trace this protein in Nicotiana benthamiana plants during the PPV infection. Immunoblot analysis with the anti-his antibody showed a diffuse band corresponding to the molecular weight about 70-80 kDa (about twice larger than expected) in the root samples from early stage of infection. This signal culminated on the sixth day post inoculation, later it rapidly disappeared. Sample denaturation by boiling in SDS before centrifugal clarification was essential, indicating strong affinity of P1-his to some plant compound sedimenting with the tissue and cell debris.

Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules.

PubMed

Han, M; Gao, X; Su, J Z; Nie, S

2001-07-01

Multicolor optical coding for biological assays has been achieved by embedding different-sized quantum dots (zinc sulfide-capped cadmium selenide nanocrystals) into polymeric microbeads at precisely controlled ratios. Their novel optical properties (e.g., size-tunable emission and simultaneous excitation) render these highly luminescent quantum dots (QDs) ideal fluorophores for wavelength-and-intensity multiplexing. The use of 10 intensity levels and 6 colors could theoretically code one million nucleic acid or protein sequences. Imaging and spectroscopic measurements indicate that the QD-tagged beads are highly uniform and reproducible, yielding bead identification accuracies as high as 99.99% under favorable conditions. DNA hybridization studies demonstrate that the coding and target signals can be simultaneously read at the single-bead level. This spectral coding technology is expected to open new opportunities in gene expression studies, high-throughput screening, and medical diagnostics.

Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xinxin; Zhang, Min; Takano, Tetsuo

Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membranemore » and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.« less

SwellGel: an affinity chromatography technology for high-capacity and high-throughput purification of recombinant-tagged proteins.

PubMed

Draveling, C; Ren, L; Haney, P; Zeisse, D; Qoronfleh, M W

2001-07-01

The revolution in genomics and proteomics is having a profound impact on drug discovery. Today's protein scientist demands a faster, easier, more reliable way to purify proteins. A high capacity, high-throughput new technology has been developed in Perbio Sciences for affinity protein purification. This technology utilizes selected chromatography media that are dehydrated to form uniform aggregates. The SwellGel aggregates will instantly rehydrate upon addition of the protein sample, allowing purification and direct performance of multiple assays in a variety of formats. SwellGel technology has greater stability and is easier to handle than standard wet chromatography resins. The microplate format of this technology provides high-capacity, high-throughput features, recovering milligram quantities of protein suitable for high-throughput screening or biophysical/structural studies. Data will be presented applying SwellGel technology to recombinant 6x His-tagged protein and glutathione-S-transferase (GST) fusion protein purification. Copyright 2001 Academic Press.

PSBinder: A Web Service for Predicting Polystyrene Surface-Binding Peptides.

PubMed

Li, Ning; Kang, Juanjuan; Jiang, Lixu; He, Bifang; Lin, Hao; Huang, Jian

2017-01-01

Polystyrene surface-binding peptides (PSBPs) are useful as affinity tags to build a highly effective ELISA system. However, they are also a quite common type of target-unrelated peptides (TUPs) in the panning of phage-displayed random peptide library. As TUP, PSBP will mislead the analysis of panning results if not identified. Therefore, it is necessary to find a way to quickly and easily foretell if a peptide is likely to be a PSBP or not. In this paper, we describe PSBinder, a predictor based on SVM. To our knowledge, it is the first web server for predicting PSBP. The SVM model was built with the feature of optimized dipeptide composition and 87.02% (MCC = 0.74; AUC = 0.91) of peptides were correctly classified by fivefold cross-validation. PSBinder can be used to exclude highly possible PSBP from biopanning results or to find novel candidates for polystyrene affinity tags. Either way, it is valuable for biotechnology community.

Affinity monolith-integrated poly(methyl methacrylate) microchips for on-line protein extraction and capillary electrophoresis.

PubMed

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T

2008-07-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with eight reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in a porogenic solvent consisting of 70% 1-dodecanol and 30% cyclohexanol. Antifluorescein isothiocyanate was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of fluorescein isothiocyanate (FITC)-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis.

Affinity Monolith-Integrated Poly(methyl Methacrylate) Microchips for On-Line Protein Extraction and Capillary Electrophoresis

PubMed Central

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T.

2008-01-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with 8 reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene dimethacrylate in a porogenic solvent consisting of 70% dodecanol and 30% hexanol. Anti-fluorescein isothiocyanate (FITC) was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of FITC-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis. PMID:18479142

Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.

PubMed

Trachman, Robert J; Abdolahzadeh, Amir; Andreoni, Alessio; Cojocaru, Razvan; Knutson, Jay R; Ryckelynck, Michael; Unrau, Peter J; Ferré-D'Amaré, Adrian R

2018-05-24

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.

Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

NASA Astrophysics Data System (ADS)

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-08-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

Quantitative Peptidomics with Five-plex Reductive Methylation labels

NASA Astrophysics Data System (ADS)

Tashima, Alexandre K.; Fricker, Lloyd D.

2017-12-01

Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.

Quantitative Peptidomics with Five-plex Reductive Methylation labels

NASA Astrophysics Data System (ADS)

Tashima, Alexandre K.; Fricker, Lloyd D.

2018-05-01

Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.

Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

PubMed

Caruccio, Nicholas

2011-01-01

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

Single-tube, non-isotopic, multiplex PCR/OLA assay and sequence-coded separation for simultaneous screening of 31 cystic fibrosis mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brinson, E.C.; Adriano, T.; Bloch, W.

1994-09-01

We have developed a rapid, single-tube, non-isotopic assay that screens a patient sample for the presence of 31 cystic fibrosis (CF) mutations. This assay can identify these mutations in a single reaction tube and a single electrophoresis run. Sample preparation is a simple, boil-and-go procedure, completed in less than an hour. The assay is composed of a 15-plex PCR, followed by a 61-plex oligonucleotide ligation assay (OLA), and incorporates a novel detection scheme, Sequence Coded Separation. Initially, the multiplex PCR amplifies 15 relevant segments of the CFTR gene, simultaneously. These PCR amplicons serve as templates for the multiplex OLA, whichmore » detects the normal or mutant allele at all loci, simultaneously. Each polymorphic site is interrogated by three oligonucleotide probes, a common probe and two allele-specific probes. Each common probe is tagged with a fluorescent dye, and the competing normal and mutant allelic probes incorporate different, non-nucleotide, mobility modifiers. These modifiers are composed of hexaethylene oxide (HEO) units, incorporated as HEO phosphoramidite monomers during automated DNA synthesis. The OLA is based on both probe hybridization and the ability of DNA ligase to discriminate single base mismatches at the junction between paired probes. Each single tube assay is electrophoresed in a single gel lane of a 4-color fluorescent DNA sequencer (Applied Biosystems, Model 373A). Each of the ligation products is identified by its unique combination of electrophoretic mobility and one of three colors. The fourth color is reserved for the in-lane size standard, used by GENESCAN{sup TM} software (Applied Biosystems) to size the OLA electrophoresis products. The Genotyper{sub TM} software (Applied Biosystems) decodes these Sequence-Coded-Separation data to create a patient summary report for all loci tested.« less

System and method for simultaneously collecting serial number information from numerous identity tags

DOEpatents

Doty, Michael A.

1997-01-01

A system and method for simultaneously collecting serial number information reports from numerous colliding coded-radio-frequency identity tags. Each tag has a unique multi-digit serial number that is stored in non-volatile RAM. A reader transmits an ASCII coded "D" character on a carrier of about 900 MHz and a power illumination field having a frequency of about 1.6 Ghz. A one MHz tone is modulated on the 1.6 Ghz carrier as a timing clock for a microprocessor in each of the identity tags. Over a thousand such tags may be in the vicinity and each is powered-up and clocked by the 1.6 Ghz power illumination field. Each identity tag looks for the "D" interrogator modulated on the 900 MHz carrier, and each uses a digit of its serial number to time a response. Clear responses received by the reader are repeated for verification. If no verification or a wrong number is received by any identity tag, it uses a second digital together with the first to time out a more extended period for response. Ultimately, the entire serial number will be used in the worst case collision environments; and since the serial numbers are defined as being unique, the final possibility will be successful because a clear time-slot channel will be available.

System and method for simultaneously collecting serial number information from numerous identity tags

DOEpatents

Doty, M.A.

1997-01-07

A system and method are disclosed for simultaneously collecting serial number information reports from numerous colliding coded-radio-frequency identity tags. Each tag has a unique multi-digit serial number that is stored in non-volatile RAM. A reader transmits an ASCII coded ``D`` character on a carrier of about 900 MHz and a power illumination field having a frequency of about 1.6 Ghz. A one MHz tone is modulated on the 1.6 Ghz carrier as a timing clock for a microprocessor in each of the identity tags. Over a thousand such tags may be in the vicinity and each is powered-up and clocked by the 1.6 Ghz power illumination field. Each identity tag looks for the ``D`` interrogator modulated on the 900 MHz carrier, and each uses a digit of its serial number to time a response. Clear responses received by the reader are repeated for verification. If no verification or a wrong number is received by any identity tag, it uses a second digital together with the first to time out a more extended period for response. Ultimately, the entire serial number will be used in the worst case collision environments; and since the serial numbers are defined as being unique, the final possibility will be successful because a clear time-slot channel will be available. 5 figs.

Investigation of triaxiality in 54122-128Xe isotopes in the framework of sdg-IBM

NASA Astrophysics Data System (ADS)

Jafarizadeh, M. A.; Ranjbar, Z.; Fouladi, N.; Ghapanvari, M.

In this paper, a transitional interacting boson model (IBM) Hamiltonian in both sd-(IBM) and sdg-IBM versions based on affine SU(1, 1) Lie algebra is employed to describe deviations from the gamma-unstable nature of Hamiltonian along the chain of Xe isotopes. sdg-IBM Hamiltonian proposed a better interpretation of this deviation which cannot be explained in the sd-boson models. The nuclei studied have well-known γ bands close to the γ-unstable limit. The energy levels, B(E2) transition rates and signature splitting of the γ -vibrational band are calculated via the affine SU(1,1) Lie algebra. An acceptable degree of agreement was achieved based on this procedure. It is shown that in these isotopes the signature splitting is better reproduced by the inclusion of sdg-IBM. In none of them, any evidence for a stable, triaxial ground state shape is found.

Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

PubMed

Gans, Jonathan; Osborne, Jonathan; Cheng, Juliet; Djapgne, Louise; Oglesby-Sherrouse, Amanda G

2018-01-01

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

Application of the accurate mass and time tag approach in studies of the human blood lipidome

PubMed Central

Ding, Jie; Sorensen, Christina M.; Jaitly, Navdeep; Jiang, Hongliang; Orton, Daniel J.; Monroe, Matthew E.; Moore, Ronald J.; Smith, Richard D.; Metz, Thomas O.

2008-01-01

We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance. PMID:18502191

Pharmacoproteomics of a Metalloproteinase Hydroxamate Inhibitor in Breast Cancer Cells: Dynamics of Membrane Type 1 Matrix Metalloproteinase-Mediated Membrane Protein Shedding ▿ ‡

PubMed Central

Butler, Georgina S.; Dean, Richard A.; Tam, Eric M.; Overall, Christopher M.

2008-01-01

Broad-spectrum matrix metalloproteinase (MMP) inhibitors (MMPI) were unsuccessful in cancer clinical trials, partly due to side effects resulting from limited knowledge of the full repertoire of MMP substrates, termed the substrate degradome, and hence the in vivo functions of MMPs. To gain further insight into the degradome of MMP-14 (membrane type 1 MMP) an MMPI, prinomastat (drug code AG3340), was used to reduce proteolytic processing and ectodomain shedding in human MDA-MB-231 breast cancer cells transfected with MMP-14. We report a quantitative proteomic evaluation of the targets and effects of the inhibitor in this cell-based system. Proteins in cell-conditioned medium (the secretome) and membrane fractions with levels that were modulated by the MMPI were identified by isotope-coded affinity tag (ICAT) labeling and tandem mass spectrometry. Comparisons of the expression of MMP-14 with that of a vector control resulted in increased MMP-14/vector ICAT ratios for many proteins in conditioned medium, indicating MMP-14-mediated ectodomain shedding. Following MMPI treatment, the MMPI/vehicle ICAT ratio was reversed, suggesting that MMP-14-mediated shedding of these proteins was blocked by the inhibitor. The reduction in shedding or the release of substrates from pericellular sites in the presence of the MMPI was frequently accompanied by the accumulation of the protein in the plasma membrane, as indicated by high MMPI/vehicle ICAT ratios. Considered together, this is a strong predictor of biologically relevant substrates cleaved in the cellular context that led to the identification of many undescribed MMP-14 substrates, 20 of which we validated biochemically, including DJ-1, galectin-1, Hsp90α, pentraxin 3, progranulin, Cyr61, peptidyl-prolyl cis-trans isomerase A, and dickkopf-1. Other proteins with altered levels, such as Kunitz-type protease inhibitor 1 and beta-2-microglobulin, were not substrates in biochemical assays, suggesting an indirect affect of the MMPI, which might be important in drug development as biomarkers or, in preclinical phases, to predict systemic drug actions and adverse side effects. Hence, this approach describes the dynamic pattern of cell membrane ectodomain shedding and its perturbation upon metalloproteinase drug treatment. PMID:18505826

ONE-STEP METAL-AFFINITY PURIFICATION OF HISTIDINE-TAGGED PROTEINS BY TEMPERATURE-TRIGGERED PRECIPITATION. (R829606)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Improvements in the MGA Code Provide Flexibility and Better Error Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruhter, W D; Kerr, J

2005-05-26

The Multi-Group Analysis (MGA) code is widely used to determine nondestructively the relative isotopic abundances of plutonium by gamma-ray spectrometry. MGA users have expressed concern about the lack of flexibility and transparency in the code. Users often have to ask the code developers for modifications to the code to accommodate new measurement situations, such as additional peaks being present in the plutonium spectrum or expected peaks being absent. We are testing several new improvements to a prototype, general gamma-ray isotopic analysis tool with the intent of either revising or replacing the MGA code. These improvements will give the user themore » ability to modify, add, or delete the gamma- and x-ray energies and branching intensities used by the code in determining a more precise gain and in the determination of the relative detection efficiency. We have also fully integrated the determination of the relative isotopic abundances with the determination of the relative detection efficiency to provide a more accurate determination of the errors in the relative isotopic abundances. We provide details in this paper on these improvements and a comparison of results obtained with current versions of the MGA code.« less

Identification of core components and transient interactors of the peroxisomal importomer by dual-track stable isotope labeling with amino acids in cell culture analysis.

PubMed

Oeljeklaus, Silke; Reinartz, Benedikt S; Wolf, Janina; Wiese, Sebastian; Tonillo, Jason; Podwojski, Katharina; Kuhlmann, Katja; Stephan, Christian; Meyer, Helmut E; Schliebs, Wolfgang; Brocard, Cécile; Erdmann, Ralf; Warscheid, Bettina

2012-04-06

The importomer complex plays an essential role in the biogenesis of peroxisomes by mediating the translocation of matrix proteins across the organellar membrane. A central part of this highly dynamic import machinery is the docking complex consisting of Pex14p, Pex13p, and Pex17p that is linked to the RING finger complex (Pex2p, Pex10p, Pex12p) via Pex8p. To gain detailed knowledge on the molecular players governing peroxisomal matrix protein import and, thus, the integrity and functionality of peroxisomes, we aimed at a most comprehensive investigation of stable and transient interaction partners of Pex14p, the central component of the importomer. To this end, we performed a thorough quantitative proteomics study based on epitope tagging of Pex14p combined with dual-track stable isotope labeling with amino acids in cell culture-mass spectrometry (SILAC-MS) analysis of affinity-purified Pex14p complexes and statistics. The results led to the establishment of the so far most extensive Pex14p interactome, comprising 9 core and further 12 transient components. We confirmed virtually all known Pex14p interaction partners including the core constituents of the importomer as well as Pex5p, Pex11p, Pex15p, and Dyn2p. More importantly, we identified new transient interaction partners (Pex25p, Hrr25p, Esl2p, prohibitin) that provide a valuable resource for future investigations on the functionality, dynamics, and regulation of the peroxisomal importomer.

Reactive transport modeling of stable carbon isotope fractionation in a multi-phase multi-component system during carbon sequestration

DOE PAGES

Zhang, Shuo; DePaolo, Donald J.; Zheng, Liange; ...

2014-12-31

Carbon stable isotopes can be used in characterization and monitoring of CO 2 sequestration sites to track the migration of the CO 2 plume and identify leakage sources, and to evaluate the chemical reactions that take place in the CO 2-water-rock system. However, there are few tools available to incorporate stable isotope information into flow and transport codes used for CO 2 sequestration problems. We present a numerical tool for modeling the transport of stable carbon isotopes in multiphase reactive systems relevant to geologic carbon sequestration. The code is an extension of the reactive transport code TOUGHREACT. The transport modulemore » of TOUGHREACT was modified to include separate isotopic species of CO 2 gas and dissolved inorganic carbon (CO 2, CO 3 2-, HCO 3 -,…). Any process of transport or reaction influencing a given carbon species also influences its isotopic ratio. Isotopic fractionation is thus fully integrated within the dynamic system. The chemical module and database have been expanded to include isotopic exchange and fractionation between the carbon species in both gas and aqueous phases. The performance of the code is verified by modeling ideal systems and comparing with theoretical results. Efforts are also made to fit field data from the Pembina CO 2 injection project in Canada. We show that the exchange of carbon isotopes between dissolved and gaseous carbon species combined with fluid flow and transport, produce isotopic effects that are significantly different from simple two-component mixing. These effects are important for understanding the isotopic variations observed in field demonstrations.« less

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

PubMed

Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

2017-03-01

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ocean Heat Content Reveals Secrets of Fish Migrations

PubMed Central

Luo, Jiangang; Ault, Jerald S.; Shay, Lynn K.; Hoolihan, John P.; Prince, Eric D.; Brown, Craig A.; Rooker, Jay R.

2015-01-01

For centuries, the mechanisms surrounding spatially complex animal migrations have intrigued scientists and the public. We present a new methodology using ocean heat content (OHC), a habitat metric that is normally a fundamental part of hurricane intensity forecasting, to estimate movements and migration of satellite-tagged marine fishes. Previous satellite-tagging research of fishes using archival depth, temperature and light data for geolocations have been too coarse to resolve detailed ocean habitat utilization. We combined tag data with OHC estimated from ocean circulation and transport models in an optimization framework that substantially improved geolocation accuracy over SST-based tracks. The OHC-based movement track provided the first quantitative evidence that many of the tagged highly migratory fishes displayed affinities for ocean fronts and eddies. The OHC method provides a new quantitative tool for studying dynamic use of ocean habitats, migration processes and responses to environmental changes by fishes, and further, improves ocean animal tracking and extends satellite-based animal tracking data for other potential physical, ecological, and fisheries applications. PMID:26484541

Designed Proteins as Novel Imaging Reagents in Living Escherichia coli.

PubMed

Pratt, Susan E; Speltz, Elizabeth B; Mochrie, Simon G J; Regan, Lynne

2016-09-02

Fluorescence imaging is a powerful tool to study protein function in living cells. Here, we introduce a novel imaging strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the protein of interest (POI). Our method was based on a set of designed tetratricopeptide repeat affinity proteins (TRAPs) that specifically and reversibly interact with a short, extended peptide tag. We co-expressed the TRAPs fused to fluorescent proteins (FPs) and the peptide tags fused to the POIs. We illustrated the method using the Escherichia coli protein FtsZ and showed that our system could track distinct FtsZ structures under both low and high expression conditions in live cells. We anticipate that our imaging strategy will be a useful tool for imaging the subcellular localization of many proteins, especially those recalcitrant to imaging by direct tagging with FPs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP.

PubMed

Chen, Ying-Hui; Wang, Gao-Yuan; Hao, Hao-Chao; Chao, Chun-Jiang; Wang, Yamei; Jin, Quan-Wen

2017-03-01

GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe , we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale. © 2017. Published by The Company of Biologists Ltd.

Single-step purification and characterization of recombinant aspartase of Aeromonas media NFB-5.

PubMed

Singh, Ram Sarup; Yadav, Mukesh

2012-07-01

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b⁺ expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K₂HPO₄ and 0.3% KH₂PO₄. Recombinant His tagged aspartase was purified using Ni-NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K(m) and V(max) values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5-9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²⁺, Mn²⁺, and Ca²⁺ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.

Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity

PubMed Central

Sastry, M S R; Zhou, Weibin; Baneyx, François

2009-01-01

Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging. PMID:19517531

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

Swarmathon 2017

NASA Image and Video Library

2017-04-19

A Swarmie robot finds a "resource" cube marked with an AprilTag, similar to a barcode. In the Swarmathon competition at the Kennedy Space Center Visitor Complex, students were asked to develop computer code for the small robots, programming them to look for "resources" in the form of cubes with AprilTags.

Evaluation of the accuracy of mono-energetic electron and beta-emitting isotope dose-point kernels using particle and heavy ion transport code system: PHITS.

PubMed

Shiiba, Takuro; Kuga, Naoya; Kuroiwa, Yasuyoshi; Sato, Tatsuhiko

2017-10-01

We assessed the accuracy of mono-energetic electron and beta-emitting isotope dose-point kernels (DPKs) calculated using the particle and heavy ion transport code system (PHITS) for patient-specific dosimetry in targeted radionuclide treatment (TRT) and compared our data with published data. All mono-energetic and beta-emitting isotope DPKs calculated using PHITS, both in water and compact bone, were in good agreement with those in literature using other MC codes. PHITS provided reliable mono-energetic electron and beta-emitting isotope scaled DPKs for patient-specific dosimetry. Copyright © 2017 Elsevier Ltd. All rights reserved.

Geochemistry and mineralogy of kimberlites from the Arkhangelsk Region, NW Russia: evidence for transitional kimberlite magma types

NASA Astrophysics Data System (ADS)

Beard, A. D.; Downes, H.; Hegner, E.; Sablukov, S. M.

2000-03-01

The Arkhangelsk kimberlite province (AKP) is situated in the north of the Baltic Shield within the buried southeastern portion of the Kola-Kuloi craton. It forms part of the extensive Devonian magmatic event of the northern Baltic Shield and Kola Peninsula. Two main groups of kimberlites can be distinguished within the province: (1) kimberlites from the diamondiferous Zolotitsa field that have geochemical and isotopic affinities with Group 2 kimberlites and lamproites; (2) diamond-poor Ti-Fe-rich kimberlites from other Arkhangelsk fields that have geochemical and isotopic affinities with Group 1 kimberlites. However, the Zolotitsa and Ti-Fe-rich kimberlites have mineralogical characteristics that are not typical for their respective assigned kimberlite group classifications. Both groups of Arkhangelsk kimberlites are apparently transitional to Group 1 kimberlites, Group 2 kimberlites and lamproites as they are defined elsewhere in the world. An associated kimberlite from the Mela Sill Complex has strong affinities with carbonatites. The low Al 2O 3, high Ni and Cr contents, and high Mg# in both groups of kimberlites indicate strongly depleted lherzolitic-harzburgitic mantle sources. Trace element patterns show a variable enrichment of incompatible elements and strong LREE enrichment. However, kimberlites from the Zolotitsa field have overall lower trace element abundances and less steep REE patterns, suggesting a higher degree of partial melt and/or a less enriched source compared to that of the Ti-Fe-rich kimberlites. A calciocarbonatite of the Mela Sill Complex has trace element and REE patterns typical of other carbonatites closely associated with kimberlites. 87Sr/ 86Sri and 143Nd/ 144Ndi isotope compositions of the Arkhangelsk kimberlites and carbonatite reveal that at least two mantle sources are required to explain the isotopic variation: (1) most of the Zolotitsa and Mela kimberlites and the Mela carbonatite are derived from an ancient enriched lithospheric source (EMI); (2) the Ti-Fe-rich kimberlites are derived from a plume-related asthenospheric mantle source with an isotopic composition close to Bulk Earth. Present-day Pb isotope compositions reveal that the Zolotitsa kimberlites have values close to Group 1 kimberlites. However, the Ti-Fe-rich kimberlites generally have slightly more radiogenic Pb isotope values.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

Rhenium-osmium and samarium-neodymium isotopic systematics of the stillwater complex

USGS Publications Warehouse

Lambert, D.D.; Morgan, J.W.; Walker, R.J.; Shirey, S.B.; Carlson, R.W.; Zientek, M.L.; Koski, M.S.

1989-01-01

Isotopic data for the Stillwater Complex, Montana , which formed about 2700 Ma (million years ago), were obtained to evaluate the role of magma mixing in the formation of strategic platinum-group element (PGE) ore deposits. Neodymium and osmium isotopic data indicate that the intrusion formed from at least two geochemically distinct magmas. Ultramafic affinity (U-type) magmas had initial ??Nd of -0.8 to -3.2 and a chondritic initial 187Os/186Os ratio of ???0.88, whereas anorthositic affinity (A-type) magmas had ??Nd of -0.7 to +1.7 and an initial 187Os/186Os ratio of ???1.13. These data suggest that U-type magmas were derived from a lithospheric mantle source containing recycled crustal materials whereas A-type magmas originated either by crustal contamination of basaltic magmas or by partial melting of basalt in the lower crust. The Nd and Os isotopic data also suggest that Os, and probably the other PGEs in ore horizons such as the J-M Reef, was derived from A-type magmas. The Nd and Os isotopic heterogeneity observed in rocks below the J-M Reef also suggests that A-type magmas were injected into the Stillwater U-type magma chamber at several stages during the development of the Ultramafic series.

Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein.

PubMed

Monteiro, Rose A; Souza, Emanuel M; Geoffrey Yates, M; Steffens, M Berenice R; Pedrosa, Fábio O; Chubatsu, Leda S

2003-02-01

The Herbaspirillum seropedicae NifA protein is responsible for nif gene expression. The C-terminal domain of the H. seropedicae NifA protein, fused to a His-Tag sequence (His-Tag-C-terminal), was over-expressed and purified by metal-affinity chromatography to yield a highly purified and active protein. Band-shift assays showed that the NifA His-Tag-C-terminal bound specifically to the H. seropedicae nifB promoter region in vitro. In vivo analysis showed that this protein inhibited the Central + C-terminal domains of NifA protein from activating the nifH promoter of K. pneumoniae in Escherichia coli, indicating that the protein must be bound to the NifA-binding site (UAS site) at the nifH promoter region to activate transcription. Copyright 2002 Elsevier Science (USA)

Comparison of magnetic carboxymethyl chitosan nanoparticles and cation exchange resin for the efficient purification of lysine-tagged small ubiquitin-like modifier protease.

PubMed

Li, Junhua; Zhang, Yang; Shen, Fei; Yang, Yanjun

2012-10-15

A fusion tag that can be purified by the cheap ion-exchanger based on the ionic binding force may provide a cost-effective scheme over other affinity fusion tags. Small ubiquitin-like modifier (SUMO) protease derived from Saccharomyces cerevisiae was fused with a poly lysine tag containing 10 lysine residues at its C-terminus and then expressed in Escherichia coli. The ionic binding force provided by the ploy lysine tag allowed the selective recovery of the small ubiquitin-like modifier protease from recombinant E. coli cell extracts. A preliminary comparative study of the adsorption and elution of poly lysine tagged SUMO protease on Amberlite Cobalamion and magnetite carboxymethyl chitosan nanoparticles was performed. Amberlite Cobalamion and magnetite nanoparticles had the similar elution profile due to the common functional groups - carboxyl groups. The maximum dynamic adsorption capacity of Amberlite Cobalamion and magnetite nanoparticles reached 36.8 and 211.4 mg/g, respectively. The lysine-tagged protease can be simply purified by magnetite nanoparticles from cell extracts with higher purity than that by Amberlite Cobalamion. The superparamagnetic nanoparticles possess the advantages of highly specific, fast and excellent binding of a larger amount of lysine tagged SUMO modifier protease, and it is also easier to separate from the crude biological process liquors compared with the conventional separation techniques of polycationic amino acids fusion proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

Recoveries of rat lymph FA after administration of specific structured 13C-TAG.

PubMed

Vistisen, Bodil; Mu, Huiling; Høy, Carl-Erik

2003-09-01

The potential of the specific structured TAG MLM [where M = caprylic acid (8:0) and L = linoleic acid (18:2n-6)] is the simultaneous delivery of energy and EFA. Compared with long-chain TAG (LLL), they may be more rapidly hydrolyzed and absorbed. This study examined the lymphatic recoveries of intragastrically administered L*L*L*, M*M*M*, ML*M, and ML*L* (where * = 13C-labeled FA) in rats. Lymph lipids were separated into lipid classes and analyzed by GC combustion isotope ratio MS. The recoveries of lymph TAG 18:2n-6 8 h after administration of L*L*L*, ML*M, and ML*L* were 38.6, 48.4, and 49.1%, respectively, whereas after 24 h the recoveries were approximately 50% in all experimental groups. The exogenous contribution to lymph TAG 18:2n-6 was approximately 80 and 60% at maximum absorption of the specific structured TAG and L*L*L*, respectively, 3-6 h after administration. The tendency toward more rapid recovery of exogenous long-chain FA following administration of specific structured TAG compared with long-chain TAG was probably due to fast hydrolysis. The lymphatic recovery of 8:0 was 2.2% 24 h after administration of M*M*M*. This minor lymphatic recovery of exogenous 8:0 was probably due to low stimulation of chylomicron formation. These results demonstrate tendencies toward faster lymphatic recovery of long-chain FA after administration of specific structured TAG compared with long-chain TAG.

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side-by-side.

PubMed

Linscheid, Michael W

2018-03-30

To understand biological processes, not only reliable identification, but quantification of constituents in biological processes play a pivotal role. This is especially true for the proteome: protein quantification must follow protein identification, since sometimes minute changes in abundance tell the real tale. To obtain quantitative data, many sophisticated strategies using electrospray and MALDI mass spectrometry (MS) have been developed in recent years. All of them have advantages and limitations. Several years ago, we started to work on strategies, which are principally capable to overcome some of these limits. The fundamental idea is to use elemental signals as a measure for quantities. We began by replacing the radioactive 32 P with the "cold" natural 31 P to quantify modified nucleotides and phosphorylated peptides and proteins and later used tagging strategies for quantification of proteins more generally. To do this, we introduced Inductively Coupled Plasma Mass Spectrometry (ICP-MS) into the bioanalytical workflows, allowing not only reliable and sensitive detection but also quantification based on isotope dilution absolute measurements using poly-isotopic elements. The detection capability of ICP-MS becomes particularly attractive with heavy metals. The covalently bound proteins tags developed in our group are based on the well-known DOTA chelate complex (1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid) carrying ions of lanthanoides as metal core. In this review, I will outline the development of this mutual assistance between molecular and elemental mass spectrometry and discuss the scope and limitations particularly of peptide and protein quantification. The lanthanoide tags provide low detection limits, but offer multiplexing capabilities due to the number of very similar lanthanoides and their isotopes. With isotope dilution comes previously unknown accuracy. Separation techniques such as electrophoresis and HPLC were used and just slightly adapted workflows, already in use for quantification in bioanalysis. Imaging mass spectrometry (MSI) with MALDI and laser ablation ICP-MS complemented the range of application in recent years. © 2018 Wiley Periodicals, Inc.

A new pH-responsive peptide tag for protein purification.

PubMed

Nonaka, Takahiro; Tsurui, Noriko; Mannen, Teruhisa; Kikuchi, Yoshimi; Shiraki, Kentaro

2018-06-01

This paper describes a new pH-responsive peptide tag that adds a protein reversible precipitation and redissolution character. This peptide tag is a part of a cell surface protein B (CspB) derived from Corynebacterium glutamicum. Proinsulin that genetically fused with a peptide of N-terminal 6, 17, 50, or 250 amino acid residues of CspB showed that the reversible precipitation and redissolution depended on the pH. The transition occurred within a physiological and narrow pH range. A CspB50 tag comprising 50 amino acid residues of N-terminal CspB was further evaluated as a representative using other pharmaceutical proteins. Below pH 6.8, almost all CspB50-Teriparatide fusion formed an aggregated state. Subsequent addition of alkali turned the cloudy protein solution transparent above pH 7.3, in which almost all the CspB50-Teriparatide fusion redissolved. The CspB50-Bivalirudin fusion showed a similar behavior with slightly different pH range. This tag is offering a new protein purification method based on liquid-solid separation which does not require an affinity ligand. This sharp response around neutral pH is useful as a pH-responsive tag for the purification of unstable proteins at a non-physiological pH. Copyright © 2018 Elsevier Inc. All rights reserved.

Muon capture on light isotopes in Double Chooz

NASA Astrophysics Data System (ADS)

Strait, M.; Double Chooz Collaboration

2017-09-01

Using the Double Chooz reactor neutrino detector, we have measured the products of µ - capture on 12C, 13C, 14N and 16O. Over a period of 490 days, we collected 2.3 × 106 stopping cosmic µ -, of which 1.8 × 105 captured on these nuclei in the inner detector. The resulting isotopes were tagged using prompt neutron emission (when applicable), the subsequent beta decays, and, in some cases, β-delayed neutrons. Production of these βn isotopes, primarily 9Li, which are {{{ν _e}} \\over {{ν _μ }}} backgrounds, was found at a significance of 5.5σ. The probability of 9Li production per capture on natC is (2.4 ± 0.9(stat) ± 0.1(syst)) × 10-4. We have made the most precise measurement of the rate of 12C(µ -, ν)12B to date, 6.57 - 0.21 + 0.11 × {10^3}{{ }}{{{s}} - 1},{{ or }}≤ft( {17.35 - 0.59 + 0.35} \\right)% of nuclear captures. By tagging excited states emitting gammas, the ground state transition rate to 12B is found to be 5.68 - 0.23 + 0.14 × {10^3}{{ }}{{{s}} - 1}.

Stable isotope labelling methods in mass spectrometry-based quantitative proteomics.

PubMed

Chahrour, Osama; Cobice, Diego; Malone, John

2015-09-10

Mass-spectrometry based proteomics has evolved as a promising technology over the last decade and is undergoing a dramatic development in a number of different areas, such as; mass spectrometric instrumentation, peptide identification algorithms and bioinformatic computational data analysis. The improved methodology allows quantitative measurement of relative or absolute protein amounts, which is essential for gaining insights into their functions and dynamics in biological systems. Several different strategies involving stable isotopes label (ICAT, ICPL, IDBEST, iTRAQ, TMT, IPTL, SILAC), label-free statistical assessment approaches (MRM, SWATH) and absolute quantification methods (AQUA) are possible, each having specific strengths and weaknesses. Inductively coupled plasma mass spectrometry (ICP-MS), which is still widely recognised as elemental detector, has recently emerged as a complementary technique to the previous methods. The new application area for ICP-MS is targeting the fast growing field of proteomics related research, allowing absolute protein quantification using suitable elemental based tags. This document describes the different stable isotope labelling methods which incorporate metabolic labelling in live cells, ICP-MS based detection and post-harvest chemical label tagging for protein quantification, in addition to summarising their pros and cons. Copyright © 2015 Elsevier B.V. All rights reserved.

Supporting Situated Learning Based on QR Codes with Etiquetar App: A Pilot Study

ERIC Educational Resources Information Center

Camacho, Miguel Olmedo; Pérez-Sanagustín, Mar; Alario-Hoyos, Carlos; Soldani, Xavier; Kloos, Carlos Delgado; Sayago, Sergio

2014-01-01

EtiquetAR is an authoring tool for supporting the design and enactment of situated learning experiences based on QR tags. Practitioners use etiquetAR for creating, managing and personalizing collections of QR codes with special properties: (1) codes can have more than one link pointing at different multimedia resources, (2) codes can be updated…

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

PubMed

Zhang, Huang-Ge; Xie, Jinfu; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Hsu, Hui-Chen; Mountz, John D

2002-12-01

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

PubMed

Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

2015-02-01

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of selected binding domains for the analysis of ubiquitinated proteomes

PubMed Central

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-01-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising, but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ∼200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle. PMID:23649778

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

PubMed

Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

2017-05-13

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

Electronic tagging and integrated product intelligence

NASA Astrophysics Data System (ADS)

Swerdlow, Martin; Weeks, Brian

1996-03-01

The advent of 'intelligent,' electronic data bearing tags is set to revolutionize the way industrial and retail products are identified and tracked throughout their life cycles. The dominant system for unique identification today is the bar code, which is based on printed symbology and regulated by the International Article Numbering Association. Bar codes provide users with significant operational advantages and generate considerable added value to packaging companies, product manufacturers, distributors and retailers, across supply chains in many different sectors, from retailing, to baggage handling and industrial components, e.g., for vehicles or aircraft. Electronic tags offer the potential to: (1) record and store more complex data about the product or any modifications which occur during its life cycle; (2) access (and up-date) stored data in real time in a way which does not involve contact with the product or article; (3) overcome the limitations imposed by systems which rely on line-of-sight access to stored data. Companies are now beginning to consider how electronic data tags can be used, not only to improve the efficiency of their supply chain processes, but also to revolutionize the way they do business. This paper reviews the applications and business opportunities for electronic tags and outlines CEST's strategy for achieving an 'open' standard which will ensure that tags from different vendors can co-exist on an international basis.

The chemical basis for the origin of the genetic code and the process of protein synthesis

NASA Technical Reports Server (NTRS)

1982-01-01

The major thrust is to understand just how the process of protein synthesis, including that very important aspect, genetic coding, came to be. Two aspects of the problem: the chemistry of active aminoacyl species; and affinities between amino acids and nucleotides, and specifically, how these affinities might affect the chemistry between the two are stressed.

Shielding from space radiations

NASA Technical Reports Server (NTRS)

Chang, C. Ken; Badavi, Forooz F.; Tripathi, Ram K.

1993-01-01

This Progress Report covering the period of December 1, 1992 to June 1, 1993 presents the development of an analytical solution to the heavy ion transport equation in terms of Green's function formalism. The mathematical development results are recasted into a highly efficient computer code for space applications. The efficiency of this algorithm is accomplished by a nonperturbative technique of extending the Green's function over the solution domain. The code may also be applied to accelerator boundary conditions to allow code validation in laboratory experiments. Results from the isotopic version of the code with 59 isotopes present for a single layer target material, for the case of an iron beam projectile at 600 MeV/nucleon in water is presented. A listing of the single layer isotopic version of the code is included.

HaloTag technology for specific and covalent labeling of fusion proteins.

PubMed

Benink, Hélène A; Urh, Marjeta

2015-01-01

Appending proteins of interest to fluorescent protein tags such as GFP has revolutionized how proteins are studied in the cellular environment. Over the last few decades many varieties of fluorescent proteins have been generated, each bringing new capability to research. However, taking full advantage of standard fluorescent proteins with advanced and differential features requires significant effort on the part of the researcher. This approach necessitates that many genetic fusions be generated and confirmed to function properly in cells with the same protein of interest. To lessen this burden, a newer category of protein fusion tags termed "self-labeling protein tags" has been developed. This approach utilizes a single protein tag, the function of which can be altered by attaching various chemical moieties (fluorescent labels, affinity handles, etc.). In this way a single genetically encoded protein fusion can easily be given functional diversity and adaptability as supplied by synthetic chemistry. Here we present protein labeling methods using HaloTag technology; comprised of HaloTag protein and the collection of small molecules designed to bind it specifically and provide it with varied functionalities. For imaging purposes these small molecules, termed HaloTag ligands, contain distinct fluorophores. Due to covalent and rapid binding between HaloTag protein and its ligands, labeling is permanent and efficient. Many of these ligands have been optimized for permeability across cellular membranes allowing for live cell labeling and imaging analysis. Nonpermeable ligands have also been developed for specific labeling of surface proteins. Overall, HaloTag is a versatile technology that empowers the end user to label a protein of interest with the choice of different fluorophores while alleviating the need for generation of multiple genetic fusions.

Effect of Isotope Mass in Simulations of JET H-mode Discharges

NASA Astrophysics Data System (ADS)

Snyder, S. E.; Onjun, T.; Kritz, A. H.; Bateman, G.; Parail, V.

2004-11-01

In JET type-I ELMy H-mode discharges, it is found that the height of the pressure pedestal increases and the frequency of the ELMs decreases with increasing isotope mass. These experimentally observed trends are obtained in these simulations only if the pedestal width increases with isotope mass. Simulations are carried out using the JETTO integrated modeling code with a dynamic model for the H-mode pedestal and the ELMs.(T. Onjun et al, Phys. Plasmas 11 (2004) 1469 and 3006.) The HELENA and MISHKA stability codes are applied to calibrate the stability criteria used to trigger ELM crashes in the JETTO code and to explore possible access to second stability in the pedestal. In the simulations, transport in the pedestal is given by the ion thermal neoclassical diffusivity, which increases with isotope mass. Consequently, as the isotope mass is increased, the pressure gradient and the bootstrap current in the pedestal rebuild more slowly after each ELM crash. Several models are explored in which the pedestal width increases with isotope mass.

A two-step strategy to visually identify molecularly imprinted polymers for tagged proteins.

PubMed

Brandis, Alexander; Partouche, Eran; Yechezkel, Tamar; Salitra, Yoseph; Shkoulev, Vladimir; Scherz, Avigdor; Grynszpan, Flavio

2017-08-01

A practical and relatively simple method to identify molecularly imprinted polymers capable of binding proteins via the molecular tagging (epitope-like) approach has been developed. In our two-step method, we first challenge a previously obtained anti-tag molecularly imprinted polymer with a small molecule including the said tag of choice (a biotin derivative as shown here or other) connected to a linker bound to a second biotin moiety. An avidin molecule partially decorated with fluorescent labels is then allowed to bind the available biotin derivative associated with the polymer matrix. At the end of this simple process, and after washing off all the low-affinity binding molecules from the polymer matrix, only suitable molecularly imprinted polymers binding avidin through its previously acquired small molecule tag (or epitope-like probe, in a general case) will remain fluorescent. For confirmation, we tested the selective performance of the anti-biotin molecularly imprinted polymer binding it to biotinylated alkaline phosphatase. Residual chemical activity of the enzyme on the molecularly imprinted polymer solid support was observed. In all cases, the corresponding nonimprinted polymer controls were inactive. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of Fe3O4@nickel-silicate core-shell nanoparticles for His-tagged enzyme immobilizing agents

NASA Astrophysics Data System (ADS)

Shin, Moo-Kwang; Kang, Byunghoon; Yoon, Nam-Kyung; Kim, Myeong-Hoon; Ki, Jisun; Han, Seungmin; Ahn, Jung-Oh; Haam, Seungjoo

2016-12-01

Immobilizing enzymes on artificially fabricated carriers for their efficient use and easy removal from reactants has attracted enormous interest for decades. Specifically, binding platforms using inorganic nanoparticles have been widely explored because of the benefits of their large surface area, easy surface modification, and high stability in various pH and temperatures. Herein, we fabricated Fe3O4 encapsulated ‘sea-urchin’ shaped nickel-silicate nanoparticles with a facile synthetic route. The enzymes were then rapidly and easily immobilized with poly-histidine tags (His-tags) and nickel ion affinity. Porous nickel silicate covered nanoparticles achieved a high immobilization capacity (85 μg mg-1) of His-tagged tobacco etch virus (TEV) protease. To investigate immobilized TEV protease enzymatic activity, we analyzed the cleaved quantity of maltose binding protein-exendin-fused immunoglobulin fusion protein, which connected with the TEV protease-specific cleavage peptide sequence. Moreover, TEV protease immobilized nanocomplexes conveniently removed and recollected from the reactant by applying an external magnetic field, maintained their enzymatic activity after reuse. Therefore, our newly developed nanoplatform for His-tagged enzyme immobilization provides advantageous features for biotechnological industries including recombinant protein processing.

Rb-Sr isotopic studies of postorogenic granites from the eastern Arabian Shield, Kingdom of Saudi Arabia

USGS Publications Warehouse

Stuckless, J.S.; Futa, Kiyoto

1987-01-01

Available data indicate that postorogenic granites tend to be older in the southern part of the Arabian Shield. This suggests that plutonism started in the south and progressed to the north. Initial 87Sr/86Sr values also form a regional pattern. These ratios tend to be higher in the eastern part of the Arabian Shield, and suggest one source of continental affinity to the east and one of oceanic affinity to the west. The distribution of initial strontium isotope ratios does not clearly discriminate between the various models for Shield evolution; however, a sedimentary source region of mixed end members seems more compatible with the data pattern than models based on discrete boundaries between unrelated accreted blocks.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent

2007-10-01

Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosismore » mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.« less

Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1.

PubMed

Tirard, Marilyn; Brose, Nils

2016-01-01

Protein SUMOylation is a posttranslational protein modification that is emerging as a key regulatory process in neurobiology. To date, however, SUMOylation in vivo has only been studied cursorily. Knock-in mice expressing His6-HA-SUMO1 from the Sumo1 locus allow for the highly specific localization and identification of endogenous SUMO1 substrates under physiological and pathophysiological conditions. By making use of the HA-tag and using wild-type mice for highly stringent negative control samples, SUMO1 targets can be specifically localized in and purified from cultured mouse nerve cells and mouse tissues.

Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements

PubMed Central

2016-01-01

Proteomics is presently dominated by the “bottom-up” strategy, in which proteins are enzymatically digested into peptides for mass spectrometric identification. Although this approach is highly effective at identifying large numbers of proteins present in complex samples, the digestion into peptides renders it impossible to identify the proteoforms from which they were derived. We present here a powerful new strategy for the identification of proteoforms and the elucidation of proteoform families (groups of related proteoforms) from the experimental determination of the accurate proteoform mass and number of lysine residues contained. Accurate proteoform masses are determined by standard LC–MS analysis of undigested protein mixtures in an Orbitrap mass spectrometer, and the lysine count is determined using the NeuCode isotopic tagging method. We demonstrate the approach in analysis of the yeast proteome, revealing 8637 unique proteoforms and 1178 proteoform families. The elucidation of proteoforms and proteoform families afforded here provides an unprecedented new perspective upon proteome complexity and dynamics. PMID:26941048

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

PubMed Central

Tulloch, Lindsay B.; Menzies, Stefanie K.; Fraser, Andrew L.; Gould, Eoin R.; King, Elizabeth F.; Zacharova, Marija K.; Florence, Gordon J.

2017-01-01

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model. PMID:28873407

Feasibility of ultra-wideband SAW RFID tags meeting FCC rules.

PubMed

Härmä, Sanna; Plessky, Victor P; Li, Xianyi; Hartogh, Paul

2009-04-01

We discuss the feasibility of surface acoustic wave (SAW) radio-frequency identification (RFID) tags that rely on ultra-wideband (UWB) technology. We propose a design of a UWB SAW tag, carry out numerical experiments on the device performance, and study signal processing in the system. We also present experimental results for the proposed device and estimate the potentially achievable reading distance. UWB SAW tags will have an extremely small chip size (<0.5 x 1 mm(2)) and a low cost. They also can provide a large number of different codes. The estimated read range for UWB SAW tags is about 2 m with a reader radiating as low as <0.1 mW power levels with an extremely low duty factor.

Phase modulation in RF tag

DOEpatents

Carrender, Curtis Lee; Gilbert, Ronald W.

2007-02-20

A radio frequency (RF) communication system employs phase-modulated backscatter signals for RF communication from an RF tag to an interrogator. The interrogator transmits a continuous wave interrogation signal to the RF tag, which based on an information code stored in a memory, phase-modulates the interrogation signal to produce a backscatter response signal that is transmitted back to the interrogator. A phase modulator structure in the RF tag may include a switch coupled between an antenna and a quarter-wavelength stub; and a driver coupled between the memory and a control terminal of the switch. The driver is structured to produce a modulating signal corresponding to the information code, the modulating signal alternately opening and closing the switch to respectively decrease and increase the transmission path taken by the interrogation signal and thereby modulate the phase of the response signal. Alternatively, the phase modulator may include a diode coupled between the antenna and driver. The modulating signal from the driver modulates the capacitance of the diode, which modulates the phase of the response signal reflected by the diode and antenna.

Peptide library synthesis on spectrally encoded beads for multiplexed protein/peptide bioassays

NASA Astrophysics Data System (ADS)

Nguyen, Huy Q.; Brower, Kara; Harink, Björn; Baxter, Brian; Thorn, Kurt S.; Fordyce, Polly M.

2017-02-01

Protein-peptide interactions are essential for cellular responses. Despite their importance, these interactions remain largely uncharacterized due to experimental challenges associated with their measurement. Current techniques (e.g. surface plasmon resonance, fluorescence polarization, and isothermal calorimetry) either require large amounts of purified material or direct fluorescent labeling, making high-throughput measurements laborious and expensive. In this report, we present a new technology for measuring antibody-peptide interactions in vitro that leverages spectrally encoded beads for biological multiplexing. Specific peptide sequences are synthesized directly on encoded beads with a 1:1 relationship between peptide sequence and embedded code, thereby making it possible to track many peptide sequences throughout the course of an experiment within a single small volume. We demonstrate the potential of these bead-bound peptide libraries by: (1) creating a set of 46 peptides composed of 3 commonly used epitope tags (myc, FLAG, and HA) and single amino-acid scanning mutants; (2) incubating with a mixture of fluorescently-labeled antimyc, anti-FLAG, and anti-HA antibodies; and (3) imaging these bead-bound libraries to simultaneously identify the embedded spectral code (and thus the sequence of the associated peptide) and quantify the amount of each antibody bound. To our knowledge, these data demonstrate the first customized peptide library synthesized directly on spectrally encoded beads. While the implementation of the technology provided here is a high-affinity antibody/protein interaction with a small code space, we believe this platform can be broadly applicable to any range of peptide screening applications, with the capability to multiplex into libraries of hundreds to thousands of peptides in a single assay.

Metal isotope coded profiling of organic ligands by mass spectrometry in aquatic environments

NASA Astrophysics Data System (ADS)

Wichard, Thomas; Deicke, Michael; Frieder Mohr, Jan; Klein, Martin

2017-04-01

Metal isotope coded profiling (MICP) introduces a universal discovery platform for metal chelating natural products that act as metallophores, ion buffers or sequestering agents. The detection of cation and oxoanion complexing ligands is facilitated by the identification of unique isotopic signatures created by the application of isotopically pure metals. We present a targeted analysis of low-molecular-weight organic ligands based on fast UHPLC-ESI-MS measurements. Replacement of, for example, natural iron or molybdenum with isotopically pure 54Fe/58Fe (ratio 1:1) or 95Mo/98Mo (ratio 1:1) causes easily detectable unique isotopic signatures in the mass spectra of potential metal-complexing ligands. This can be achieved under laboratory conditions not only in growth media, but also by spiking directly aqueous samples or solid-phase extracts. Importantly, as the relative affinity of the metallophores for e.g., Mo or Fe is dependent on the pH, all experiments needs to be conducted under pH-controlled conditions. The improved ionization efficiency of some metal complexes helps to enhance the signal-to-noise ratio compared to the free ligand at the same chromatographic conditions. The methodology does not necessarily depend on HR-ESI-MS measurements (e.g., Q-Exactive Orbitrap) and can be applied to any mass spectrometer. With MICP, two birds can be killed with one stone: (i) the identification of metallophores (e.g., siderophores, molybdophores) for metal uptake by any organism and (ii) organic ligands which solely work as metal buffer in dissolved organic matter (DOM). We currently address following two main research lines: First, DOM has often been used as a proxy for bio-productivity in terms of a carbon source; however, the specific impact of DOM as a "metal buffer" for biological processes is still under-investigated. Upon the administration of individual isotopes or isotopic pairs, for example, 54Fe/58Fe, 63Cu/65Cu, 66Zn/68Zn, or 95Mo/98Mo and subsequent measurements by mass spectrometry, organic ligands can be identified that contribute potentially to the regulation of bioavailability and toxicity of metals in the water column. Secondly, metal acquisition by ligand exchange processes is vital for macroalgae-bacteria interactions. Both alga and bacteria contribute to the "soup of organic ligands" which potentially increases the bioavailability of metals due to various ligand exchanges with DOM. In particular, we will outline the analytical process resulting in the identification of ligands in the chemosphere of the marine macroalga Ulva (Chlorophyta) and its associated bacteria. In summary, the identification of metal organic ligands in natural samples will pave the way for understanding the organic ligand-mediated metal acquisition shuttled from DOM to alga. (References: Wichard T. (2016) Front. Mar. Sci. 3:131; Deicke M., Mohr J.M., Wichard T. (2014) Analyst, 139, 6096-6099)

Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

NASA Astrophysics Data System (ADS)

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-coordinating function impaired was used in replace of the original full length dockerin domain. The truncated dockerin domain maintained its functionality as an effective affinity tag, and efficient EDTA mediated dissociation of the bound dockerin-intein tag was also realized. The regenerated ELP capturing scaffold was reused for additional purification cycles without any decrease in efficiency. The second objective was to assemble biocatalysts for biofuel cells. Three beta-NAD dependent dehydrogenases, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FALDH) and formate dehydrogenase (FDH), were site-specifically co-localized onto the scaffolds displayed on the yeast surface based on the high-affinity interactions between three orthogonal cohesin/dockerin pairs. The assembled multi-enzyme cascades, which can completely convert methanol to CO2, showed improved production yield compared with that of the non-complexed enzyme mixture, indicating efficient substrate channeling among the three enzymes. This strategy can be easily extended to other complex cascade reactions for enzymatic fuel cell applications. To further explore the role of biotechnology toward environmental sustainability, Escherichia coli was engineered to express phytochelatin synthase, which converted glutathione into the metal-binding peptide phytochelatin (PC). PCs served as peptide scaffolds and mediated synthesis of CdS nanocrystals. This approach may be generalized to guide the in vitro self-assembly of a wide range of nanocrystals with different compositions and sizes.

Dynamic optical tags

NASA Astrophysics Data System (ADS)

Griggs, Steven P.; Mark, Martin B.; Feldman, Barry J.

2004-07-01

The goal of the DARPA Dynamic Optical Tags (DOTs) program is to develop a small, robust, persistent, 2-way tagging, tracking and locating device that also supports communications at data rates greater than 100 kbps and can be interrogated at significant range. These tags will allow for two-way data exchange and tagging operations in friendly and denied areas. The DOTs will be passive and non-RF. To accomplish this, the DOTs program will develop small, thin, retro-reflecting modulators. The tags will operate for long periods of time (greater than two months) in real-world environmental conditions (-40° to +70° C) and allow for a wide interrogation angle (+/-60°). The tags will be passive (in the sleep mode) for most of the time and only become active when interrogated by a laser with the correct code. Once correctly interrogated, the tags will begin to modulate and retro-reflect the incoming beam. The program will also develop two tag specific transceiver systems that are eye-safe, employ automated scanning algorithms, and are capable of short search and interrogate times.

Simultaneous quantification of endogenous and exogenous plasma glucose by isotope dilution LC-MS/MS with indirect MRM of the derivative tag.

PubMed

Yu, Lingling; Wen, Chao; Li, Xing; Fang, Shiqi; Yang, Lichuan; Wang, Tony; Hu, Kaifeng

2018-03-01

Quantification of endogenous and exogenous plasma glucose can help more comprehensively evaluate the glucose metabolic status. A ratio-based approach using isotope dilution liquid chromatography tandem mass spectrometry (ID LC-MS/MS) with indirect multiple reaction monitoring (MRM) of the derivative tag was developed to simultaneously quantify endo-/exogenous plasma glucose. Using diluted D-[ 13 C 6 ] glucose as tracer of exogenous glucose, 12 C 6 / 13 C 6 glucoses were first derivatized and then data were acquired in MRM mode. The metabolism of exogenous glucose can be tracked and the concentration ratio of endo/exo-genous glucose can be measured by calculating the endo-/exo-genous glucose concentrations from peak area ratio of specific daughter ions. Joint application of selective derivatization and MRM analysis not only improves the sensitivity but also minimizes the interference from the background of plasma, which warrants the accuracy and reproducibility. Good agreement between the theoretical and calculated concentration ratios was obtained with a linear correlation coefficient (R) of 0.9969 in the range of D-glucose from 0.5 to 20.0 mM, which covers the healthy and diabetic physiological scenarios. Satisfactory reproducibility was obtained by evaluation of the intra- and inter-day precisions with relative standard deviations (RSDs) less than 5.16%, and relative recoveries of 85.96 to 95.92% were obtained at low, medium, and high concentration, respectively. The method was successfully applied to simultaneous determination of the endo-/exogenous glucose concentration in plasma of non-diabetic and type II diabetic cynomolgus monkeys. Graphical Abstract The scheme of the proposed ratio-based approach using isotope dilution LC-MS/MS with indirect MRM of the derivative tag for simultaneous quantification of endogenous and exogenous plasma glucose.

Aptamer facilitated purification of functional proteins.

PubMed

Beloborodov, Stanislav S; Bao, Jiayin; Krylova, Svetlana M; Shala-Lawrence, Agnesa; Johnson, Philip E; Krylov, Sergey N

2018-01-15

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER.

PubMed

Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco; Hannibal-Bach, Hans K; Ejsing, Christer S; Rapoport, Tom A

2014-01-16

Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins.

PubMed

Hu, Pingzhao; Janga, Sarath Chandra; Babu, Mohan; Díaz-Mejía, J Javier; Butland, Gareth; Yang, Wenhong; Pogoutse, Oxana; Guo, Xinghua; Phanse, Sadhna; Wong, Peter; Chandran, Shamanta; Christopoulos, Constantine; Nazarians-Armavil, Anaies; Nasseri, Negin Karimi; Musso, Gabriel; Ali, Mehrab; Nazemof, Nazila; Eroukova, Veronika; Golshani, Ashkan; Paccanaro, Alberto; Greenblatt, Jack F; Moreno-Hagelsieb, Gabriel; Emili, Andrew

2009-04-28

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a "systems-wide" functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages

PubMed Central

Daniel, Jaiyanth; Sirakova, Tatiana D.; Kolattukudy, Pappachan E.

2011-01-01

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism. PMID:21731490

Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

NASA Astrophysics Data System (ADS)

Borjigin, Jimo; Nathans, Jeremy

1993-01-01

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

Doclet To Synthesize UML

NASA Technical Reports Server (NTRS)

Barry, Matthew R.; Osborne, Richard N.

2005-01-01

The RoseDoclet computer program extends the capability of Java doclet software to automatically synthesize Unified Modeling Language (UML) content from Java language source code. [Doclets are Java-language programs that use the doclet application programming interface (API) to specify the content and format of the output of Javadoc. Javadoc is a program, originally designed to generate API documentation from Java source code, now also useful as an extensible engine for processing Java source code.] RoseDoclet takes advantage of Javadoc comments and tags already in the source code to produce a UML model of that code. RoseDoclet applies the doclet API to create a doclet passed to Javadoc. The Javadoc engine applies the doclet to the source code, emitting the output format specified by the doclet. RoseDoclet emits a Rose model file and populates it with fully documented packages, classes, methods, variables, and class diagrams identified in the source code. The way in which UML models are generated can be controlled by use of new Javadoc comment tags that RoseDoclet provides. The advantage of using RoseDoclet is that Javadoc documentation becomes leveraged for two purposes: documenting the as-built API and keeping the design documentation up to date.

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

DOE PAGES

Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; ...

2015-03-31

Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstratemore » reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.« less

IsoMS: automated processing of LC-MS data generated by a chemical isotope labeling metabolomics platform.

PubMed

Zhou, Ruokun; Tseng, Chiao-Li; Huan, Tao; Li, Liang

2014-05-20

A chemical isotope labeling or isotope coded derivatization (ICD) metabolomics platform uses a chemical derivatization method to introduce a mass tag to all of the metabolites having a common functional group (e.g., amine), followed by LC-MS analysis of the labeled metabolites. To apply this platform to metabolomics studies involving quantitative analysis of different groups of samples, automated data processing is required. Herein, we report a data processing method based on the use of a mass spectral feature unique to the chemical labeling approach, i.e., any differential-isotope-labeled metabolites are detected as peak pairs with a fixed mass difference in a mass spectrum. A software tool, IsoMS, has been developed to process the raw data generated from one or multiple LC-MS runs by peak picking, peak pairing, peak-pair filtering, and peak-pair intensity ratio calculation. The same peak pairs detected from multiple samples are then aligned to produce a CSV file that contains the metabolite information and peak ratios relative to a control (e.g., a pooled sample). This file can be readily exported for further data and statistical analysis, which is illustrated in an example of comparing the metabolomes of human urine samples collected before and after drinking coffee. To demonstrate that this method is reliable for data processing, five (13)C2-/(12)C2-dansyl labeled metabolite standards were analyzed by LC-MS. IsoMS was able to detect these metabolites correctly. In addition, in the analysis of a (13)C2-/(12)C2-dansyl labeled human urine, IsoMS detected 2044 peak pairs, and manual inspection of these peak pairs found 90 false peak pairs, representing a false positive rate of 4.4%. IsoMS for Windows running R is freely available for noncommercial use from www.mycompoundid.org/IsoMS.

HIV–host interactome revealed directly from infected cells

PubMed Central

Luo, Yang; Jacobs, Erica Y.; Greco, Todd M.; Mohammed, Kevin D.; Tong, Tommy; Keegan, Sarah; Binley, James M.; Cristea, Ileana M.; Fenyö, David; Rout, Michael P.; Chait, Brian T.; Muesing, Mark A.

2016-01-01

Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection. Importantly, interacting proteins were recovered without bias, regardless of their potential for positive, negative or neutral impact on viral replication. We identified specific host associations made with trimerized Env during its biosynthesis, at virological synapses, with innate immune effectors (such as HLA-E) and with certain cellular signalling pathways (for example, Notch1). We also defined Vif associations with host proteins involved in the control of nuclear transcription and nucleoside biosynthesis as well as those interacting stably or transiently with the cytoplasmic protein degradation apparatus. Our approach is broadly applicable to elucidating pathogen–host interactomes, providing high-certainty identification of interactors by their direct access during cycling infection. Understanding the pathophysiological consequences of these associations is likely to provide strategic targets for antiviral intervention. PMID:27375898

Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli.

PubMed

Borgnia, M J; Agre, P

2001-02-27

A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family-a water channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification. Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg(2+) stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl(2). Our studies demonstrate that, unlike AqpZ, a water-selective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

Coded wire tag recoveries from pink salmon in Prince William sound salmon fisheries, 1993. Restoration project 93067. Exxon Valdez oil spill restoration project final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharr, S.; Peckham, C.J.; Sharp, D.G.

1995-11-01

Coded wire tags applied to pink salmon fry in 1992 at four hatcheries in Prince William Sound were recovered in the commercial catch of 1993 and used to provide inseason estimates of hatchery contributions. These estimates were used by fishery managers to target the numerically superior hatchery returns, and reduce the pressure on oil-damaged wild stocks. Inseason estimates were made in two stages. The postseason analysis revealed that of a catch of 3.51 million pink salmon, 1.12 million were estimated to be of wild origin.

Semantic Annotation of Complex Text Structures in Problem Reports

NASA Technical Reports Server (NTRS)

Malin, Jane T.; Throop, David R.; Fleming, Land D.

2011-01-01

Text analysis is important for effective information retrieval from databases where the critical information is embedded in text fields. Aerospace safety depends on effective retrieval of relevant and related problem reports for the purpose of trend analysis. The complex text syntax in problem descriptions has limited statistical text mining of problem reports. The presentation describes an intelligent tagging approach that applies syntactic and then semantic analysis to overcome this problem. The tags identify types of problems and equipment that are embedded in the text descriptions. The power of these tags is illustrated in a faceted searching and browsing interface for problem report trending that combines automatically generated tags with database code fields and temporal information.

Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

PubMed

Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

2015-10-21

Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.

Tail proteins of phage T5: investigation of the effect of the His6-tag position, from expression to crystallisation.

PubMed

Noirclerc-Savoye, Marjolaine; Flayhan, Ali; Pereira, Cindy; Gallet, Benoit; Gans, Pierre; Ebel, Christine; Breyton, Cécile

2015-05-01

Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality. Copyright © 2015 Elsevier Inc. All rights reserved.

Simultaneous detection of assembly and disassembly of multivalent HA tag and anti-HA antibody in single in-capillary assay.

PubMed

Wang, Jianhao; Qin, Yuqin; Qin, Haifang; Liu, Li; Ding, Shumin; Teng, Yiwan; Ji, Junling; Qiu, Lin; Jiang, Pengju

2016-08-01

Herein, we have developed an in-capillary assay for simultaneous detection of the assembly and disassembly of the multivalent HA tag peptide and antibody. HA tag with hexahistidine at C terminus (YPYDVPDYAG4 H6 , termed YPYDH6 ) was conjugated with quantum dots (QDs) by metal-affinity force to form a multivalent HA tag (QD-YPYDH6 ). QD-YPYDH6 and monoclonal anti-HA antibody (anti-HA) were sequentially injected into the capillary. They were mixed and assembled inside the capillary. The reaction products were online discriminated and detected by fluorescence coupled capillary electrophoresis (CE-FL). For the in-capillary assay, the binding efficiency of the multivalent HA tag and antibody on was influenced by the molar ratio and injection time. Such novel assay could even give out the self-assembly kinetic constant of QDs and YPYDH6 as KD of 34.1 μM with n (binding cooperativeness) of 2.2 by Hill equation. More importantly, the simultaneous detection of the assembly and imidazole (Im) induced disassembly of the QD-YPYDH6 -anti-HA complex was achieved in a single in-capillary assay. Our study demonstrated a new method for the online detection of antigen-antibody interactions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stable isotope reactive transport modeling in water-rock interactions during CO2 injection

NASA Astrophysics Data System (ADS)

Hidalgo, Juan J.; Lagneau, Vincent; Agrinier, Pierre

2010-05-01

Stable isotopes can be of great usefulness in the characterization and monitoring of CO2 sequestration sites. Stable isotopes can be used to track the migration of the CO2 plume and identify leakage sources. Moreover, they provide unique information about the chemical reactions that take place on the CO2-water-rock system. However, there is a lack of appropriate tools that help modelers to incorporate stable isotope information into the flow and transport models used in CO2 sequestration problems. In this work, we present a numerical tool for modeling the transport of stable isotopes in groundwater reactive systems. The code is an extension of the groundwater single-phase flow and reactive transport code HYTEC [2]. HYTEC's transport module was modified to include element isotopes as separate species. This way, it is able to track isotope composition of the system by computing the mixing between the background water and the injected solution accounting for the dependency of diffusion on the isotope mass. The chemical module and database have been expanded to included isotopic exchange with minerals and the isotope fractionation associated with chemical reactions and mineral dissolution or precipitation. The performance of the code is illustrated through a series of column synthetic models. The code is also used to model the aqueous phase CO2 injection test carried out at the Lamont-Doherty Earth Observatory site (Palisades, New York, USA) [1]. References [1] N. Assayag, J. Matter, M. Ader, D. Goldberg, and P. Agrinier. Water-rock interactions during a CO2 injection field-test: Implications on host rock dissolution and alteration effects. Chemical Geology, 265(1-2):227-235, July 2009. [2] Jan van der Lee, Laurent De Windt, Vincent Lagneau, and Patrick Goblet. Module-oriented modeling of reactive transport with HYTEC. Computers & Geosciences, 29(3):265-275, April 2003.

Water Isotopes in the GISS GCM: History, Applications and Potential

NASA Astrophysics Data System (ADS)

Schmidt, G. A.; LeGrande, A. N.; Field, R. D.; Nusbaumer, J. M.

2017-12-01

Water isotopes have been incorporated in the GISS GCMs since the pioneering work of Jean Jouzel in the 1980s. Since 2005, this functionality has been maintained within the master branch of the development code and has been usable (and used) in all subsequent versions. This has allowed a wide variety of applications, across multiple time-scales and interests, to be tackled coherently. Water isotope tracers have been used to debug the atmospheric model code, tune parameterisations of moist processes, assess the isotopic fingerprints of multiple climate drivers, produce forward models for remotely sensed isotope products, and validate paleo-climate interpretations from the last millennium to the Eocene. We will present an overview of recent results involving isotope tracers, including improvements in models for the isotopic fractionation processes themselves, and demonstrate the potential for using these tracers and models more systematically in paleo-climate reconstructions and investigations of the modern hydrological cycle.

Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.

PubMed

Mittermayr, Stefan; Albrecht, Simone; Váradi, Csaba; Millán-Martín, Silvia; Bones, Jonathan

2017-01-01

Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromatogram/electropherogram ratios generated from the light and heavy isotope clusters.

A Laboratory Exercise to Illustrate Protein-Membrane Interactions

ERIC Educational Resources Information Center

Weers, Paul M. M.; Prenner, Elmar J.; Curic, Spomenka; Lohmeier-Vogel, Elke M.

2016-01-01

The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6-tagged human apolipoprotein A-I (apoA-I) with Ni-NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre-dried phospholipid films. When apoA-I is added to the MLV,…

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging

PubMed Central

Liu, Wenhao; Howarth, Mark; Greytak, Andrew B.; Zheng, Yi; Nocera, Daniel G.; Ting, Alice Y.; Bawendi, Moungi G.

2009-01-01

We present a family of water-soluble quantum dots (QDs) that exhibit low nonspecific binding to cells, small hydrodynamic diameter, tunable surface charge, high quantum yield, and good solution stability across a wide pH range. These QDs are amenable to covalent modification via simple carbodiimide coupling chemistry, which is achieved by functionalizing the surface of QDs with a new class of heterobifunctional ligands incorporating dihydrolipoic acid, a short poly(ethylene glycol) (PEG) spacer, and an amine or carboxylate terminus. The covalent attachment of molecules is demonstrated by appending a rhodamine dye to form a QD-dye conjugate exhibiting fluorescence resonance energy transfer (FRET). High-affinity labeling is demonstrated by covalent attachment of streptavidin, thus enabling the tracking of biotinylated epidermal growth factor (EGF) bound to EGF receptor on live cells. In addition, QDs solubilized with the heterobifunctional ligands retain their metal-affinity driven conjugation chemistry with polyhistidine-tagged proteins. This dual functionality is demonstrated by simultaneous covalent attachment of a rhodamine FRET acceptor and binding of polyhistidine-tagged streptavidin on the same nanocrystal to create a targeted QD, which exhibits dual-wavelength emission. Such emission properties could serve as the basis for ratiometric sensing of the cellular receptor’s local chemical environment. PMID:18177042

Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae.

PubMed

Twerdochlib, Adriana L; Chubatsu, Leda S; Souza, Emanuel M; Pedrosa, Fábio O; Steffens, M Berenice R; Yates, M Geoffrey; Rigo, Liu U

2003-07-01

NtrC is a bacterial enhancer-binding protein (EBP) that activates transcription by the sigma54 RNA polymerase holoenzyme. NtrC has a three domain structure typical of EBP family. In Herbaspirillum seropedicae, an endophytic diazotroph, NtrC regulates several operons involved in nitrogen assimilation, including glnAntrBC. In order to over-express and purify the NtrC protein, DNA fragments containing the complete structural gene for the whole protein, and for the N-terminal+Central and Central+C-terminal domains were cloned into expression vectors. The NtrC and NtrC(N-terminal+Central) proteins were over-expressed as His-tag fusion proteins upon IPTG addition, solubilized using N-lauryl-sarcosyl and purified by metal affinity chromatography. The over-expressed His-tag-NtrC(Central+C-terminal) fusion protein was partially soluble and was also purified by affinity chromatography. DNA band-shift assays showed that the NtrC protein and the Central+C-terminal domains bound specifically to the H. seropedicae glnA promoter region. The C-terminal domain is presumably necessary for DNA-protein interaction and DNA-binding does not require a phosphorylated protein.

Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

PubMed Central

Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

2018-01-01

Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

Comparison of deterministic and stochastic approaches for isotopic concentration and decay heat uncertainty quantification on elementary fission pulse

NASA Astrophysics Data System (ADS)

Lahaye, S.; Huynh, T. D.; Tsilanizara, A.

2016-03-01

Uncertainty quantification of interest outputs in nuclear fuel cycle is an important issue for nuclear safety, from nuclear facilities to long term deposits. Most of those outputs are functions of the isotopic vector density which is estimated by fuel cycle codes, such as DARWIN/PEPIN2, MENDEL, ORIGEN or FISPACT. CEA code systems DARWIN/PEPIN2 and MENDEL propagate by two different methods the uncertainty from nuclear data inputs to isotopic concentrations and decay heat. This paper shows comparisons between those two codes on a Uranium-235 thermal fission pulse. Effects of nuclear data evaluation's choice (ENDF/B-VII.1, JEFF-3.1.1 and JENDL-2011) is inspected in this paper. All results show good agreement between both codes and methods, ensuring the reliability of both approaches for a given evaluation.

Vehicle autonomous localization in local area of coal mine tunnel based on vision sensors and ultrasonic sensors

PubMed Central

Yang, Wei; You, Kaiming; Li, Wei; Kim, Young-il

2017-01-01

This paper presents a vehicle autonomous localization method in local area of coal mine tunnel based on vision sensors and ultrasonic sensors. Barcode tags are deployed in pairs on both sides of the tunnel walls at certain intervals as artificial landmarks. The barcode coding is designed based on UPC-A code. The global coordinates of the upper left inner corner point of the feature frame of each barcode tag deployed in the tunnel are uniquely represented by the barcode. Two on-board vision sensors are used to recognize each pair of barcode tags on both sides of the tunnel walls. The distance between the upper left inner corner point of the feature frame of each barcode tag and the vehicle center point can be determined by using a visual distance projection model. The on-board ultrasonic sensors are used to measure the distance from the vehicle center point to the left side of the tunnel walls. Once the spatial geometric relationship between the barcode tags and the vehicle center point is established, the 3D coordinates of the vehicle center point in the tunnel’s global coordinate system can be calculated. Experiments on a straight corridor and an underground tunnel have shown that the proposed vehicle autonomous localization method is not only able to quickly recognize the barcode tags affixed to the tunnel walls, but also has relatively small average localization errors in the vehicle center point’s plane and vertical coordinates to meet autonomous unmanned vehicle positioning requirements in local area of coal mine tunnel. PMID:28141829

Vehicle autonomous localization in local area of coal mine tunnel based on vision sensors and ultrasonic sensors.

PubMed

Xu, Zirui; Yang, Wei; You, Kaiming; Li, Wei; Kim, Young-Il

2017-01-01

This paper presents a vehicle autonomous localization method in local area of coal mine tunnel based on vision sensors and ultrasonic sensors. Barcode tags are deployed in pairs on both sides of the tunnel walls at certain intervals as artificial landmarks. The barcode coding is designed based on UPC-A code. The global coordinates of the upper left inner corner point of the feature frame of each barcode tag deployed in the tunnel are uniquely represented by the barcode. Two on-board vision sensors are used to recognize each pair of barcode tags on both sides of the tunnel walls. The distance between the upper left inner corner point of the feature frame of each barcode tag and the vehicle center point can be determined by using a visual distance projection model. The on-board ultrasonic sensors are used to measure the distance from the vehicle center point to the left side of the tunnel walls. Once the spatial geometric relationship between the barcode tags and the vehicle center point is established, the 3D coordinates of the vehicle center point in the tunnel's global coordinate system can be calculated. Experiments on a straight corridor and an underground tunnel have shown that the proposed vehicle autonomous localization method is not only able to quickly recognize the barcode tags affixed to the tunnel walls, but also has relatively small average localization errors in the vehicle center point's plane and vertical coordinates to meet autonomous unmanned vehicle positioning requirements in local area of coal mine tunnel.

Preliminary results of oxygen isotope ratio measurement with a particle-gamma coincidence method

NASA Astrophysics Data System (ADS)

Borysiuk, Maciek; Kristiansson, Per; Ros, Linus; Abdel, Nassem S.; Elfman, Mikael; Nilsson, Charlotta; Pallon, Jan

2015-04-01

The possibility to study variations in the oxygen isotopic ratio with photon tagged nuclear reaction analysis (pNRA) is evaluated in the current work. The experiment described in the article was performed at Lund Ion Beam Analysis Facility (LIBAF) with a 2 MeV deuteron beam. Isotopic fractionation of light elements such as carbon, oxygen and nitrogen is the basis of many analytical tools in hydrology, geology, paleobiology and paleogeology. IBA methods provide one possible tool for measurement of isotopic content. During this experimental run we focused on measurement of the oxygen isotopic ratio. The measurement of stable isotopes of oxygen has a number of applications; the particular one driving the current investigation belongs to the field of astrogeology and specifically evaluation of fossil extraterrestrial material. There are three stable isotopes of oxygen: 16O, 17O and 18O. We procured samples highly enriched with all three isotopes. Isotopes 16O and 18O were easily detected in the enriched samples, but no significant signal from 17O was detected in the same samples. The measured yield was too low to detect 18O in a sample with natural abundances of oxygen isotopes, at least in the current experimental setup, but the spectral line from the reaction with 16O was clearly visible.

Determination of the NPP Kr\\vsko spent fuel decay heat

NASA Astrophysics Data System (ADS)

Kromar, Marjan; Kurinčič, Bojan

2017-07-01

Nuclear fuel is designed to support fission process in a reactor core. Some of the isotopes, formed during the fission, decay and produce decay heat and radiation. Accurate knowledge of the nuclide inventory producing decay heat is important after reactor shut down, during the fuel storage and subsequent reprocessing or disposal. In this paper possibility to calculate the fuel isotopic composition and determination of the fuel decay heat with the Serpent code is investigated. Serpent is a well-known Monte Carlo code used primarily for the calculation of the neutron transport in a reactor. It has been validated for the burn-up calculations. In the calculation of the fuel decay heat different set of isotopes is important than in the neutron transport case. Comparison with the Origen code is performed to verify that the Serpent is taking into account all isotopes important to assess the fuel decay heat. After the code validation, a sensitivity study is carried out. Influence of several factors such as enrichment, fuel temperature, moderator temperature (density), soluble boron concentration, average power, burnable absorbers, and burnup is analyzed.

High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

PubMed

Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

2016-01-01

Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bio-inorganic synthesis of ZnO powders using recombinant His-tagged ZnO binding peptide as a promoter.

PubMed

Song, Lei; Liu, Yingying; Zhang, Zhifang; Wang, Xi; Chen, Jinchun

2010-10-01

Inorganic-binding peptides termed as genetically engineered polypeptides for inorganics (GEPIs), are small peptide sequences selected via combinatorial biology-based protocols of phage or cell surface display technologies. Recent advances in nanotechnology and molecular biology allow the engineering of these peptides with specific affinity to inorganics, often used as molecular linkers or assemblers, to facilitate materials synthesis, which provides a new insight into the material science and engineering field. As a case study on this biomimetic application, here we report a novel biosynthetic ZnO binding protein and its application in promoting bio-inorganic materials synthesis. In brief, the gene encoding a ZnO binding peptide(ZBP) was genetically fused with His(6)-tag and GST-tag using E.coli expression vector pET-28a (+) and pGEX-4T-3. The recombinant protein GST-His-ZBP was expressed, purified with Ni-NTA system, identified by SDS-PAGE electrophoresis and Western blot analysis and confirmed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Affinity adsorption test demonstrated that the fusion protein had a specific avidity for ZnO nanoparticles (NPs). Results from the bio-inorganic synthesis experiment indicated that the new protein played a promoting part in grain refinement and accelerated precipitation during the formation of the ultra-fine precursor powders in the Zn(OH)(2) sol. X-ray diffraction (XRD) analysis on the final products after calcining the precursor powders showed that hexagonal wurtzite ZnO crystals were obtained. Our work suggested a novel approach to the application about the organic-inorganic interactions.

Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

PubMed

Sarpong, Kwabena; Bose, Ron

2017-03-15

A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

Simulated shifts in the mid-latitude storm tracks over the western US detected through isotopes in precipitation and vapor

NASA Astrophysics Data System (ADS)

Buenning, N. H.; Stott, L. D.; Kanner, L.; Yoshimura, K.

2013-12-01

One of the most robust features of climate model projections for the 21st century includes a poleward shift of middle latitude storm tracks in response to enhanced radiative forcing. This study evaluates how shifts in the middle latitude storm tracks over the North Pacific has been expressed in the stable isotopic composition of atmospheric vapor and precipitation in the past 60 years. Previous work has demonstrated how the isotopic composition of precipitation (δ18Op) in the Pacific Northwest and in atmospheric vapor (δ18Ov) across the western US reflects the large-scale atmospheric circulation. Thus, it is possible to use the isotopic composition of water in these regions to detect shifts in mid-latitude storm tracks. Results from the Isotope-incorporated Global Spectral Model (IsoGSM) are presented to better understand the recent low frequency variations in δ18O values over the western US. The IsoGSM simulations presented here were spectrally nudged every six hours to the NCEP/NCAR Reanalysis wind and temperature fields. The spectral nudging technique allows for realistic isotopic simulations that are consistent with observed large-scale mid-latitude storm systems. Model results suggest that δ18Op has risen over the Pacific Northwest and δ18Ov has increased across the western US since the 1950s (see Figure), an indication of more moisture advection from the tropics and less moisture transported from the middle latitudes. Water tagging simulations reveal that as δ18Ov increased in the western US, the fraction of vapor from the subtropics had also increased, while the fraction from the middle latitudes had decreased. Similarly, the tagging simulations resulted in increased subtropical precipitation falling in the Pacific Northwest and decreased precipitation from the middle latitudes. These model simulations suggest that a northward shift in storm tracks has already taken place over the last 60 years in the western US. Furthermore, the results underscore the potential of using isotopes in vapor and precipitation in certain regions of western North America to monitor middle latitude storm behavior as the climate warms.

Microparticles: Facile and High-Throughput Synthesis of Functional Microparticles with Quick Response Codes (Small 24/2016).

PubMed

Ramirez, Lisa Marie S; He, Muhan; Mailloux, Shay; George, Justin; Wang, Jun

2016-06-01

Microparticles carrying quick response (QR) barcodes are fabricated by J. Wang and co-workers on page 3259, using a massive coding of dissociated elements (MiCODE) technology. Each microparticle can bear a special custom-designed QR code that enables encryption or tagging with unlimited multiplexity, and the QR code can be easily read by cellphone applications. The utility of MiCODE particles in multiplexed DNA detection and microtagging for anti-counterfeiting is explored. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tectonic affinities of the accreted basalts in southern Taiwan

NASA Astrophysics Data System (ADS)

Chen, Hsin-Yu; Yang, Huai-Jen; Liu, Yung-Hsin; Huang, Kuo-Fang; Takazawa, Eiichi

2018-06-01

Tectonic affinities of accreted basalts provide constraints on mass transport in convergent boundaries, improving our understandings on the evolution of regional geology. In this study, nineteen accreted basalts from the southernmost tip of Taiwan Island, which is on the convergent boundary between the Eurasian and Philippine Sea Plates, were analyzed for element concentrations as well as Sr, Nd, Hf, and Pb isotope ratios to investigate their tectonic affinities. All the samples contain > 3% LOI, reflecting post-magmatic alteration. LOI and Nb variation diagrams together with comparisons to oceanic basalt compositions indicated that the concentrations of most major elements and Rb, Sr, and Ba were modified by post-magmatic processes to varying extents, while P2O5, REE and HFSE remained immobile. Although some samples show Pb loss, most samples have Pb concentrations not affected by post-magmatic processes. Isotope ratios of Pb, Nd and Hf, generally reflect the mantle source characteristics. The εNd-εHf relationship and trace element abundance ratios indicated that the LREE-depleted samples were mostly scraped off the subducting South China Sea floor, reflecting the volumetric dominance of N-MORB on ocean floors. The overriding Philippine Sea Plate contributed both N-MORB and E-MORB to the accretionary prism. The tectonic affinities of the LREE-enriched samples, however, could not be unambiguously determined for the large geochemical variability of OIB from both subducting and overlying slabs. Based on our results, it is proposed that the tectonic affinity of the basalts in an accretionary prism can indicate the subduction polarity of the associated convergent boundary, providing a constraint for regional geology evolution.

The Effects of Computerized Auditory Feedback on Electronic Article Surveillance Tag Placement in an Auto-Parts Distribution Center

ERIC Educational Resources Information Center

Goomas, David T.

2008-01-01

In this report from the field, computerized auditory feedback was used to inform order selectors and order selector auditors in a distribution center to add an electronic article surveillance (EAS) adhesive tag. This was done by programming handheld computers to emit a loud beep for high-priced items upon scanning the item's bar-coded Universal…

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

PubMed Central

Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen JT; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

2015-01-01

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001 PMID:25824290

Functionalized Nano-adsorbent for Affinity Separation of Proteins

NASA Astrophysics Data System (ADS)

Zou, Xueyan; Yang, Fengbo; Sun, Xin; Qin, Mingming; Zhao, Yanbao; Zhang, Zhijun

2018-05-01

Thiol-functionalized silica nanospheres (SiO2-SH NSs) with an average diameter of 460 nm were synthesized through a hydrothermal route. Subsequently, the prepared SiO2-SH NSs were modified by SnO2 quantum dots to afford SnO2/SiO2 composite NSs possessing obvious fluorescence, which could be used to trace the target protein. The SnO2/SiO2 NSs were further modified by reduced glutathione (GSH) to obtain SnO2/SiO2-GSH NSs, which can specifically separate glutathione S-transferase-tagged (GST-tagged) protein. Moreover, the peroxidase activity of glutathione peroxidase 3 (GPX3) separated from SnO2/SiO2-GSH NSs in vitro was evaluated. Results show that the prepared SnO2/SiO2-GSH NSs exhibit negligible nonspecific adsorption, high concentration of protein binding (7.4 mg/g), and good reused properties. In the meantime, the GST-tagged GPX3 separated by these NSs can retain its redox state and peroxidase activity. Therefore, the prepared SnO2/SiO2-GSH NSs might find promising application in the rapid separation and purification of GST-tagged proteins.

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

PubMed

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

2016-10-01

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.

Construction of stabilized and tagged foot-and-mouth disease virus.

PubMed

Park, Jeong-Nam; Ko, Mi-Kyeong; Kim, Rae-Hyung; Park, Min-Eun; Lee, Seo-Yong; Yoon, Ji-Eun; Choi, Joo-Hyung; You, Su-Hwa; Park, Jung-Won; Lee, Kwang-Nyeong; Chun, Ji-Eun; Kim, Su-Mi; Tark, Dongseob; Lee, Hyang-Sim; Ko, Young-Joon; Kim, Byounghan; Lee, Myoung-Heon; Park, Jong-Hyeon

2016-11-01

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease that affects cloven-hoofed animals worldwide. Construction and purification of stable antigen for vaccine are necessary but technically difficult and laborious. Here, we have tried to investigate an alternative method by inserting a hexa-histidine tag (6xHIS) in the VP1 C-terminal for easy purification and replacing two amino acids of VP1/VP2 to enhance the stability of the capsid of the FMD virus (FMDV) Asia1/MOG/05. In addition, infectious 6xHIS-tagged stable (S/T) FMDVs were maintained under acidic conditions (pH 6.0) and were readily purified from small-scale cultures using a commercial metal-affinity column. The groups vaccinated with the S/T FMDV antigen showed complete protection comparing to low survival rate in the group vaccinated with non-S/T FMDV against lethal challenge with Asia1 Shamir in mice. Therefore, the present findings indicate that the stabilized and tagged antigen offers an alternative to using the current methods for antigen purification and enhancement of stability and has potential for the development of a new FMD vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

Nickel nanoparticle decorated graphene for highly selective isolation of polyhistidine-tagged proteins

NASA Astrophysics Data System (ADS)

Liu, Jia-Wei; Yang, Ting; Ma, Lin-Yu; Chen, Xu-Wei; Wang, Jian-Hua

2013-12-01

Nickel nanoparticle decorated graphene (GP-Ni) is prepared by one-pot hydrothermal reduction of graphene oxide and nickel cations by hydrazine hydrate in the presence of poly(sodium-p-styrenesulfonate) (PSS). The GP-Ni hybrid is characterized by XRD, TEM, SEM, XPS, Raman and FT-IR spectra, demonstrating the formation of poly-dispersed nickel nanoparticles with an average size of 83 nm attached on the surface of graphene sheets. The GP-Ni hybrid exhibits ferromagnetic behavior with a magnetization saturation of 31.1 emu g-1 at 10 000 Oersted (Oe). The GP-Ni also possesses favorable stability in aqueous medium and rapid magnetic response to an external magnetic field. These make it a novel magnetic adsorbent for the separation/isolation of His6-tagged recombinant proteins from a complex sample matrix (cell lysate). The targeted protein species is captured onto the surface of the GP-Ni hybrid via specific metal affinity force between polyhistidine groups and nickel nanoparticles. The SDS-PAGE assay indicates highly selective separation of His6-tagged Smt A from cell lysate. The GP-Ni hybrid displays favorable performance on the separation/isolation of His6-tagged recombinant proteins with respect to the commercial NTA-Ni2+ column.

A New Aloha Anti-Collision Algorithm Based on CDMA

NASA Astrophysics Data System (ADS)

Bai, Enjian; Feng, Zhu

The tags' collision is a common problem in RFID (radio frequency identification) system. The problem has affected the integrity of the data transmission during the process of communication in the RFID system. Based on analysis of the existing anti-collision algorithm, a novel anti-collision algorithm is presented. The new algorithm combines the group dynamic frame slotted Aloha algorithm with code division multiple access technology. The algorithm can effectively reduce the collision probability between tags. Under the same number of tags, the algorithm is effective in reducing the reader recognition time and improve overall system throughput rate.

Printed dose-recording tag based on organic complementary circuits and ferroelectric nonvolatile memories

PubMed Central

Nga Ng, Tse; Schwartz, David E.; Mei, Ping; Krusor, Brent; Kor, Sivkheng; Veres, Janos; Bröms, Per; Eriksson, Torbjörn; Wang, Yong; Hagel, Olle; Karlsson, Christer

2015-01-01

We have demonstrated a printed electronic tag that monitors time-integrated sensor signals and writes to nonvolatile memories for later readout. The tag is additively fabricated on flexible plastic foil and comprises a thermistor divider, complementary organic circuits, and two nonvolatile memory cells. With a supply voltage below 30 V, the threshold temperatures can be tuned between 0 °C and 80 °C. The time-temperature dose measurement is calibrated for minute-scale integration. The two memory bits are sequentially written in a thermometer code to provide an accumulated dose record. PMID:26307438

Wireless SAW passive tag temperature measurement in the collision case

NASA Astrophysics Data System (ADS)

Sorokin, A.; Shepeta, A.; Wattimena, M.

2018-04-01

This paper describes temperature measurement in the multisensor systems based on the radio-frequency identification SAW passive tags which are currently applied in the electric power systems and the switchgears. Different approaches of temperature measurement in the collision case are shown here. The study is based on the tag model with specific topology, which allows us to determine temperature through the response signal with time-frequency information. This research considers the collision case for several passive tags as the temperature sensors which are placed in the switchgear. This research proposal is to analyze the possibility of using several SAW passive sensors in the collision case. We consider the using of the different typical elements for passive surface acoustic wave tag which applies as an anticollision passive sensor. These wireless sensors based on the surface acoustic waves tags contain specifically coded structures. This topology makes possible the reliability of increasing tag identification and the temperature measurement in the collision case. As the results for this case we illustrate simultaneous measurement of at least six sensors.

Sensitive Carbohydrate Detection using Surface Enhanced Raman Tagging

PubMed Central

Vangala, Karthikeshwar; Yanney, Michael; Hsiao, Cheng-Te; Wu, Wells W.; Shen, Rong-Fong; Zou, Sige; Sygula, Andrzej; Zhang, Dongmao

2010-01-01

Glycomic analysis is an increasingly important field in biological and biomedical research as glycosylation is one of the most important protein post-translational modifications. We have developed a new technique to detect carbohydrates using surface enhanced Raman spectroscopy (SERS) by designing and applying a Rhodamine B derivative as the SERS tag. Using a reductive amination reaction, the Rhodamine-based tag (RT) was successfully conjugated to three model carbohydrates (glucose, lactose and glucuronic acid). SERS detection limits obtained with 632 nm HeNe laser were ~1 nM in concentration for all the RT-carbohydrate conjugates and ~10 fmol in total sample consumption. The dynamic range of the SERS method is about 4 orders of magnitude, spanning from 1 nM to 5 µM. Ratiometric SERS quantification using isotope-substituted SERS internal references also allows comparative quantifications of carbohydrates labeled with RT and deuterium/hydrogen substituted RT tags, respectively. In addition to enhancing the SERS detection of the tagged carbohydrates, the Rhodamine tagging facilitates fluorescence and mass spectrometric detection of carbohydrates. Current fluorescence sensitivity of RT-carbohydrates is ~ 3 nM in concentration while the mass spectrometry (MS) sensitivity is about 1 fmol that was achieved with linear ion trap electrospray ionization (ESI)-MS instrument. Potential applications that take advantage of the high SERS, fluorescence and MS sensitivity of this SERS tagging strategy are discussed for practical glycomic analysis where carbohydrates may be quantified with a fluorescence and SERS technique, and then identified with ESI-MS techniques. PMID:21082777

Analysis of Ethylene Receptor Interactions by Co-immunoprecipitation Assays.

PubMed

Gao, Zhiyong; Schaller, G Eric

2017-01-01

Ethylene receptors are predominantly localized to the endoplasmic reticulum (ER) membrane, and coordinate ethylene signal output through protein-protein interactions with each other and additional signaling components. Here, we describe a co-immunoprecipitation (Co-IP) assay based on the use of the Tandem Affinity Purification (TAP) tag to examine the interactions of ethylene receptors in plant extracts. Human IgG-agarose beads are used to pull down TAP-tagged versions of the protein of interest from detergent extracts of Arabidopsis membranes, and the precipitate then is analyzed immunologically for co-purification of the ethylene receptors. This method has been successfully used to examine interactions of the receptors with each other as well as with the Raf-like kinase CTR1.

Stable Isotope Applications for Understanding Shark Ecology in the Northeast Pacific Ocean.

PubMed

Reum, Jonathan C P; Williams, Gregory D; Harvey, Chris J

Stable isotopes are used to address a wide range of ecological questions and can help researchers and managers better understand the movement and trophic ecology of sharks. Here, we review how shark studies from the Northeast Pacific Ocean (NEP) have employed stable isotopes to estimate trophic level and diet composition and infer movement and habitat-use patterns. To date, the number of NEP shark studies that have used stable isotopes is limited, suggesting that the approach is underutilized. To aid shark researchers in understanding the strengths and limitations of the approach, we provide a brief overview of carbon and nitrogen stable isotope trophic discrimination properties (e.g., change in δ 15 N between predator and prey), tissue sample preparation methods specific to elasmobranchs, and methodological considerations for the estimation of trophic level and diet composition. We suggest that stable isotopes are a potentially powerful tool for addressing basic questions about shark ecology and are perhaps most valuable when combined and analysed with other data types (e.g., stomach contents, tagging data, or other intrinsic biogeochemical markers). © 2017 Elsevier Ltd. All rights reserved.

Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

PubMed Central

Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

2016-01-01

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

Recombinant glycoprotein G analog for determination of specific immunoglobulins to herpes simplex virus type 2 by ELISA.

PubMed

Korshun, Ludmila; Vudmaska, Mariya; Moysa, Larissa; Kovtonjuk, Galina; Mikhalap, Svetlana; Ganova, Larissa; Spivak, Nikolay

2013-12-01

In order to the detection of type-specific IgG to herpes simplex virus type 2 (HSV-2) in human serum or plasma the recombinant analog of HSV-2 glycoprotein G (gG2) was created. To construct an expression vector the DNA fragment with a sequence identical to immunodominant regions of HSV-2 gG2 was cloned into modified vector pET28a containing of the glutation-S-transferase sequence (pET28-GST). Escherichia coli BL21 (DE3) were transformed with the recombinant plasmid. The target protein was expressed mainly in soluble form. Chromatographic purification of soluble GST-gG2 protein was performed taking into account the features of its primary structure that are 6His-tag and GST-tag. To determine the affinity constant of the specific IgG to GST-gG2 we used the method proposed by Friguet et al. (1985). The affinity constants were within the range of 10(7)-10(8)M(-1) proving their high-affinity. The purified recombinant HSV-2 antigen was used to design a diagnostic ELISA kit, which was evaluated with referent controls and standard panels of sera containing and/or not containing anti-HSV-2 IgG. Comparative evaluation of this kit and the commercially available "HSV-Type 2 IgG-ELISA" (NovaTec, Dietzenbach, Germany) kit was performed. There was no significant difference (P>0.05). It allows to use developed ELISA kit for clinical diagnosis of HSV-2 infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

PubMed

Roelofs, Anke J; Stewart, Charlotte A; Sun, Shuting; Błażewska, Katarzyna M; Kashemirov, Boris A; McKenna, Charles E; Russell, R Graham G; Rogers, Michael J; Lundy, Mark W; Ebetino, Frank H; Coxon, Fraser P

2012-04-01

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton. Copyright © 2012 American Society for Bone and Mineral Research.

Use of isotopically-tagged isolates of E. coli for tracking bacterial movement in karst environments

NASA Astrophysics Data System (ADS)

Bandy, A.; Fryar, A. E.; Macko, S. A.; Cook, K.

2014-12-01

Because of limited filtration and turbulent flow, karst aquifers are more susceptible to microbial contamination than clastic aquifers. Assessment of microbial transport in groundwater is complicated by the need to identify tracers that have a low detection limit, have minimal background concentrations, behave like the organisms of interest, and are non-pathogenic. We are assessing transport of two non-pathogenic isolates of Escherichia coli (E. coli) compared to traditional groundwater tracers in epikarst above Cave Springs Cavern near Bowling Green, KY, and in a karst conduit that emerges at Royal Spring in Georgetown, KY. The E. coli isolate exhibiting higher attachment efficiency in saturated granular columns contains the iha gene, while the isolate exhibiting lower attachment efficiency contains the kps gene. For the field experiments, bacteria are being grown on media enriched in 13C or 15N. Isotopically-tagged bacteria will be injected with rhodamine WT as a solute tracer and fluorescent microspheres as an abiotic particulate tracer. We will monitor breakthrough of the tracers in the cave and at the spring; based on a previous field test, we anticipate that particulate tracers may be remobilized during subsequent storm events. E. coli will be quantified by molecular methods (qPCR) and dual isotope analysis. Preliminary findings suggest that these two methods may be complementary, with each method having detection limitations.

Identification and characterization of polydimethylsiloxane-binding peptides (PDMS-tag) for oriented immobilization of functional protein on a PDMS surface.

PubMed

Kumada, Yoichi; Otsuki, Ryoko; Sakoda, Yumiko; Akai, Ryota; Matoba, Kazutaka; Katayama, Junko; Kishimoto, Michimasa; Horiuchi, Jun-Ichi

2016-10-20

In this study we focused on identifying and characterizing polydimethylsiloxane-binding peptides (PDMS-tags) that show a strong binding affinity towards a PDMS surface. Three kinds of E. coli host proteins (ELN, OMC and TPA) that were preferentially adsorbed onto a PDMS surface were identified from the E. coli cell lysate via 2-D electrophoresis and MALDI TOF MS. Digestion of these PDMS-binding proteins by 3 types of proteases (trypsin, chymotrypsin and V8 protease) resulted in the production of a wide variety of peptide fragments with different amino acid biases. Nine types of peptide fragments showing binding affinities to a PDMS surface were identified, and they were genetically fused at the C-terminal region of glutathione S-transferase (GST). The adsorption kinetics of peptide-fused GSTs to a PDMS surface were evaluated using a quartz crystal microbalance (QCM) sensor equipped with a sensor chip coated with a PDMS thin film. Consequently, all GSTs fused with the peptides adsorbed at a level higher than that of wild-type GST. In particular, the adsorption levels of GSTs fused with ELN-V81, TPA-V81, and OMC-V81 peptides were 8- to 10-fold higher than that of the wild-type GST. These results indicated that the selected peptides possessed a strong binding affinity towards a PDMS surface even in cases where they were introduced to the C-terminal region of a model protein. The remaining activities of GSTs with PDMS-binding peptides were also greater than that of the wild-type GST. Almost a third (30%) of enzymatic activity was maintained by genetic fusion of the peptide ELN-V81, compared with only 1.5% of wild-type GST in the adsorption state. Thus, the PDMS-binding peptides (PDMS-tags) identified in this study will be considerably useful for the site-specific immobilization of functional proteins to a PDMS surface, which will be a powerful tool in the fabrication of protein-based micro-reactors and biosearation chips. Copyright © 2016 Elsevier B.V. All rights reserved.

Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: biochemical characterization and identification of interacting partners through proteomic approaches.

PubMed

Lima, Analía; Durán, Rosario; Schujman, Gustavo Enrique; Marchissio, María Julia; Portela, María Magdalena; Obal, Gonzalo; Pritsch, Otto; de Mendoza, Diego; Cerveñansky, Carlos

2011-08-24

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thr-phosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His(6)-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes. Copyright © 2011 Elsevier B.V. All rights reserved.

Molecular cloning, expression and characterization of 100K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome.

PubMed

Shah, M S; Ashraf, A; Khan, M I; Rahman, M; Habib, M; Qureshi, J A

2016-01-01

Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage. Copyright © 2015 The International Alliance for Biological Standardization. All rights reserved.

Chemical derivatization for enhancing sensitivity during LC/ESI-MS/MS quantification of steroids in biological samples: a review.

PubMed

Higashi, Tatsuya; Ogawa, Shoujiro

2016-09-01

Sensitive and specific methods for the detection, characterization and quantification of endogenous steroids in body fluids or tissues are necessary for the diagnosis, pathological analysis and treatment of many diseases. Recently, liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been widely used for these purposes due to its specificity and versatility. However, the ESI efficiency and fragmentation behavior of some steroids are poor, which lead to a low sensitivity. Chemical derivatization is one of the most effective methods to improve the detection characteristics of steroids in ESI-MS/MS. Based on this background, this article reviews the recent advances in chemical derivatization for the trace quantification of steroids in biological samples by LC/ESI-MS/MS. The derivatization in ESI-MS/MS is based on tagging a proton-affinitive or permanently charged moiety on the target steroid. Introduction/formation of a fragmentable moiety suitable for the selected reaction monitoring by the derivatization also enhances the sensitivity. The stable isotope-coded derivatization procedures for the steroid analysis are also described. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris.

PubMed

Kim, Tae-Kang; Zhang, Rundong; Feng, Wenke; Cai, Jian; Pierce, William; Song, Zhao-Hui

2005-03-01

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.

High-Affinity Rb Binding, p53 Inhibition, Subcellular Localization, and Transformation by Wild-Type or Tumor-Derived Shortened Merkel Cell Polyomavirus Large T Antigens

PubMed Central

Borchert, Sophie; Czech-Sioli, Manja; Neumann, Friederike; Schmidt, Claudia; Wimmer, Peter; Dobner, Thomas

2014-01-01

ABSTRACT Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ∼90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential. PMID:24371076

Exploiting Diffusion Barrier and Chemical Affinity of Metal-Organic Frameworks for Efficient Hydrogen Isotope Separation.

PubMed

Kim, Jin Yeong; Balderas-Xicohténcatl, Rafael; Zhang, Linda; Kang, Sung Gu; Hirscher, Michael; Oh, Hyunchul; Moon, Hoi Ri

2017-10-25

Deuterium plays a pivotal role in industrial and scientific research, and is irreplaceable for various applications such as isotope tracing, neutron moderation, and neutron scattering. In addition, deuterium is a key energy source for fusion reactions. Thus, the isolation of deuterium from a physico-chemically almost identical isotopic mixture is a seminal challenge in modern separation technology. However, current commercial approaches suffer from extremely low separation efficiency (i.e., cryogenic distillation, selectivity of 1.5 at 24 K), requiring a cost-effective and large-scale separation technique. Herein, we report a highly effective hydrogen isotope separation system based on metal-organic frameworks (MOFs) having the highest reported separation factor as high as ∼26 at 77 K by maximizing synergistic effects of the chemical affinity quantum sieving (CAQS) and kinetic quantum sieving (KQS). For this purpose, the MOF-74 system having high hydrogen adsorption enthalpies due to strong open metal sites is chosen for CAQS functionality, and imidazole molecules (IM) are employed to the system for enhancing the KQS effect. To the best of our knowledge, this work is not only the first attempt to implement two quantum sieving effects, KQS and CAQS, in one system, but also provides experimental validation of the utility of this system for practical industrial usage by isolating high-purity D 2 through direct selective separation studies using 1:1 D 2 /H 2 mixtures.

Laser photodetachment of radioactive 128 I -

DOE PAGES

Rothe, Sebastian; Sundberg, Julia; Welander, Jakob; ...

2017-08-31

The first experimental investigation of the electron affinity (EA) of a radioactive isotope has been conducted at the CERN-ISOLDE radioactive ion beam facility. The EA of the radioactive iodine isotope 128I ($t$ 1/2 = 25 min) was determined to be 3.059 052(38) eV. The experiment was conducted using the newly developed Gothenburg ANion Detector for Affinity measurements by Laser PHotodetachment (GANDALPH) apparatus, connected to a CERN-ISOLDE experimental beamline. 128I was produced in fission induced by 1.4 GeV protons striking a thorium/tantalum foil target and then extracted as singly charged negative ions at a beam energy of 20 keV. Laser photodetachmentmore » of the fast ion beam was performed in a collinear geometry inside the GANDALPH chamber. Neutral atoms produced in the photodetachment process were detected by allowing them to impinge on a glass surface, creating secondary electrons which were then detected using a channel electron multiplier. The photon energy of the laser was tuned across the threshold of the photodetachment process and the detachment threshold data were fitted to a Wigner law function in order to extract the EA. In conclusion, this first successful demonstration of photodetachment at an isotope separator on line facility opens up the opportunity for future studies of the fundamental properties of negatively charged radioactive isotopes such as the EA of astatine and polonium.« less

Comprehensive analytical methodology to determine hydrocarbons in marine waters using extraction disks coupled to glass fiber filters and compound-specific isotope analyses.

PubMed

Ternon, Eva; Tolosa, Imma

2015-07-24

Solid-phase extraction of both aliphatic (AHs) and aromatic polycyclic hydrocarbons (PAHs) from seawater samples was evaluated using a GFF filter stacked upon an octadecyl bonded silica (C18) disk. Stable-isotope measurements were developed on hydrocarbons extracted from both GFF and C18-disks in order to characterize the source of hydrocarbons. A clear partition of hydrocarbon compounds between the dissolved and the particulate phase was highlighted. PAHs showed a higher affinity with the dissolved phase (recoveries efficiency of 48-71%) whereas AHs presented strong affinity with the particulate phase (up to 76% of extraction efficiency). Medium volumes of seawater samples were tested and no breakthrough was observed for a 5L sample. Isotopic fractionation was investigated within all analytical steps but none was evidenced. This method has been applied to harbor seawater samples and very low AH and PAH concentrations were achieved. Due to the low concentration levels of hydrocarbons in the samples, the source of hydrocarbons was determined by molecular indices rather than isotopic measurements and a pyrolytic origin was evidenced. The aliphatic profile also revealed the presence of long-chain linear alkylbenzenes (LABs). The methodology presented here would better fit to polluted coastal environments affected by recent oil spills. Copyright © 2015 Elsevier B.V. All rights reserved.

Focused Logistics, Joint Vision 2010: A Joint Logistics Roadmap

DTIC Science & Technology

2010-01-01

AIS). AIT devices include bar codes for individual items, optical memory cards for multipacks and containers, radio frequency tags for containers and...Fortezza Card and Firewall technologies are being developed to prevent unau- thorized access. As for infrastructure, DISA has already made significant in...radio frequency tags and optical memory cards , to continuously update the JTAV database. By September 1998, DSS will be deployed in all wholesale

SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution.

PubMed

Kaltenbach, Miriam; Stein, Viktor; Hollfelder, Florian

2011-09-19

Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pasek, Marta; Boeggeman, Elizabeth; Ramakrishnan, Boopathy

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of amore » large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.« less

Evidence that coded-wire-tagging procedures can enhance transmission of Renibacterium salmoninarum in chinook salmon

USGS Publications Warehouse

Elliott, D.G.; Pascho, R.J.

2001-01-01

Binary coded wire tags (CWTs) are used extensively for identification and management of anadromous salmonid populations. A study of bacterial kidney disease (BKD) in two brood year groups of hatchery-reared spring chinook salmon Oncorhynchus tshawytscha provided strong evidence that horizontal transmission of Renibacterium salmoninarum, the causative agent of BKD, might be enhanced by CWT-marking procedures. About 4 months after CWTs were implanted in the snouts of juvenile fish, 14-16 different tissues were sampled from each of 60 fish per brood year group for histological analysis. Of the fish that were positive for R. salmoninarum by histological examination, 41% (7 of 17) of the 1988 brood year fish and 24% (10 of 42) of the 1989 brood year fish had BKD lesions confined to the head near the site of tag implantation. These lesions often resulted in the destruction of tissues of one or both olfactory organs. No focal snout infections were observed in fish that had not been marked with CWTs. Further data obtained from tissue analyses by use of an enzyme-linked immunosorbent assay and a fluorescent antibody test for detection of R. salmoninarum supported the hypothesis that infections of R. salmoninarum can be initiated in the snout tissues of CWT-marked fish and then spread to other organs. The tagging procedures might promote transmission of the pathogen among fish via contaminated tagging needles, by facilitating the entry of pathogens through the injection wound, or both. Limited evidence from this study suggested that implantation of passive integrated transponder tags in the peritoneal cavities of fish might also promote the transmission of R. salmoninarum or exacerbate existing infections. The results indicated a need for strict sanitary procedures during the tagging of fish in populations positive for R. salmoninarum to reduce the probability of enhanced horizontal transmission of the pathogen.

A porewater - based stable isotope approach for the investigation of subsurface hydrological processes

NASA Astrophysics Data System (ADS)

Garvelmann, J.; Külls, C.; Weiler, M.

2011-10-01

Predicting and understanding subsurface flowpaths is still a crucial issue in hydrological research. We present an experimental approach to reveal present and past subsurface flowpaths of water in the unsaturated and saturated zone. Two hillslopes in a humid moutainous catchment have been investigated. The H2O(liquid) - H2O(vapor) equilibration laser spectroscopy method was used to obtain high resolution δ2H vertical depth profiles of porewater at various points along a fall line of a pasture hillslope in the southern Black Forest, Germany. The Porewater Stable Isotope Profile (PSIP) approach was developed to use the integrated information of several vertical depth profiles of deuterium along two transects at the hillslopes. Different shapes of depth profiles were observed in relation to hillslope position. The statistical variability (inter-quartile range and standard deviation) of each profile was used to characterize different types of depth profiles. The profiles upslope or with a weak affinity for saturation as indicated by a low topographic wetness index preserve the isotopic input signal by precipitation with a distinct seasonal variability. These observations indicate mainly vertical movement of soil water in the upper part of the hillslope before sampling. The profiles downslope or at locations with a strong affinity for saturation do not show a similar seasonal isotopic signal. The input signal is erased in the foothills and a large proportion of pore water samples are close to the isotopic values of δ2H in stream water during base flow. Near the stream indications for efficient mixing of water from lateral subsurface flow paths with vertical percolation are found.

An Analysis of Information Assurance Relating to the Department of Defense Radio Frequency Identification (RFID) Passive Network

DTIC Science & Technology

2005-03-01

codes speed up consumer shopping, package shipping, and inventory tracking. RFID offers many advantages over bar codes, as the table below shows...sunlight” (Accenture, 2001, p. 4). Finally, one of the most significant advantages of RFID is the advent of anti-collision. Anti-collision allows an...RFID reader to read and/or write to multiple tags at one time, which is not possible for bar codes. Despite the many advantages RFID over bar codes

High-Resolution Enabled 12-Plex DiLeu Isobaric Tags for Quantitative Proteomics

PubMed Central

2015-01-01

Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of 13C, 15N, and 2H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography–tandem mass spectrometry (nanoLC–MS2) analysis on an Orbitrap Elite mass spectrometer. PMID:25405479

Publicizing Your Web Resources for Maximum Exposure.

ERIC Educational Resources Information Center

Smith, Kerry J.

2001-01-01

Offers advice to librarians for marketing their Web sites on Internet search engines. Advises against relying solely on spiders and recommends adding metadata to the source code and delivering that information directly to the search engines. Gives an overview of metadata and typical coding for meta tags. Includes Web addresses for a number of…

Server-Side Includes Made Simple.

ERIC Educational Resources Information Center

Fagan, Jody Condit

2002-01-01

Describes server-side include (SSI) codes which allow Webmasters to insert content into Web pages without programming knowledge. Explains how to enable the codes on a Web server, provides a step-by-step process for implementing them, discusses tags and syntax errors, and includes examples of their use on the Web site for Southern Illinois…

A review of automatic patient identification options for public health care centers with restricted budgets.

PubMed

García-Betances, Rebeca I; Huerta, Mónica K

2012-01-01

A comparative review is presented of available technologies suitable for automatic reading of patient identification bracelet tags. Existing technologies' backgrounds, characteristics, advantages and disadvantages, are described in relation to their possible use by public health care centers with budgetary limitations. A comparative assessment is presented of suitable automatic identification systems based on graphic codes, both one- (1D) and two-dimensional (2D), printed on labels, as well as those based on radio frequency identification (RFID) tags. The analysis looks at the tradeoffs of these technologies to provide guidance to hospital administrator looking to deploy patient identification technology. The results suggest that affordable automatic patient identification systems can be easily and inexpensively implemented using 2D code printed on low cost bracelet labels, which can then be read and automatically decoded by ordinary mobile smart phones. Because of mobile smart phones' present versatility and ubiquity, the implantation and operation of 2D code, and especially Quick Response® (QR) Code, technology emerges as a very attractive alternative to automate the patients' identification processes in low-budget situations.

A Review of Automatic Patient Identification Options for Public Health Care Centers with Restricted Budgets

PubMed Central

García-Betances, Rebeca I.; Huerta, Mónica K.

2012-01-01

A comparative review is presented of available technologies suitable for automatic reading of patient identification bracelet tags. Existing technologies’ backgrounds, characteristics, advantages and disadvantages, are described in relation to their possible use by public health care centers with budgetary limitations. A comparative assessment is presented of suitable automatic identification systems based on graphic codes, both one- (1D) and two-dimensional (2D), printed on labels, as well as those based on radio frequency identification (RFID) tags. The analysis looks at the tradeoffs of these technologies to provide guidance to hospital administrator looking to deploy patient identification technology. The results suggest that affordable automatic patient identification systems can be easily and inexpensively implemented using 2D code printed on low cost bracelet labels, which can then be read and automatically decoded by ordinary mobile smart phones. Because of mobile smart phones’ present versatility and ubiquity, the implantation and operation of 2D code, and especially Quick Response® (QR) Code, technology emerges as a very attractive alternative to automate the patients’ identification processes in low-budget situations. PMID:23569629

A secure RFID authentication protocol adopting error correction code.

PubMed

Chen, Chien-Ming; Chen, Shuai-Min; Zheng, Xinying; Chen, Pei-Yu; Sun, Hung-Min

2014-01-01

RFID technology has become popular in many applications; however, most of the RFID products lack security related functionality due to the hardware limitation of the low-cost RFID tags. In this paper, we propose a lightweight mutual authentication protocol adopting error correction code for RFID. Besides, we also propose an advanced version of our protocol to provide key updating. Based on the secrecy of shared keys, the reader and the tag can establish a mutual authenticity relationship. Further analysis of the protocol showed that it also satisfies integrity, forward secrecy, anonymity, and untraceability. Compared with other lightweight protocols, the proposed protocol provides stronger resistance to tracing attacks, compromising attacks and replay attacks. We also compare our protocol with previous works in terms of performance.

A Secure RFID Authentication Protocol Adopting Error Correction Code

PubMed Central

Zheng, Xinying; Chen, Pei-Yu

2014-01-01

RFID technology has become popular in many applications; however, most of the RFID products lack security related functionality due to the hardware limitation of the low-cost RFID tags. In this paper, we propose a lightweight mutual authentication protocol adopting error correction code for RFID. Besides, we also propose an advanced version of our protocol to provide key updating. Based on the secrecy of shared keys, the reader and the tag can establish a mutual authenticity relationship. Further analysis of the protocol showed that it also satisfies integrity, forward secrecy, anonymity, and untraceability. Compared with other lightweight protocols, the proposed protocol provides stronger resistance to tracing attacks, compromising attacks and replay attacks. We also compare our protocol with previous works in terms of performance. PMID:24959619

Expression and purification of a functional recombinant aspartate aminotransferase (AST) from Escherichia coli.

PubMed

Zou, Lihui; Zhao, Haijian; Wang, Daguang; Wang, Meng; Zhang, Chuanbao; Xiao, Fei

2014-07-01

Aspartate aminotransferase (AST; E.C. 2.6.1.1), a vitamin B6-dependent enzyme, preferentially promotes the mutual transformation of aspartate and α-ketoglutarate to oxaloacetate and glutamate. It plays a key role in amino acid metabolism and has been widely recommended as a biomarker of liver and heart damage. Our study aimed to evaluate the extensive preparation of AST and its application in quality control in clinical laboratories. We describe a scheme to express and purify the 6His-AST fusion protein. An optimized sequence coding AST was synthesized and transformed into Escherichia coli BL21 (DE3) strain for protein expression. Ideally, the fusion protein has a volumetric productivity achieving 900 mg/l cultures. After affinity chromatography, the enzyme activity of purified AST reached 150,000 U/L. Commutability assessment between the engineered AST and standard AST from Roche suggested that the engineered AST was the better candidate for the reference material. Moreover, the AST showed high stability during long-term storage at -20ºC. In conclusion, the highly soluble 6His-tagged AST can become a convenient tool for supplying a much better and cheaper standard or reference material for the clinical laboratory.

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, andmore » enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.« less

Improvement of Modeling HTGR Neutron Physics by Uncertainty Analysis with the Use of Cross-Section Covariance Information

NASA Astrophysics Data System (ADS)

Boyarinov, V. F.; Grol, A. V.; Fomichenko, P. A.; Ternovykh, M. Yu

2017-01-01

This work is aimed at improvement of HTGR neutron physics design calculations by application of uncertainty analysis with the use of cross-section covariance information. Methodology and codes for preparation of multigroup libraries of covariance information for individual isotopes from the basic 44-group library of SCALE-6 code system were developed. A 69-group library of covariance information in a special format for main isotopes and elements typical for high temperature gas cooled reactors (HTGR) was generated. This library can be used for estimation of uncertainties, associated with nuclear data, in analysis of HTGR neutron physics with design codes. As an example, calculations of one-group cross-section uncertainties for fission and capture reactions for main isotopes of the MHTGR-350 benchmark, as well as uncertainties of the multiplication factor (k∞) for the MHTGR-350 fuel compact cell model and fuel block model were performed. These uncertainties were estimated by the developed technology with the use of WIMS-D code and modules of SCALE-6 code system, namely, by TSUNAMI, KENO-VI and SAMS. Eight most important reactions on isotopes for MHTGR-350 benchmark were identified, namely: 10B(capt), 238U(n,γ), ν5, 235U(n,γ), 238U(el), natC(el), 235U(fiss)-235U(n,γ), 235U(fiss).

One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

PubMed Central

Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

2008-01-01

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

PubMed

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

PubMed Central

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

Mass spectrometry-based methods for detection and differentiation of botulinum neurotoxins

DOEpatents

Schmidt, Jurgen G [Los Alamos, NM; Boyer, Anne E [Atlanta, GA; Kalb, Suzanne R [Atlanta, GA; Moura, Hercules [Tucker, GA; Barr, John R [Suwannee, GA; Woolfitt, Adrian R [Atlanta, GA

2009-11-03

The present invention is directed to a method for detecting the presence of clostridial neurotoxins in a sample by mixing a sample with a peptide that can serve as a substrate for proteolytic activity of a clostridial neurotoxin; and measuring for proteolytic activity of a clostridial neurotoxin by a mass spectroscopy technique. In one embodiment, the peptide can have an affinity tag attached at two or more sites.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

PubMed

Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

2015-02-23

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

GLUT-3 expression in human skeletal muscle

NASA Technical Reports Server (NTRS)

Stuart, C. A.; Wen, G.; Peng, B. H.; Popov, V. L.; Hudnall, S. D.; Campbell, G. A.

2000-01-01

Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions

PubMed Central

Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

2015-01-01

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

Graphene Field-Effect Transistors for the Sensitive and Selective Detection of Escherichia coli Using Pyrene-Tagged DNA Aptamer.

PubMed

Wu, Guangfu; Dai, Ziwen; Tang, Xin; Lin, Zihong; Lo, Pik Kwan; Meyyappan, M; Lai, King Wai Chiu

2017-10-01

This study reports biosensing using graphene field-effect transistors with the aid of pyrene-tagged DNA aptamers, which exhibit excellent selectivity, affinity, and stability for Escherichia coli (E. coli) detection. The aptamer is employed as the sensing probe due to its advantages such as high stability and high affinity toward small molecules and even whole cells. The change of the carrier density in the probe-modified graphene due to the attachment of E. coli is discussed theoretically for the first time and also verified experimentally. The conformational change of the aptamer due to the binding of E. coli brings the negatively charged E. coli close to the graphene surface, increasing the hole carrier density efficiently in graphene and achieving electrical detection. The binding of negatively charged E. coli induces holes in graphene, which are pumped into the graphene channel from the contact electrodes. The carrier mobility, which correlates the gate voltage to the electrical signal of the APG-FETs, is analyzed and optimized here. The excellent sensing performance such as low detection limit, high sensitivity, outstanding selectivity and stability of the graphene biosensor for E. coli detection paves the way to develop graphene biosensors for bacterial detection. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of coded wire tags for marking lake trout

USGS Publications Warehouse

Elrod, Joseph H.; Schneider, Clifford P.

1986-01-01

Among hatchery-reared lake trout (Salvelinus namaycush) of the 1979-1982 year classes stocked in New York waters of Lake Ontario, more than 3 million fish were marked with a coded wire tag (CWT) plus an adipose fin clip, and 1.5 million with only conventional fin clips. Altogether, 7,640 tags were recovered from fish collected with bottom trawls and gill nets or caught by anglers during 1980-1983. One person was able to extract and decipher 200 or more CWTs per day with about a 1% error rate in reading and recording codes. Presence of the CWT did not affect growth. The adipose fin clip did not regenerate. The occurrence of fish with an adipose fin clip but no CWT resulted primarily from the regeneration of paired fins among fish marked with a combination of the adipose fin and a paired fin. Loss of CWTs between marking and stocking (generally 4-5 months for fish stocked in spring and 1-8 d for fish stocked in fall) declined from nearly 11% for the 1979 year class stocked as fall fingerlings to less than 3% for the 1981 and 1982 year classes - a difference that primarily reflected improvements in instrumentation and tagging technique. The rate of CWT loss after the marked fish were stocked was probably less than 1% per year. The CWT is a reliable method for marking hatchery-reared lake trout. A large number of experimental groups can be uniquely marked, and fish from each group can be accurately identified throughout their life. Use of this technique should greatly facilitate evaluations of genetic strain, hatchery experience, condition at time of stocking, season of stocking, size at stocking, method of stocking and other factors that affect poststocking survival and performance of lake trout stocked in the Great Lakes.

Habitat and diet differentiation by two strains of rainbow trout in Lake Superior based on archival tags, stable isotopes, and bioenergetics

EPA Science Inventory

Two strains of potamodromous rainbow trout Oncorhynchus mykiss in the Minnesota waters of Lake Superior exhibited differences in behavior and temperature tolerance at egg and fry stages, but the extent of these differences was not well understood in adult fish. To gain a better u...

An introduction to mass cytometry: fundamentals and applications.

PubMed

Tanner, Scott D; Baranov, Vladimir I; Ornatsky, Olga I; Bandura, Dmitry R; George, Thaddeus C

2013-05-01

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

Synthesis of poly(N-isopropylacrylamide) particles for metal affinity binding of peptides

PubMed Central

Tsai, Hsin-Yi; Lee, Alexander; Peng, Wei; Yates, Matthew Z.

2013-01-01

Temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgel particles with metal affinity ligands were prepared for selective binding of peptides containing the His6-tag (six consecutive histidine residues). The PNIPAM particles were copolymerized with the functional ligand vinylbenzyl iminodiacetic acid (VBIDA) through a two-stage dispersion polymerization using poly(N-vinyl pyrrolidone) (PVP) as a steric stabilizer. The resulting particles were monodisperse in size and colloidally stable over a wide range of temperature and ionic strength due to chemically grafted PVP chains. The particle size was also found to be sensitive to ionic strength and pH of the aqueous environment, likely due to the electrostatic repulsion between ionized VBIDA groups. Divalent nickel ions were chelated to the VBIDA groups, allowing selective metal affinity attachment of a His6-Cys peptide. The peptide was released upon the addition of the competitive ligand imidazole, demonstrating that the peptide attachment to the particles is reversible and selective. PMID:24176889

Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

NASA Astrophysics Data System (ADS)

Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

2017-07-01

The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin.

PubMed

de Aquino, Luciana Cristina Lins; de Sousa, Heloisa Ribeiro Tunes; Miranda, Everson Alves; Vilela, Luciano; Bueno, Sônia Maria Alves

2006-04-13

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).

A genome-wide resource for the analysis of protein localisation in Drosophila

PubMed Central

Sarov, Mihail; Barz, Christiane; Jambor, Helena; Hein, Marco Y; Schmied, Christopher; Suchold, Dana; Stender, Bettina; Janosch, Stephan; KJ, Vinay Vikas; Krishnan, RT; Krishnamoorthy, Aishwarya; Ferreira, Irene RS; Ejsmont, Radoslaw K; Finkl, Katja; Hasse, Susanne; Kämpfer, Philipp; Plewka, Nicole; Vinis, Elisabeth; Schloissnig, Siegfried; Knust, Elisabeth; Hartenstein, Volker; Mann, Matthias; Ramaswami, Mani; VijayRaghavan, K; Tomancak, Pavel; Schnorrer, Frank

2016-01-01

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts. DOI: http://dx.doi.org/10.7554/eLife.12068.001 PMID:26896675

Radar transponder apparatus and signal processing technique

DOEpatents

Axline, Jr., Robert M.; Sloan, George R.; Spalding, Richard E.

1996-01-01

An active, phase-coded, time-grating transponder and a synthetic-aperture radar (SAR) and signal processor means, in combination, allow the recognition and location of the transponder (tag) in the SAR image and allow communication of information messages from the transponder to the SAR. The SAR is an illuminating radar having special processing modifications in an image-formation processor to receive an echo from a remote transponder, after the transponder receives and retransmits the SAR illuminations, and to enhance the transponder's echo relative to surrounding ground clutter by recognizing special transponder modulations from phase-shifted from the transponder retransmissions. The remote radio-frequency tag also transmits information to the SAR through a single antenna that also serves to receive the SAR illuminations. Unique tag-modulation and SAR signal processing techniques, in combination, allow the detection and precise geographical location of the tag through the reduction of interfering signals from ground clutter, and allow communication of environmental and status information from said tag to be communicated to said SAR.

Radar transponder apparatus and signal processing technique

DOEpatents

Axline, R.M. Jr.; Sloan, G.R.; Spalding, R.E.

1996-01-23

An active, phase-coded, time-grating transponder and a synthetic-aperture radar (SAR) and signal processor means, in combination, allow the recognition and location of the transponder (tag) in the SAR image and allow communication of information messages from the transponder to the SAR. The SAR is an illuminating radar having special processing modifications in an image-formation processor to receive an echo from a remote transponder, after the transponder receives and retransmits the SAR illuminations, and to enhance the transponder`s echo relative to surrounding ground clutter by recognizing special transponder modulations from phase-shifted from the transponder retransmissions. The remote radio-frequency tag also transmits information to the SAR through a single antenna that also serves to receive the SAR illuminations. Unique tag-modulation and SAR signal processing techniques, in combination, allow the detection and precise geographical location of the tag through the reduction of interfering signals from ground clutter, and allow communication of environmental and status information from said tag to be communicated to said SAR. 4 figs.

Isotope-coded ESI-enhancing derivatization reagents for differential analysis, quantification and profiling of metabolites in biological samples by LC/MS: A review.

PubMed

Higashi, Tatsuya; Ogawa, Shoujiro

2016-10-25

The analysis of the qualitative and quantitative changes of metabolites in body fluids and tissues yields valuable information for the diagnosis, pathological analysis and treatment of many diseases. Recently, liquid chromatography/electrospray ionization-(tandem) mass spectrometry [LC/ESI-MS(/MS)] has been widely used for these purposes due to the high separation capability of LC, broad coverage of ESI for various compounds and high specificity of MS(/MS). However, there are still two major problems to be solved regarding the biological sample analysis; lack of sensitivity and limited availability of stable isotope-labeled analogues (internal standards, ISs) for most metabolites. Stable isotope-coded derivatization (ICD) can be the answer for these problems. By the ICD, different isotope-coded moieties are introduced to the metabolites and one of the resulting derivatives can serve as the IS, which minimize the matrix effects. Furthermore, the derivatization can improve the ESI efficiency, fragmentation property in the MS/MS and chromatographic behavior of the metabolites, which lead to a high sensitivity and specificity in the various detection modes. Based on this background, this article reviews the recently-reported isotope-coded ESI-enhancing derivatization (ICEED) reagents, which are key components for the ICD-based LC/MS(/MS) studies, and their applications to the detection, identification, quantification and profiling of metabolites in human and animal samples. The LC/MS(/MS) using the ICEED reagents is the powerful method especially for the differential analysis (relative quantification) of metabolites in two comparative samples, simultaneous quantification of multiple metabolites whose stable isotope-labeled ISs are not available, and submetabolome profiling. Copyright © 2016 Elsevier B.V. All rights reserved.

Nassi-Schneiderman Diagram in HTML Based on AML

ERIC Educational Resources Information Center

Menyhárt, László

2013-01-01

In an earlier work I defined an extension of XML called Algorithm Markup Language (AML) for easy and understandable coding in an IDE which supports XML editing (e.g. NetBeans). The AML extension contains annotations and native language (English or Hungarian) tag names used when coding our algorithm. This paper presents a drawing tool with which…

Recombinant expression of Intrepicalcin from the scorpion Vaejovis intrepidus and its effect on skeletal ryanodine receptors.

PubMed

Vargas-Jaimes, Leonel; Xiao, Liang; Zhang, Jing; Possani, Lourival D; Valdivia, Héctor H; Quintero-Hernández, Verónica

2017-04-01

Scorpion venoms contain toxins that modulate ionic channels, among which are the calcins, a small group of short, basic peptides with an Inhibitor Cystine Knot (ICK) motif that target calcium release channels/ryanodine receptors (RyRs) with high affinity and selectivity. Here we describe the heterologous expression of Intrepicalcin, identified by transcriptomic analysis of venomous glands from Vaejovis intrepidus. Recombinant Intrepicalcin was obtained in Escherichia coli BL21-DE3 (periplasm) by fusing the Intrepicalcin gene to sequences coding for signal-peptide, thioredoxin, His-tag and enterokinase cleavage site. [ 3 H]Ryanodine binding, used as a functional index of RyR activity, revealed that recombinant Intrepicalcin activates skeletal RyR (RyR1) dose-dependently with K d =17.4±4.0nM. Intrepicalcin significantly augments the bell-shaped [Ca 2+ ]-[ 3 H]ryanodine binding curve at all [Ca 2+ ] ranges, as is characteristic of the calcins. In single channel recordings, Intrepicalcin induces the appearance of a subconductance state in RyR1 with a fractional value ∼55% of the full conductance state, very close to that of Vejocalcin. Furthermore, Intrepicalcin stimulates Ca 2+ release at an initial dose=45.3±2.5nM, and depletes ~50% of Ca 2+ load from skeletal sarcoplasmic reticulum vesicles. We conclude that active recombinant Intrepicalcin was successfully obtained without the need of manual oxidation, enabling it to target RyR1s with high affinity. This is the first calcin heterologously expressed in the periplasma of Escherichia coli BL21-DE3, shown to be pharmacologically effective, thus paving the way for the generation of Intrepicalcin variants that are required for structure-function relationship studies of calcins and RyRs. Copyright © 2017 Elsevier B.V. All rights reserved.

[Methods of quantitative proteomics].

PubMed

Kopylov, A T; Zgoda, V G

2007-01-01

In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.

Quantitative charge-tags for sterol and oxysterol analysis.

PubMed

Crick, Peter J; William Bentley, T; Abdel-Khalik, Jonas; Matthews, Ian; Clayton, Peter T; Morris, Andrew A; Bigger, Brian W; Zerbinati, Chiara; Tritapepe, Luigi; Iuliano, Luigi; Wang, Yuqin; Griffiths, William J

2015-02-01

Global sterol analysis is challenging owing to the extreme diversity of sterol natural products, the tendency of cholesterol to dominate in abundance over all other sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative charge-tags for the LC-MS analysis of sterols including oxysterols. Sterols/oxysterols in plasma were extracted in ethanol containing deuterated internal standards, separated by C18 solid-phase extraction, and derivatized with GP, with or without prior oxidation of 3β-hydroxy to 3-oxo groups. By use of different isotope-labeled GPs, it was possible to analyze in a single LC-MS analysis both sterols/oxysterols that naturally possess a 3-oxo group and those with a 3β-hydroxy group. Intra- and interassay CVs were <15%, and recoveries for representative oxysterols and cholestenoic acids were 85%-108%. By adopting a multiplex approach to isotope labeling, we analyzed up to 4 different samples in a single run. Using plasma samples, we could demonstrate the diagnosis of inborn errors of metabolism and also the export of oxysterols from brain via the jugular vein. This method allows the profiling of the widest range of sterols/oxysterols in a single analytical run and can be used to identify inborn errors of cholesterol synthesis and metabolism. © 2014 American Association for Clinical Chemistry.

Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling

PubMed Central

Fujishiro, Mitsuhiro; Okamura, Tomohisa; Fujio, Keishi; Okazaki, Hiroaki; Nomura, Seitaro; Takeda, Norifumi; Harada, Mutsuo; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Yamamoto, Kazuhiko; Komuro, Issei; Yanagisawa, Masashi

2015-01-01

G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays. PMID:26020647

Microbially Mediated Kinetic Sulfur Isotope Fractionation: Reactive Transport Modeling Benchmark

NASA Astrophysics Data System (ADS)

Wanner, C.; Druhan, J. L.; Cheng, Y.; Amos, R. T.; Steefel, C. I.; Ajo Franklin, J. B.

2014-12-01

Microbially mediated sulfate reduction is a ubiquitous process in many subsurface systems. Isotopic fractionation is characteristic of this anaerobic process, since sulfate reducing bacteria (SRB) favor the reduction of the lighter sulfate isotopologue (S32O42-) over the heavier isotopologue (S34O42-). Detection of isotopic shifts have been utilized as a proxy for the onset of sulfate reduction in subsurface systems such as oil reservoirs and aquifers undergoing uranium bioremediation. Reactive transport modeling (RTM) of kinetic sulfur isotope fractionation has been applied to field and laboratory studies. These RTM approaches employ different mathematical formulations in the representation of kinetic sulfur isotope fractionation. In order to test the various formulations, we propose a benchmark problem set for the simulation of kinetic sulfur isotope fractionation during microbially mediated sulfate reduction. The benchmark problem set is comprised of four problem levels and is based on a recent laboratory column experimental study of sulfur isotope fractionation. Pertinent processes impacting sulfur isotopic composition such as microbial sulfate reduction and dispersion are included in the problem set. To date, participating RTM codes are: CRUNCHTOPE, TOUGHREACT, MIN3P and THE GEOCHEMIST'S WORKBENCH. Preliminary results from various codes show reasonable agreement for the problem levels simulating sulfur isotope fractionation in 1D.

Fractionation of Fe isotopes by soil microbes and organic acids

USGS Publications Warehouse

Brantley, Susan L.; Liermann, Laura; Bullen, Thomas D.

2001-01-01

Small natural variations in Fe isotopes have been attributed to biological cycling. However, without understanding the mechanism of fractionation, it is impossible to interpret such variations. Here we show that the δ56Fe of Fe dissolved from a silicate soil mineral by siderophore-producing bacteria is as much as 0.8% lighter than bulk Fe in the mineral. A smaller isotopic shift is observed for Fe released abiotically by two chelates, and the magnitude of the shift increases with affinity of the ligand for Fe, consistent with a kinetic isotope effect during hydrolysis of Fe at the mineral surface. Fe dissolved abiotically without chelates shows no isotopic shift. The δ56Fe of the exchange fraction on soil grains is also lighter by ~0.6%-1% than Fe from both hornblende and iron oxyhydroxides. The kinetic isotope effect is therefore preserved in open systems such as soils. when recorded in the rock record, Fe isotopic fractionation could document Fe transport by organic molecules or by microbes where such entities were present in the geologic past.

V isotope composition in modern marine hydrothermal sediments

NASA Astrophysics Data System (ADS)

Wu, F.; Owens, J. D.; Nielsen, S.; German, C. R.; Rachel, M.

2017-12-01

Vanadium is multivalence transition metal with two isotopes (51V and 50V). Recent work has shown that large V isotope variations occur with oxygen variations in modern sediments (Wu et al., 2016 and 2017 Goldschmidt Abstracts), providing its potential as a promising proxy for determining low oxygen conditions. However, the development of V isotopes as a proxy to probe past redox conditions requires a comprehensive understanding of the modern oceanic isotopic mass balance. Therein, the scavenging of V from the hydrous iron oxides in hydrothermal fluid has been shown to be an important removal process from seawater (Rudnicki and Elderfield, 1993 GCA) but remains unquantified. In this study, we analyzed V isotopic compositions of metalliferous sediments around the active TAG hydrothermal mound from the mid-Atlantic Ridge (26° degrees North) and the Eastern Pacific Zonal Transect (GEOTRACES EPZT cruise GP16). The TAG sediments deposited as Fe oxyhydroxides from plume fall-out, and have δ51V values between -0.3 to 0‰. The good correlation between Fe and V for these metalliferous sediments indicate that the accumulation of V in these samples is directly related to the deposition of Fe oxyhydroxides, which also control their V isotope signature. The EPZT samples cover 8,000 km in the South Pacific Ocean with sedimentary areas that underlie the Peru upwelling region and the well-oxygenated deep South Pacific Ocean influenced by hydtorthermal plume material from southern East Pacific Rise (EPR). The sediments collected at the east of the EPR have δ51V values between -1.2 to -0.7‰, similar to previous δ51V of oxic sediments. In contrast, the sediments from the west of the EPR have δ51V values (-0.4 to 0‰) similar to hydrothermal sediments from the mid-Atlantic Ridge, indicating the long transportation (more than 4,000 km, Fitzsimmons et al., 2017 NG) of Fe and Mn from hydrothermal plume and their incorporation into sediments have a major impact on the cycle of V in the ocean. The fingerprint of δ51V between oxic sediments and hydrothermal flux are significantly different and should be easily discernible in the geologic record. Consequently, our results show that the removal of V from hydrothermal sediments has an important influence on the marine V cycle, which needs to be considered for future modern and paleoclimatic studies.

Identification of novel lysosomal matrix proteins by proteome analysis.

PubMed

Kollmann, Katrin; Mutenda, Kudzai E; Balleininger, Martina; Eckermann, Ellen; von Figura, Kurt; Schmidt, Bernhard; Lübke, Torben

2005-10-01

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.

A rapid and fluorogenic TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells.

PubMed

Liu, Wei; Li, Fu; Chen, Xi; Hou, Jian; Yi, Long; Wu, Yao-Wen

2014-03-26

Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

A Suite of "Minimalist" Photo-Crosslinkers for Live-Cell Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors.

PubMed

Pan, Sijun; Jang, Se-Young; Wang, Danyang; Liew, Si Si; Li, Zhengqiu; Lee, Jun-Seok; Yao, Shao Q

2017-09-18

Affinity-based probes (AfBPs) provide a powerful tool for large-scale chemoproteomic studies of drug-target interactions. The development of high-quality probes capable of recapitulating genuine drug-target engagement, however, could be challenging. "Minimalist" photo-crosslinkers, which contain an alkyl diazirine group and a chemically tractable tag, could alleviate such challenges, but few are currently available. Herein, we have developed new alkyl diazirine-containing photo-crosslinkers with different bioorthogonal tags. They were subsequently used to create a suite of AfBPs based on GW841819X (a small molecule inhibitor of BRD4). Through in vitro and in situ studies under conditions that emulated native drug-target interactions, we have obtained better insights into how a tag might affect the probe's performance. Finally, SILAC-based chemoproteomic studies have led to the discovery of a novel off-target, APEX1. Further studies showed GW841819X binds to APEX1 and caused up-regulation of endogenous DNMT1 expression under normoxia conditions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Profiling global kinome signatures of the radioresistant MCF-7/C6 breast cancer cells using MRM-based targeted proteomics.

PubMed

Guo, Lei; Xiao, Yongsheng; Fan, Ming; Li, Jian Jian; Wang, Yinsheng

2015-01-02

Ionizing radiation is widely used in cancer therapy; however, cancer cells often develop radioresistance, which compromises the efficacy of cancer radiation therapy. Quantitative assessment of the alteration of the entire kinome in radioresistant cancer cells relative to their radiosensitive counterparts may provide important knowledge to define the mechanism(s) underlying tumor adaptive radioresistance and uncover novel target(s) for effective prevention and treatment of tumor radioresistance. By employing a scheduled multiple-reaction monitoring analysis in conjunction with isotope-coded ATP affinity probes, we assessed the global kinome of radioresistant MCF-7/C6 cells and their parental MCF-7 human breast cancer cells. We rigorously quantified 120 kinases, of which (1)/3 exhibited significant differences in expression levels or ATP binding affinities. Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase. The elevated expression of CHK1, CDK1, and CDK2 in MCF-7/C6 cells was further validated by Western blot analysis. Thus, the altered kinome profile of radioresistant MCF-7/C6 cells suggests the involvement of kinases on cell cycle progression and DNA repair in tumor adaptive radioresistance. The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.

Biochemical localization of a protein involved in Gluconacetobacter hansenii cellulose synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iyer, Prashanti R; Catchmark, Jeffrey M; Brown, Nicole Robitaille

2011-02-08

Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.

Extraction and Quantitation of Ketones and Aldehydes from Mammalian Cells Using Fluorous Tagging and Capillary LC-MS.

PubMed

Yuan, Wei; Li, Shuwei; Edwards, James L

2015-08-04

The extraction and quantitation of carbonyl metabolites from cell lysate was accomplished using a carbonyl-reactive fluorous tag and capillary liquid chromatography coupled to mass spectrometry (capLC-MS). Selective fluorous tagging for ketones and aldehydes provided a 30-fold increase in sensitivity using electrospray ionization MS. Separation of fluorous tagged carbonyl resulted in good separation of all components, and tandem MS was able to differentiate structural carbonyl isomers. The average limit of detection for carbonyl standards was 37 nM (range 1.5-250 nM), with linearity of R(2) > 0.99. Reproducibility for metabolites in cell lysate averaged 9% RSD. Human aortic endothelial cells (HAECs) were exposed to varying levels of glucose, and their carbonyl metabolite levels were quantified. Significant metabolite changes were seen in glycolysis and the propanoate pathway from a glucose challenge. Using an untargeted approach, 120 carbonyl metabolites were found to change in hyperglycemic HAECs. From this list of compounds, multiple metabolites from the pentose phosphate and tryptophan metabolic pathways were discovered. This system provides excellent sensitivity and quantitation of carbonyl metabolites without the need for isotope standards or labels.

A porewater-based stable isotope approach for the investigation of subsurface hydrological processes

NASA Astrophysics Data System (ADS)

Garvelmann, J.; Külls, C.; Weiler, M.

2012-02-01

Predicting and understanding subsurface flowpaths is still a crucial issue in hydrological research. We present an experimental approach to reveal present and past subsurface flowpaths of water in the unsaturated and saturated zone. Two hillslopes in a humid mountainous catchment have been investigated. The H2O(liquid) - H2O(vapor) equilibration laser spectroscopy method was used to obtain high resolution δ2H vertical depth profiles of pore water at various points along two fall lines of a pasture hillslope in the southern Black Forest, Germany. The Porewater-based Stable Isotope Profile (PSIP) approach was developed to use the integrated information of several vertical depth profiles of deuterium along transects at the hillslope. Different shapes of depth profiles were observed in relation to hillslope position. The statistical variability (inter-quartile range and standard deviation) of each profile was used to characterize different types of depth profiles. The profiles upslope or with a weak affinity for saturation as indicated by a low topographic wetness index preserve the isotopic input signal by precipitation with a distinct seasonal variability. These observations indicate mainly vertical movement of soil water in the upper part of the hillslope before sampling. The profiles downslope or at locations with a strong affinity for saturation do not show a similar seasonal isotopic signal. The input signal is erased in the foothills and a large proportion of pore water samples are close to the isotopic values of δ2H in streamwater during base flow conditions indicating the importance of the groundwater component in the catchment. Near the stream indications for efficient mixing of water from lateral subsurface flow paths with vertical percolation are found.

Unravelling biocultural population structure in 4th/3rd century BC Monterenzio Vecchio (Bologna, Italy) through a comparative analysis of strontium isotopes, non-metric dental evidence, and funerary practices.

PubMed

Sorrentino, Rita; Bortolini, Eugenio; Lugli, Federico; Mancuso, Giuseppe; Buti, Laura; Oxilia, Gregorio; Vazzana, Antonino; Figus, Carla; Serrangeli, Maria Cristina; Margherita, Cristiana; Penzo, Annachiara; Gruppioni, Giorgio; Gottarelli, Antonio; Jochum, Klaus Peter; Belcastro, Maria Giovanna; Cipriani, Anna; Feeney, Robin N M; Benazzi, Stefano

2018-01-01

The 4th century BC marks the main entrance of Celtic populations in northern Italy. Their arrival has been suggested based on the presence of Celtic customs in Etruscan mortuary contexts, yet up to now few bioarchaeological data have been examined to support or reject the arrival of these newcomers. Here we use strontium isotopes, non-metric dental traits and funerary patterns to unravel the biocultural structure of the necropolis of Monterenzio Vecchio (Bologna, Italy). Subsamples of our total sample of 38 individuals were analyzed based on different criteria characterizing the following analyses: 1) strontium isotope analysis to investigate migratory patterns and provenance; 2) non-metric dental traits to establish biological relationships between Monterenzio Vecchio, 13 Italian Iron age necropolises and three continental and non-continental Celtic necropolises; 3) grave goods which were statistically explored to detect possible patterns of cultural variability. The strontium isotopes results indicate the presence of local and non-local individuals, with some revealing patterns of mobility. The dental morphology reveals an affinity between Monterenzio Vecchio and Iron Age Italian samples. However, when the Monterenzio Vecchio sample is separated by isotopic results into locals and non-locals, the latter share affinity with the sample of non-continental Celts from Yorkshire (UK). Moreover, systematic analyses demonstrate that ethnic background does not retain measurable impact on the distribution of funerary elements. Our results confirm the migration of Celtic populations in Monterenzio as archaeologically hypothesized on the basis of the grave goods, followed by a high degree of cultural admixture between exogenous and endogenous traits. This contribution shows that combining different methods offers a more comprehensive perspective for the exploration of biocultural processes in past and present populations.

Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag

PubMed Central

Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

2015-01-01

With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant. PMID:26539718

Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.

PubMed

Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

2015-01-01

With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant.

HEC Applications on Columbia Project

NASA Technical Reports Server (NTRS)

Taft, Jim

2004-01-01

NASA's Columbia system consists of a cluster of twenty 512 processor SGI Altix systems. Each of these systems is 3 TFLOP/s in peak performance - approximately the same as the entire compute capability at NAS just one year ago. Each 512p system is a single system image machine with one Linunx O5, one high performance file system, and one globally shared memory. The NAS Terascale Applications Group (TAG) is chartered to assist in scaling NASA's mission critical codes to at least 512p in order to significantly improve emergency response during flight operations, as well as provide significant improvements in the codes. and rate of scientific discovery across the scientifc disciplines within NASA's Missions. Recent accomplishments are 4x improvements to codes in the ocean modeling community, 10x performance improvements in a number of computational fluid dynamics codes used in aero-vehicle design, and 5x improvements in a number of space science codes dealing in extreme physics. The TAG group will continue its scaling work to 2048p and beyond (10240 cpus) as the Columbia system becomes fully operational and the upgrades to the SGI NUMAlink memory fabric are in place. The NUMlink uprades dramatically improve system scalability for a single application. These upgrades will allow a number of codes to execute faster at higher fidelity than ever before on any other system, thus increasing the rate of scientific discovery even further

Heat-mediated activation of affinity-immobilized Taq DNA polymerase.

PubMed

Nilsson, J; Bosnes, M; Larsen, F; Nygren, P A; Uhlén, M; Lundeberg, J

1997-04-01

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.

Identification of Small RNA-Protein Partners in Plant Symbiotic Bacteria.

PubMed

Robledo, Marta; Matia-González, Ana M; García-Tomsig, Natalia I; Jiménez-Zurdo, José I

2018-01-01

The identification of the protein partners of bacterial small noncoding RNAs (sRNAs) is essential to understand the mechanistic principles and functions of riboregulation in prokaryotic cells. Here, we describe an optimized affinity chromatography protocol that enables purification of in vivo formed sRNA-protein complexes in Sinorhizobium meliloti, a genetically tractable nitrogen-fixing plant symbiotic bacterium. The procedure requires the tagging of the desired sRNA with the MS2 aptamer, which is affinity-captured by the MS2-MBP protein conjugated to an amylose resin. As proof of principle, we show recovery of the RNA chaperone Hfq associated to the strictly Hfq-dependent AbcR2 trans-sRNA. This method can be applied for the investigation of sRNA-protein interactions on a broad range of genetically tractable α-proteobacteria.

Impact of liver fat on the differential partitioning of hepatic triacylglycerol into VLDL subclasses on high and low sugar diets.

PubMed

Umpleby, A Margot; Shojaee-Moradie, Fariba; Fielding, Barbara; Li, Xuefei; Marino, Andrea; Alsini, Najlaa; Isherwood, Cheryl; Jackson, Nicola; Ahmad, Aryati; Stolinski, Michael; Lovegrove, Julie A; Johnsen, Sigurd; Jeewaka R Mendis, A S; Wright, John; Wilinska, Malgorzata E; Hovorka, Roman; Bell, Jimmy D; Thomas, E Louise; Frost, Gary S; Griffin, Bruce A

2017-11-01

Dietary sugars are linked to the development of non-alcoholic fatty liver disease (NAFLD) and dyslipidaemia, but it is unknown if NAFLD itself influences the effects of sugars on plasma lipoproteins. To study this further, men with NAFLD ( n = 11) and low liver fat 'controls' ( n = 14) were fed two iso-energetic diets, high or low in sugars (26% or 6% total energy) for 12 weeks, in a randomised, cross-over design. Fasting plasma lipid and lipoprotein kinetics were measured after each diet by stable isotope trace-labelling.There were significant differences in the production and catabolic rates of VLDL subclasses between men with NAFLD and controls, in response to the high and low sugar diets. Men with NAFLD had higher plasma concentrations of VLDL 1 -triacylglycerol (TAG) after the high ( P <0.02) and low sugar ( P <0.0002) diets, a lower VLDL 1 -TAG fractional catabolic rate after the high sugar diet ( P <0.01), and a higher VLDL 1 -TAG production rate after the low sugar diet ( P <0.01), relative to controls. An effect of the high sugar diet, was to channel hepatic TAG into a higher production of VLDL 1 -TAG ( P <0.02) in the controls, but in contrast, a higher production of VLDL 2 -TAG ( P <0.05) in NAFLD. These dietary effects on VLDL subclass kinetics could be explained, in part, by differences in the contribution of fatty acids from intra-hepatic stores, and de novo lipogenesis. The present study provides new evidence that liver fat accumulation leads to a differential partitioning of hepatic TAG into large and small VLDL subclasses, in response to high and low intakes of sugars. © 2017 The Author(s).

Nd, Pb, Sr, and O isotopic characterization of Saudi Arabian Shield terranes

USGS Publications Warehouse

Stoeser, D.B.; Frost, C.D.

2006-01-01

New Nd, Sr and O isotopic data for granitoid rocks of the Saudi Arabian Shield are presented together with published Nd, Pb, Sr and O isotopic data and all available geologic and geochronologic information to re-evaluate the terranes defined for the Saudi Arabian part of the Arabian-Nubian Shield. Three groups of terranes are identified: 1) the western arc terranes, 2) the eastern arc terranes, and 3) the Khida terrane. The Khida terrane is the only terrane composed of pre-Neoproterozoic continental crust. The western arc terranes are of oceanic arc affinity, and have the least radiogenic Pb and Sr and most radiogenic Nd isotopic compositions and some of the lowest ??18O values of any rocks of the Saudi Arabian Shield. Although some previous studies have characterized the eastern arc terranes as of continental affinity, this study shows that they too are composed of Neoproterozoic oceanic arcs, although their sources have slightly elevated 208Pb/204Pb, Nd, Sri, and ??18O values compared to the western arc terranes. These data suggest that either the isotopic composition of the mantle source for the western arc terranes is more depleted than that of the eastern arc terranes or the eastern arc terranes have been mixed with a small amount of cratonic source material, or both. We further elaborate on the Hulayfah-Ad Dafinah fault zone as a major boundary within the Saudi Arabian portion of the East African Orogen. With further study, its northern extension may be shown to pass through what has been defined as the Hail terrane, and its southern extension appears to lie under cover east of the Tathlith-Malahah terrane and extend into Yemen. It may represent the collision zone between East and West Gondwana, and at the very least it is an important suture between groups of arc terranes of contrasting isotopic composition caught between two converging continents.

UHF wearable battery free sensor module for activity and falling detection.

PubMed

Nam Trung Dang; Thang Viet Tran; Wan-Young Chung

2016-08-01

Falling is one of the most serious medical and social problems in aging population. Therefore taking care of the elderly by detecting activity and falling for preventing and mitigating the injuries caused by falls needs to be concerned. This study proposes a wearable, wireless, battery free ultra-high frequency (UHF) smart sensor tag module for falling and activity detection. The proposed tag is powered by UHF RF wave from reader and read by a standard UHF Electronic Product Code (EPC) Class-1 Generation-2 reader. The battery free sensor module could improve the wearability of the wireless device. The combination of accelerometer signal and received signal strength indication (RSSI) from a reader in the passive smart sensor tag detect the activity and falling of the elderly very successfully. The fabricated smart sensor tag module has an operating range of up to 2.5m and conducting in real-time activity and falling detection.

Isotopic Dependence of GCR Fluence behind Shielding

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Wilson, John W.; Saganti, Premkumar; Kim, Myung-Hee Y.; Cleghorn, Timothy; Zeitlin, Cary; Tripathi, Ram K.

2006-01-01

In this paper we consider the effects of the isotopic composition of the primary galactic cosmic rays (GCR), nuclear fragmentation cross-sections, and isotopic-grid on the solution to transport models used for shielding studies. Satellite measurements are used to describe the isotopic composition of the GCR. For the nuclear interaction data-base and transport solution, we use the quantum multiple-scattering theory of nuclear fragmentation (QMSFRG) and high-charge and energy (HZETRN) transport code, respectively. The QMSFRG model is shown to accurately describe existing fragmentation data including proper description of the odd-even effects as function of the iso-spin dependence on the projectile nucleus. The principle finding of this study is that large errors (+/-100%) will occur in the mass-fluence spectra when comparing transport models that use a complete isotopic-grid (approx.170 ions) to ones that use a reduced isotopic-grid, for example the 59 ion-grid used in the HZETRN code in the past, however less significant errors (<+/-20%) occur in the elemental-fluence spectra. Because a complete isotopic-grid is readily handled on small computer workstations and is needed for several applications studying GCR propagation and scattering, it is recommended that they be used for future GCR studies.

Decoration of silicon nanostructures with copper particles for simultaneous selective capture and mass spectrometry detection of His-tagged model peptide.

PubMed

Coffinier, Yannick; Kurylo, Ievgen; Drobecq, Hervé; Szunerits, Sabine; Melnyk, Oleg; Zaitsev, Vladimir N; Boukherroub, Rabah

2014-10-21

We present in this work a simple and fast preparation method of a new affinity surface-assisted laser/desorption ionization mass spectrometry (SALDI-MS) substrate based on silicon nanostructures decorated with copper particles. The silicon nanostructures were fabricated by the metal-assisted chemical etching (MACE) method. Then, superhydrophilic areas surrounded by superhydrophobic regions were formed through hydrosilylation reaction of 1-octadecene, followed by local degradation of the octadecyl layer. After that, copper particles were deposited in the hydrophilic areas by using the electroless method. We have demonstrated that these surfaces were able to perform high selective capture of model His-tag peptide even in a complex mixture such as serum solution. Then, the captured peptide was detected by mass spectrometry at a femtomolar level without the need of organic matrix.

Lower Granite Dam Smolt Monitoring Program, 2003-2004 Annual Report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mensik, Fred; Rapp, Shawn; Ross, Doug

2004-08-01

The 2003 fish collection season at Lower Granite Dam Juvenile Fish Facility (LGR) was characterized by water temperatures, total flows and spill that were below the five year average, low levels of debris, and increased smolt collection numbers compared to 2002 with the exception of unclipped sockeye/kokanee. There were 6,183,825 juvenile salmonids collected. Of these, 6,054,167 were transported to release sites below Bonneville Dam, 5,957,885 by barge and 96,282 by truck. An additional 102,340 fish were bypassed back to the river, primarily due to research projects with another 62,122 bypassed through the PIT-tag bypass system. According to the PTAGIS database,more » 152,268 PIT-tagged fish were detected at Lower Granite Dam. Of these, Smolt Monitoring Staff recorded 345 PIT-tagged raceway and sample mortalities. Of the 6,183,825 total fish collected, 113,290 were PIT-tagged or radio tagged and 380 were sacrificed by researchers. The collection included 836,885 fish that had hatchery marks other than clipped fins (elastomer, freeze brands or Coded Wire Tags). An estimated 54,857 incidental fish were collected with an additional 8,730 adult salmonids removed from the separator.« less

Burnup calculations and chemical analysis of irradiated fuel samples studied in LWR-PROTEUS phase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grimm, P.; Guenther-Leopold, I.; Berger, H. D.

2006-07-01

The isotopic compositions of 5 UO{sub 2} samples irradiated in a Swiss PWR power plant, which were investigated in the LWR-PROTEUS Phase II programme, were calculated using the CASMO-4 and BOXER assembly codes. The burnups of the samples range from 50 to 90 MWd/kg. The results for a large number of actinide and fission product nuclides were compared to those of chemical analyses performed using a combination of chromatographic separation and mass spectrometry. A good agreement of calculated and measured concentrations is found for many of the nuclides investigated with both codes. The concentrations of the Pu isotopes are mostlymore » predicted within {+-}10%, the two codes giving quite different results, except for {sup 242}Pu. Relatively significant deviations are found for some isotopes of Cs and Sm, and large discrepancies are observed for Eu and Gd. The overall quality of the predictions by the two codes is comparable, and the deviations from the experimental data do not generally increase with burnup. (authors)« less

Regulation of the catalytic behavior of pullulanases chelated onto nickel (II)-modified magnetic nanoparticles.

PubMed

Wang, Jianfeng; Liu, Zhongmei; Zhou, Zhemin

2017-06-01

Chelating of pullulanases onto nickel (II)-modified magnetic nanoparticles results in one-step purification and immobilization of pullulanase, and facilitates the commercial application of pullulanase in industrial scale. To improve the catalytic behavior, especially the operational stability, of the nanocatalyst in consecutive batch reactions, we prepared various iminodiacetic acid-modified magnetic nanoparticles differed in surface polarity and spacer length, on which the His6-tagged pullulanases were chelated via nickel ions, and then studied the correlation between the MNPs surface property and the corresponding catalyst behavior. When pullulanases were chelated onto the surface-modified MNPs, the thermostability of all pullulanase derivatives were lower than that of free counterpart, being not relevant to the protein orientation guided by the locality of the His6-tag, but related to the MNPs basal surface polarity and the grafted spacer length. After chelating of pullulanases onto MNPs, there were changes observed in the pH-activity profile and the apparent Michaelis constant toward pullulan. The changing tendencies were mainly dependent on the His6-tagged pullulanase orientation, and the changing extents were tuned by the spacer length. The reusability of pullulanase immobilized by N-terminal His6-tag was higher than that of pullulanase immobilized by C-terminal His6-tag. Moreover, the reusability of the immobilized pullulanase tested increased till grafting polyether amine-400 as spacer-arm, therefore the N-terminal His6-tagged pullulanase chelating MNPs grafted polyether amine-400 gave the best reusability, which retained 60% of initial activity after 18 consecutive cycles with a total reaction time of 9h. Additionally, the correlation analysis of the catalyst behaviors indicated that the reusability was independent from other catalytic properties such as thermostability and substrate affinity. All the results revealed that the catalyst behavior can be mainly controlled by the His6-tagged pullulanase orientation than by the MNPs surface property which can tune the catalyst function. Copyright © 2017. Published by Elsevier Inc.

I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions.

PubMed

Tackett, Alan J; DeGrasse, Jeffrey A; Sekedat, Matthew D; Oeffinger, Marlene; Rout, Michael P; Chait, Brian T

2005-01-01

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Obiero, Josiah; Bonderoff, Sara A.; Goertzen, Meghan M.

2006-08-01

Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. Deinococcus radiodurans, a Gram-positive bacterium capable of withstanding extreme ionizing radiation, contains two thioredoxins (Trx and Trx1) and a single thioredoxin reductase (TrxR) as part of its response to oxidative stress. Thioredoxin reductase is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Recombinant D. radiodurans TrxR with amore » His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. X-ray diffraction data were collected on a cryocooled crystal to a resolution of 1.9 Å using a synchrotron-radiation source. The space group was determined to be P3{sub 2}21, with unit-cell parameters a = b = 84.33, c = 159.88 Å. The structure of the enzyme has been solved by molecular-replacement methods and structure refinement is in progress.« less

Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.

PubMed

Guillén-Chable, Francisco; Arenas-Sosa, Iván; Islas-Flores, Ignacio; Corzo, Gerardo; Martinez-Liu, Cynthia; Estrada, Georgina

2017-08-01

The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa. Copyright © 2017 Elsevier Inc. All rights reserved.

Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

PubMed

Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee; Jeong, Dabin; Kim, Donghak

2017-05-28

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP + in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni 2+ -affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

PubMed Central

Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

2016-01-01

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

PubMed

Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

2016-01-01

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

PubMed Central

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

2014-01-01

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms. PMID:24457629

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography

PubMed Central

Nomura, Yayoi; Sato, Yumi; Suno, Ryoji; Horita, Shoichiro

2016-01-01

Abstract Fv antibody fragments have been used as co‐crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large‐scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (VL) domain, an intervening removable affinity tag (iRAT), and a variable heavy (VH) domain is expressed by a Gram‐positive bacterial secretion system. This method ensures stoichiometric expression of VL and VH from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site‐specific protease during the purification process to yield tag‐free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT‐based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA‐approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies. PMID:27595817

High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad Javad; Forouzandeh-Moghadam, Mehdi; Allameh, Abdol-Amir

2005-11-01

In contrast to the murine and human VHs, camels' single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.

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

PubMed

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

2014-01-15

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

High-spin level structure and Ground-state phase transition in the odd-mass 103-109Rh isotopes in the framework of exactly solvable sdg interacting boson-fermion model

NASA Astrophysics Data System (ADS)

Ghapanvari, M.; Ghorashi, A. H.; Ranjbar, Z.; Jafarizadeh, M. A.

2018-03-01

In this article, the negative-parity states in the odd-mass 103 - 109Rh isotopes in terms of the sd and sdg interacting-boson fermion models were studied. The transitional interacting boson-fermion model Hamiltonians in sd and sdg-IBFM versions based on affine SU (1 , 1) Lie Algebra were employed to describe the evolution from the spherical to deformed gamma unstable shapes along with the chain of Rh isotopes. In this method, sdg-IBFM Hamiltonian, which is a three level pairing Hamiltonian was determined easily via the exactly solvable method. Some observables of the shape phase transitions such as energy levels, the two neutron separation energies, signature splitting of the γ-vibrational band, the α-decay and double β--decay energies were calculated and examined for these isotopes. The present calculation correctly reproduces the spherical to gamma-soft phase transition in the Rh isotopes. Some comparisons were made with sd-IBFM.

Image Location Estimation by Salient Region Matching.

PubMed

Qian, Xueming; Zhao, Yisi; Han, Junwei

2015-11-01

Nowadays, locations of images have been widely used in many application scenarios for large geo-tagged image corpora. As to images which are not geographically tagged, we estimate their locations with the help of the large geo-tagged image set by content-based image retrieval. In this paper, we exploit spatial information of useful visual words to improve image location estimation (or content-based image retrieval performances). We proposed to generate visual word groups by mean-shift clustering. To improve the retrieval performance, spatial constraint is utilized to code the relative position of visual words. We proposed to generate a position descriptor for each visual word and build fast indexing structure for visual word groups. Experiments show the effectiveness of our proposed approach.

Large-scale identification of target proteins of a glycosyltransferase isozyme by Lectin-IGOT-LC/MS, an LC/MS-based glycoproteomic approach

PubMed Central

Sugahara, Daisuke; Kaji, Hiroyuki; Sugihara, Kazushi; Asano, Masahide; Narimatsu, Hisashi

2012-01-01

Model organisms containing deletion or mutation in a glycosyltransferase-gene exhibit various physiological abnormalities, suggesting that specific glycan motifs on certain proteins play important roles in vivo. Identification of the target proteins of glycosyltransferase isozymes is the key to understand the roles of glycans. Here, we demonstrated the proteome-scale identification of the target proteins specific for a glycosyltransferase isozyme, β1,4-galactosyltransferase-I (β4GalT-I). Although β4GalT-I is the most characterized glycosyltransferase, its distinctive contribution to β1,4-galactosylation has been hardly described so far. We identified a large number of candidates for the target proteins specific to β4GalT-I by comparative analysis of β4GalT-I-deleted and wild-type mice using the LC/MS-based technique with the isotope-coded glycosylation site-specific tagging (IGOT) of lectin-captured N-glycopeptides. Our approach to identify the target proteins in a proteome-scale offers common features and trends in the target proteins, which facilitate understanding of the mechanism that controls assembly of a particular glycan motif on specific proteins. PMID:23002422

Advances in Quantitative Proteomics of Microbes and Microbial Communities

NASA Astrophysics Data System (ADS)

Waldbauer, J.; Zhang, L.; Rizzo, A. I.

2015-12-01

Quantitative measurements of gene expression are key to developing a mechanistic, predictive understanding of how microbial metabolism drives many biogeochemical fluxes and responds to environmental change. High-throughput RNA-sequencing can afford a wealth of information about transcript-level expression patterns, but it is becoming clear that expression dynamics are often very different at the protein level where biochemistry actually occurs. These divergent dynamics between levels of biological organization necessitate quantitative proteomic measurements to address many biogeochemical questions. The protein-level expression changes that underlie shifts in the magnitude, or even the direction, of metabolic and biogeochemical fluxes can be quite subtle and test the limits of current quantitative proteomics techniques. Here we describe methodologies for high-precision, whole-proteome quantification that are applicable to both model organisms of biogeochemical interest that may not be genetically tractable, and to complex community samples from natural environments. Employing chemical derivatization of peptides with multiple isotopically-coded tags, this strategy is rapid and inexpensive, can be implemented on a wide range of mass spectrometric instrumentation, and is relatively insensitive to chromatographic variability. We demonstrate the utility of this quantitative proteomics approach in application to both isolates and natural communities of sulfur-metabolizing and photosynthetic microbes.

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.

PubMed

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.

Identification of Crosslinked Peptides after Click-based Enrichment Using Sequential CID and ETD Tandem Mass Spectrometry

PubMed Central

Chowdhury, Saiful M.; Du, Xiuxia; Tolić, Nikola; Wu, Si; Moore, Ronald J.; Mayer, M. Uljana; Smith, Richard D.; Adkins, Joshua N.

2010-01-01

Chemical crosslinking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of crosslinked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact crosslinkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under condition where the native complex structure is stable. In this study, we present a novel compact crosslinker that contains two distinct features: 1) an alkyne tag and 2) a small molecule detection tag (NO2-) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the crosslinked peptide after proteolytic cleavage after coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO2- moiety provides a secondary means of detecting crosslinked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this crosslinker, which we termed CLIP (Click-enabled Linker for Interacting Proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for crosslinked ubiquitin in highly complex E. coli cell lysates. Sequential CID-MS/MS and ETD-MS/MS of inter-crosslinked peptides (two peptides connected with a crosslinker) are also demonstrated for improved automated identification of crosslinked peptides. PMID:19496583

Studying Nuclear Receptor Complexes in the Cellular Environment.

PubMed

Schaufele, Fred

2016-01-01

The ligand-regulated structure and biochemistry of nuclear receptor complexes are commonly determined by in vitro studies of isolated receptors, cofactors, and their fragments. However, in the living cell, the complexes that form are governed not just by the relative affinities of isolated cofactors for the receptor but also by the cell-specific sequestration or concentration of subsets of competing or cooperating cofactors, receptors, and other effectors into distinct subcellular domains and/or their temporary diversion into other cellular activities. Most methods developed to understand nuclear receptor function in the cellular environment involve the direct tagging of the nuclear receptor or its cofactors with fluorescent proteins (FPs) and the tracking of those FP-tagged factors by fluorescence microscopy. One of those approaches, Förster resonance energy transfer (FRET) microscopy, quantifies the transfer of energy from a higher energy "donor" FP to a lower energy "acceptor" FP attached to a single protein or to interacting proteins. The amount of FRET is influenced by the ligand-induced changes in the proximities and orientations of the FPs within the tagged nuclear receptor complexes, which is an indicator of the structure of the complexes, and by the kinetics of the interaction between FP-tagged factors. Here, we provide a guide for parsing information about the structure and biochemistry of nuclear receptor complexes from FRET measurements in living cells.

Experimental studies related to the origin of the genetic code and the process of protein synthesis - A review

NASA Technical Reports Server (NTRS)

Lacey, J. C., Jr.; Mullins, D. W., Jr.

1983-01-01

A survey is presented of the literature on the experimental evidence for the genetic code assignments and the chemical reactions involved in the process of protein synthesis. In view of the enormous number of theoretical models that have been advanced to explain the origin of the genetic code, attention is confined to experimental studies. Since genetic coding has significance only within the context of protein synthesis, it is believed that the problem of the origin of the code must be dealt with in terms of the origin of the process of protein synthesis. It is contended that the answers must lie in the nature of the molecules, amino acids and nucleotides, the affinities they might have for one another, and the effect that those affinities must have on the chemical reactions that are related to primitive protein synthesis. The survey establishes that for the bulk of amino acids, there is a direct and significant correlation between the hydrophobicity rank of the amino acids and the hydrophobicity rank of their anticodonic dinucleotides.

Carbon source-dependent expansion of the genetic code in bacteria

PubMed Central

Prat, Laure; Heinemann, Ilka U.; Aerni, Hans R.; Rinehart, Jesse; O’Donoghue, Patrick; Söll, Dieter

2012-01-01

Despite the fact that the genetic code is known to vary between organisms in rare cases, it is believed that in the lifetime of a single cell the code is stable. We found Acetohalobium arabaticum cells grown on pyruvate genetically encode 20 amino acids, but in the presence of trimethylamine (TMA), A. arabaticum dynamically expands its genetic code to 21 amino acids including pyrrolysine (Pyl). A. arabaticum is the only known organism that modulates the size of its genetic code in response to its environment and energy source. The gene cassette pylTSBCD, required to biosynthesize and genetically encode UAG codons as Pyl, is present in the genomes of 24 anaerobic archaea and bacteria. Unlike archaeal Pyl-decoding organisms that constitutively encode Pyl, we observed that A. arabaticum controls Pyl encoding by down-regulating transcription of the entire Pyl operon under growth conditions lacking TMA, to the point where no detectable Pyl-tRNAPyl is made in vivo. Pyl-decoding archaea adapted to an expanded genetic code by minimizing TAG codon frequency to typically ∼5% of ORFs, whereas Pyl-decoding bacteria (∼20% of ORFs contain in-frame TAGs) regulate Pyl-tRNAPyl formation and translation of UAG by transcriptional deactivation of genes in the Pyl operon. We further demonstrate that Pyl encoding occurs in a bacterium that naturally encodes the Pyl operon, and identified Pyl residues by mass spectrometry in A. arabaticum proteins including two methylamine methyltransferases. PMID:23185002

High data density and capacity in chipless radiofrequency identification (chipless-RFID) tags based on double-chains of S-shaped split ring resonators (S-SRRs)

NASA Astrophysics Data System (ADS)

Herrojo, Cristian; Mata-Contreras, Javier; Paredes, Ferran; Martín, Ferran

2017-11-01

The data density per surface (DPS) is a figure of merit in chipless radiofrequency identification (chipless-RFID) tags. In this paper, it is demonstrated that chipless-RFID tags with high DPS can be implemented by using double-chains of S-shaped split ring resonators (S-SRRs). Tag reading is achieved by near-field coupling between the tag and the reader, a CPW transmission line fed by a harmonic signal tuned to the resonance frequency of the S-SRRs. By transversally displacing the tag over the CPW, the transmission coefficient of the line is modulated by tag motion. This effectively modulates the amplitude of the injected (carrier) signal at the output port of the line, and the identification (ID) code, determined by the presence or absence of S-SRRs at predefined and equidistant positions in the chains, is contained in the envelope function. The DPS is determined by S-SRR dimensions and by the distance between S-SRRs in the chains. However, by using two chains of S-SRRs, the number of bits per unit length that can be accommodated is very high. This chipless-RFID system is of special interest in applications where the reading distance can be sacrificed in favor of data capacity (e.g., security and authentication). Encoding of corporate documents, ballots, exams, etc., by directly printing the proposed tags on the item product to prevent counterfeiting is envisaged.

The Borexino nylon film and the third counting test facility

NASA Astrophysics Data System (ADS)

McCarty, Kevin B.

The Borexino solar neutrino detector should begin operations in late 2006. This scintillation-based detector will observe low-energy neutrinos, in real time, down to about 250 keV. The experiment should further tighten constraints on the neutrino oscillation parameters, and confirm the Standard Solar Model of solar neutrino production. It may also observe geoneutrinos; supernova neutrinos, should the timing of the experiment be fortunate; and perhaps other processes beyond the scope of the Standard Model of particle physics. At the heart of Borexino lie 300 tons of organic scintillator fluid, contained by a spherical vessel composed of transparent nylon film. Roughly 300 tons of passive buffer fluid lie between this inner vessel and a second outer nylon vessel. Both vessels are located inside a steel sphere that also supports over 2000 inward-pointing photomultiplier tubes. The two most vital components of Borexino are these nylon vessels and the scintillator itself. Numerous measurements made at Princeton of the physical and radiochemical properties of the vessel film are reported in this thesis. A 4-ton prototype of Borexino, the CTF, has been used to study scintillator radiopurity for over ten years. However, certain peculiarities of its design make determining the spatial positions of radioactive decays within the detector difficult. The development of a new position reconstruction code that takes these problems into account is reported herein. Several studies of radiopurity in the latest version of CTF were made using this new code. These include a proposal for individually tagging decays of radon and four daughter isotopes; an attempt to detect convection using the radon daughters; a hypothesis to explain peculiar behavior of the crucial isotope 210 Po; and an analysis of 40 K contamination inside the detector based on models of the spatial distribution of external g rays, leading to a somewhat concerning result. A final distillation test of the scintillator will take place shortly, and will match as closely as possible the procedure used to purify scintillator for the full Borexino detector. Analyses run on CTF data collected after this test should prove vital in understanding the detector sensitivity of Borexino.

Cooperation on Improved Isotopic Identification and Analysis Software for Portable, Electrically Cooled High-Resolution Gamma Spectrometry Systems Final Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dreyer, Jonathan G.; Wang, Tzu-Fang; Vo, Duc T.

Under a 2006 agreement between the Department of Energy (DOE) of the United States of America and the Institut de Radioprotection et de Sûreté Nucléaire (IRSN) of France, the National Nuclear Security Administration (NNSA) within DOE and IRSN initiated a collaboration to improve isotopic identification and analysis of nuclear material [i.e., plutonium (Pu) and uranium (U)]. The specific aim of the collaborative project was to develop new versions of two types of isotopic identification and analysis software: (1) the fixed-energy response-function analysis for multiple energies (FRAM) codes and (2) multi-group analysis (MGA) codes. The project is entitled Action Sheet 4more » – Cooperation on Improved Isotopic Identification and Analysis Software for Portable, Electrically Cooled, High-Resolution Gamma Spectrometry Systems (Action Sheet 4). FRAM and MGA/U235HI are software codes used to analyze isotopic ratios of U and Pu. FRAM is an application that uses parameter sets for the analysis of U or Pu. MGA and U235HI are two separate applications that analyze Pu or U, respectively. They have traditionally been used by safeguards practitioners to analyze gamma spectra acquired with high-resolution gamma spectrometry (HRGS) systems that are cooled by liquid nitrogen. However, it was discovered that these analysis programs were not as accurate when used on spectra acquired with a newer generation of more portable, electrically cooled HRGS (ECHRGS) systems. In response to this need, DOE/NNSA and IRSN collaborated to update the FRAM and U235HI codes to improve their performance with newer ECHRGS systems. Lawrence Livermore National Laboratory (LLNL) and Los Alamos National Laboratory (LANL) performed this work for DOE/NNSA.« less

Expression and affinity purification of recombinant proteins from plants

NASA Technical Reports Server (NTRS)

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).

Systems Biology of Recombinant Protein Production in Bacillus megaterium

NASA Astrophysics Data System (ADS)

Biedendieck, Rebekka; Bunk, Boyke; Fürch, Tobias; Franco-Lara, Ezequiel; Jahn, Martina; Jahn, Dieter

Over the last two decades the Gram-positive bacterium Bacillus megaterium was systematically developed to a useful alternative protein production host. Multiple vector systems for high yield intra- and extracellular protein production were constructed. Strong inducible promoters were combined with DNA sequences for optimised ribosome binding sites, various leader peptides for protein export and N- as well as C-terminal affinity tags for affinity chromatographic purification of the desired protein. High cell density cultivation and recombinant protein production were successfully tested. For further system biology based control and optimisation of the production process the genomes of two B. megaterium strains were completely elucidated, DNA arrays designed, proteome, fluxome and metabolome analyses performed and all data integrated using the bioinformatics platform MEGABAC. Now, solid theoretical and experimental bases for primary modeling attempts of the production process are available.

Interactome Analyses of Mature γ-Secretase Complexes Reveal Distinct Molecular Environments of Presenilin (PS) Paralogs and Preferential Binding of Signal Peptide Peptidase to PS2*

PubMed Central

Jeon, Amy Hye Won; Böhm, Christopher; Chen, Fusheng; Huo, Hairu; Ruan, Xueying; Ren, Carl He; Ho, Keith; Qamar, Seema; Mathews, Paul M.; Fraser, Paul E.; Mount, Howard T. J.; St George-Hyslop, Peter; Schmitt-Ulms, Gerold

2013-01-01

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptides (Aβ) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature γ-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the γ-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H+-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing γ-secretase complexes and was observed to influence Aβ production. PMID:23589300

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress

PubMed Central

Judge, Luke M.; Perez-Bermejo, Juan A.; Truong, Annie; Ribeiro, Alexandre J.S.; Yoo, Jennie C.; Jensen, Christina L.; Mandegar, Mohammad A.; Huebsch, Nathaniel; Kaake, Robyn M.; So, Po-Lin; Srivastava, Deepak; Krogan, Nevan J.

2017-01-01

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity. PMID:28724793

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

PubMed

Judge, Luke M; Perez-Bermejo, Juan A; Truong, Annie; Ribeiro, Alexandre Js; Yoo, Jennie C; Jensen, Christina L; Mandegar, Mohammad A; Huebsch, Nathaniel; Kaake, Robyn M; So, Po-Lin; Srivastava, Deepak; Pruitt, Beth L; Krogan, Nevan J; Conklin, Bruce R

2017-07-20

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

Cloning of a Novel Aldo-Keto Reductase Gene from Klebsiella sp. Strain F51-1-2 and Its Functional Expression in Escherichia coli▿

PubMed Central

Jiang, Hong; Yang, Chao; Qu, Hong; Liu, Zheng; Fu, Q. S.; Qiao, Chuanling

2007-01-01

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P=S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His6-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the Km and kcat values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 ± 47.0 μΜ and 25.7 ± 1.7 min−1, respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling. PMID:17575004

Design and Development of a Technology Platform for DNA-Encoded Library Production and Affinity Selection.

PubMed

Castañón, Jesús; Román, José Pablo; Jessop, Theodore C; de Blas, Jesús; Haro, Rubén

2018-06-01

DNA-encoded libraries (DELs) have emerged as an efficient and cost-effective drug discovery tool for the exploration and screening of very large chemical space using small-molecule collections of unprecedented size. Herein, we report an integrated automation and informatics system designed to enhance the quality, efficiency, and throughput of the production and affinity selection of these libraries. The platform is governed by software developed according to a database-centric architecture to ensure data consistency, integrity, and availability. Through its versatile protocol management functionalities, this application captures the wide diversity of experimental processes involved with DEL technology, keeps track of working protocols in the database, and uses them to command robotic liquid handlers for the synthesis of libraries. This approach provides full traceability of building-blocks and DNA tags in each split-and-pool cycle. Affinity selection experiments and high-throughput sequencing reads are also captured in the database, and the results are automatically deconvoluted and visualized in customizable representations. Researchers can compare results of different experiments and use machine learning methods to discover patterns in data. As of this writing, the platform has been validated through the generation and affinity selection of various libraries, and it has become the cornerstone of the DEL production effort at Lilly.

Using Stable Isotope Analysis to Understand the Migration and Trophic Ecology of Northeastern Pacific White Sharks (Carcharodon carcharias)

PubMed Central

Carlisle, Aaron B.; Kim, Sora L.; Semmens, Brice X.; Madigan, Daniel J.; Jorgensen, Salvador J.; Perle, Christopher R.; Anderson, Scot D.; Chapple, Taylor K.; Kanive, Paul E.; Block, Barbara A.

2012-01-01

The white shark (Carcharodon carcharias) is a wide-ranging apex predator in the northeastern Pacific (NEP). Electronic tagging has demonstrated that white sharks exhibit a regular migratory pattern, occurring at coastal sites during the late summer, autumn and early winter and moving offshore to oceanic habitats during the remainder of the year, although the purpose of these migrations remains unclear. The purpose of this study was to use stable isotope analysis (SIA) to provide insight into the trophic ecology and migratory behaviors of white sharks in the NEP. Between 2006 and 2009, 53 white sharks were biopsied in central California to obtain dermal and muscle tissues, which were analyzed for stable isotope values of carbon (δ13C) and nitrogen (δ15N). We developed a mixing model that directly incorporates movement data and tissue incorporation (turnover) rates to better estimate the relative importance of different focal areas to white shark diet and elucidate their migratory behavior. Mixing model results for muscle showed a relatively equal dietary contribution from coastal and offshore regions, indicating that white sharks forage in both areas. However, model results indicated that sharks foraged at a higher relative rate in coastal habitats. There was a negative relationship between shark length and muscle δ13C and δ15N values, which may indicate ontogenetic changes in habitat use related to onset of maturity. The isotopic composition of dermal tissue was consistent with a more rapid incorporation rate than muscle and may represent more recent foraging. Low offshore consumption rates suggest that it is unlikely that foraging is the primary purpose of the offshore migrations. These results demonstrate how SIA can provide insight into the trophic ecology and migratory behavior of marine predators, especially when coupled with electronic tagging data. PMID:22355313

TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

PubMed Central

Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

2012-01-01

Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media.

PubMed

Massiah, Michael A; Wright, Katharine M; Du, Haijuan

2016-04-01

This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay. Copyright © 2016 John Wiley & Sons, Inc.

Recombinant antigens for immunodiagnosis of cystic echinococcosis

PubMed Central

Li, Jun; Zhang, Wen-Bao

2004-01-01

Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE) and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections. PMID:15188015

Spillover of dietary fatty acids and use of serum nonesterified fatty acids for the synthesis of VLDL-triacylglycerol under two different feeding regimens.

PubMed

Barrows, Brian R; Timlin, Maureen T; Parks, Elizabeth J

2005-09-01

The present study quantified dietary fatty acid flux in healthy men (n = 6) who were fed a liquid formula through a duodenal feeding tube (continuous feeding group) or who consumed the same formula in meals (meal feeding group). A triacylglycerol (TAG) stable isotope was added to the formula to determine the entry of dietary fatty acids into the serum and its clearance to the liver and resecretion into serum via VLDL. The contribution of dietary fatty acids to serum nonesterified fatty acids (NEFAs) was higher with meal feeding (24.4 +/- 2.6%) compared with continuous feeding (10.8 +/- 2.9%, P < 0.01) and, when multiplied by the NEFA concentration, resulted in similar absolute fatty acid spillover. Diet-derived NEFAs subsequently represented 10.6 +/- 1.2% and 4.7 +/- 1.3% of hepatic VLDL-TAG (meal feeding vs. continuous feeding, respectively, P = 0.004). Chylomicron remnant uptake by the liver contributed 9.3 +/- 1.9% of fatty acids to hepatic VLDL-TAG synthesis with meal feeding compared with continuous feeding (4.4 +/- 0.8%, P < 0.03). These data suggest that the extent of dietary fatty acid recycling via serum NEFAs and VLDL-TAG is determined by the rate of delivery of dietary fat to the intestine. The inefficient removal of dietary fat from the circulation may maintain VLDL-TAG production but may also result in prolonged postprandial lipemia.

Toward biophysical probes for the 5-HT3 receptor: structure-activity relationship study of granisetron derivatives.

PubMed

Vernekar, Sanjeev Kumar V; Hallaq, Hasan Y; Clarkson, Guy; Thompson, Andrew J; Silvestri, Linda; Lummis, Sarah C R; Lochner, Martin

2010-03-11

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT(3)A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT(3)A receptors in mammalian cells.

Toward Biophysical Probes for the 5-HT3 Receptor: Structure−Activity Relationship Study of Granisetron Derivatives

PubMed Central

2010-01-01

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT3A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT3A receptors in mammalian cells. PMID:20146481

Targeting the UPR to Circumvent Endocrine Resistance in Breast Cancer

DTIC Science & Technology

2015-10-01

were submitted for the screen. Kinase assay protocol (DiscoverX): For most assays, kinase-tagged T7 phage strains were grown in parallel in 24-well...blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of...unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test

Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

PubMed

Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

2013-12-01

The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA. © 2013.

Off-Range Beaked Whale Studies (ORBS): Baseline Data and Tagging Development for Northern Bottlenose Whales (Hyperoodon ampulatus) off Jan Mayen, Norway

DTIC Science & Technology

2015-09-30

02.003’N, 07°01.981’W) To be recovered in 2016 Ranging code #08D1; releasing code #0803 In collaboration with Rune Hansen of the University of...the animal with PTT 134760 was tracked moving all the way south to the Azores Archipelago. Figure courtesy of Rune Hansen. Objective 4. conduct

Study of phase transition of even and odd nuclei based on q-deforme SU(1,1) algebraic model

NASA Astrophysics Data System (ADS)

Jafarizadeh, M. A.; Amiri, N.; Fouladi, N.; Ghapanvari, M.; Ranjbar, Z.

2018-04-01

The q-deformed Hamiltonian for the SO (6) ↔ U (5) transitional case in s, d interaction boson model (IBM) can be constructed by using affine SUq (1 , 1) Lie algebra in the both IBM-1 and 2 versions and IBFM. In this research paper, we have studied the energy spectra of 120-128Xe isotopes and 123-131Xe isotopes and B(E2) transition probabilities of 120-128Xe isotopes in the shape phase transition region between the spherical and gamma unstable deformed shapes of the theory of quantum deformation. The theoretical results agree with the experimental data fairly well. It is shown that the q-deformed SO (6) ↔ U (5) transitional dynamical symmetry remains after deformation.

General Framework for Animal Food Safety Traceability Using GS1 and RFID

NASA Astrophysics Data System (ADS)

Cao, Weizhu; Zheng, Limin; Zhu, Hong; Wu, Ping

GS1 is global traceability standard, which is composed by the encoding system (EAN/UCC, EPC), the data carriers identified automatically (bar codes, RFID), electronic data interchange standards (EDI, XML). RFID is a non-contact, multi-objective automatic identification technique. Tracing of source food, standardization of RFID tags, sharing of dynamic data are problems to solve urgently for recent traceability systems. The paper designed general framework for animal food safety traceability using GS1 and RFID. This framework uses RFID tags encoding with EPCglobal tag data standards. Each information server has access tier, business tier and resource tier. These servers are heterogeneous and distributed, providing user access interfaces by SOAP or HTTP protocols. For sharing dynamic data, discovery service and object name service are used to locate dynamic distributed information servers.

Amino acid analysis in physiological samples by GC-MS with propyl chloroformate derivatization and iTRAQ-LC-MS/MS.

PubMed

Dettmer, Katja; Stevens, Axel P; Fagerer, Stephan R; Kaspar, Hannelore; Oefner, Peter J

2012-01-01

Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography-quadrupole mass spectrometry (GC-qMS) analysis in single-ion monitoring mode. Derivatization is carried out directly in aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC-MS. The method delivers the quantification of 26 amino acids. The isobaric tagging for relative and absolute quantification (iTRAQ) method employs the labeling of amino acids with isobaric iTRAQ tags. The tags contain two different cleavable reporter ions, one for the sample and one for the standard, which are detected by fragmentation in a tandem mass spectrometer. Reversed-phase liquid chromatography of the labeled amino acids is performed prior to mass spectrometric analysis to separate isobaric amino acids. The commercial iTRAQ kit allows for the analysis of 42 physiological amino acids with a respective isotope-labeled standard for each of these 42 amino acids.

Use of the QR Reader to Provide Real-Time Evaluation of Residents' Skills Following Surgical Procedures.

PubMed

Reynolds, Kellin; Barnhill, Danny; Sias, Jamie; Young, Amy; Polite, Florencia Greer

2014-12-01

A portable electronic method of providing instructional feedback and recording an evaluation of resident competency immediately following surgical procedures has not previously been documented in obstetrics and gynecology. This report presents a unique electronic format that documents resident competency and encourages verbal communication between faculty and residents immediately following operative procedures. The Microsoft Tag system and SurveyMonkey platform were linked by a 2-D QR code using Microsoft QR code generator. Each resident was given a unique code (TAG) embedded onto an ID card. An evaluation form was attached to each resident's file in SurveyMonkey. Postoperatively, supervising faculty scanned the resident's TAG with a smartphone and completed the brief evaluation using the phone's screen. The evaluation was reviewed with the resident and automatically submitted to the resident's educational file. The evaluation system was quickly accepted by residents and faculty. Of 43 residents and faculty in the study, 38 (88%) responded to a survey 8 weeks after institution of the electronic evaluation system. Thirty (79%) of the 38 indicated it was superior to the previously used handwritten format. The electronic system demonstrated improved utilization compared with paper evaluations, with a mean of 23 electronic evaluations submitted per resident during a 6-month period versus 14 paper assessments per resident during an earlier period of 6 months. This streamlined portable electronic evaluation is an effective tool for direct, formative feedback for residents, and it creates a longitudinal record of resident progress. Satisfaction with, and use of, this evaluation system was high.

Use of the QR Reader to Provide Real-Time Evaluation of Residents' Skills Following Surgical Procedures

PubMed Central

Reynolds, Kellin; Barnhill, Danny; Sias, Jamie; Young, Amy; Polite, Florencia Greer

2014-01-01

Background A portable electronic method of providing instructional feedback and recording an evaluation of resident competency immediately following surgical procedures has not previously been documented in obstetrics and gynecology. Objective This report presents a unique electronic format that documents resident competency and encourages verbal communication between faculty and residents immediately following operative procedures. Methods The Microsoft Tag system and SurveyMonkey platform were linked by a 2-D QR code using Microsoft QR code generator. Each resident was given a unique code (TAG) embedded onto an ID card. An evaluation form was attached to each resident's file in SurveyMonkey. Postoperatively, supervising faculty scanned the resident's TAG with a smartphone and completed the brief evaluation using the phone's screen. The evaluation was reviewed with the resident and automatically submitted to the resident's educational file. Results The evaluation system was quickly accepted by residents and faculty. Of 43 residents and faculty in the study, 38 (88%) responded to a survey 8 weeks after institution of the electronic evaluation system. Thirty (79%) of the 38 indicated it was superior to the previously used handwritten format. The electronic system demonstrated improved utilization compared with paper evaluations, with a mean of 23 electronic evaluations submitted per resident during a 6-month period versus 14 paper assessments per resident during an earlier period of 6 months. Conclusions This streamlined portable electronic evaluation is an effective tool for direct, formative feedback for residents, and it creates a longitudinal record of resident progress. Satisfaction with, and use of, this evaluation system was high. PMID:26140128

A new algorithm to handle finite nuclear mass effects in electronic calculations: the ISOTOPE program.

PubMed

Gonçalves, Cristina P; Mohallem, José R

2004-11-15

We report the development of a simple algorithm to modify quantum chemistry codes based on the LCAO procedure, to account for the isotope problem in electronic structure calculations. No extra computations are required compared to standard Born-Oppenheimer calculations. An upgrade of the Gamess package called ISOTOPE is presented, and its applicability is demonstrated in some examples.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rowe, Nathan C; Younkin, James R; Pickett, Chris A

Radio-Frequency (RF) devices have revolutionized many aspects of modern industrial processes. RF technology can enable wireless communication for tag identification, sensor communication, and asset tracking. Radio-frequency identification (RFID) is a technology that utilizes wireless communication to interrogate and identify an electronic tag attached to an item in order to identify the item. The technology can come in many forms: passive or active tags, low to ultra-wideband frequencies, small paper-thin tags to brick-sized units, and simple tags or highly integrated sensor packages. RF technology, and specifically RFID, has been applied widely in commercial markets for inventory, supply chain management, and assetmore » tracking. Several recent studies have demonstrated the safeguards benefits of utilizing RFID versus conventional inventory tagging methods for tracking nuclear material. These studies have indicated that the RF requirements for safeguards functions are more stringent than the RF requirements for other inventory tracking and accounting applications. Additionally, other requirements must be addressed, including environmental and operating conditions, authentication, and tag location and attachment. Facility restrictions on radio spectrum, method of tag attachment, and sensitivity of the data collected impact the tag selection and system design. More important, the intended use of the system must be considered. The requirements for using RF to simply replace or supplement container identifiers such as bar codes that facilitate the inventory function will differ greatly from the requirements for deploying RF for unattended monitoring applications. Several studies have investigated these considerations to advance commercial RF devices for safeguards use, and a number of system concepts have been developed. This paper will provide an overview of past studies and current technologies, and will investigate the requirements, existing gaps, and several potential next steps for advancing RF techniques for safeguards use.« less

How the brain assigns a neural tag to arbitrary points in a high-dimensional space

NASA Astrophysics Data System (ADS)

Stevens, Charles

Brains in almost all organisms need to deal with very complex stimuli. For example, most mammals are very good at face recognition, and faces are very complex objects indeed. For example, modern face recognition software represents a face as a point in a 10,000 dimensional space. Every human must be able to learn to recognize any of the 7 billion faces in the world, and can recognize familiar faces after a display of the face is viewed for only a few hundred milliseconds. Because we do not understand how faces are assigned locations in a high-dimensional space by the brain, attacking the problem of how face recognition is accomplished is very difficult. But a much easier problem of the same sort can be studied for odor recognition. For the mouse, each odor is assigned a point in a 1000 dimensional space, and the fruit fly assigns any odor a location in only a 50 dimensional space. A fly has about 50 distinct types of odorant receptor neurons (ORNs), each of which produce nerve impulses at a specific rate for each different odor. This pattern of firing produced across 50 ORNs is called `a combinatorial odor code', and this code assigns every odor a point in a 50 dimensional space that is used to identify the odor. In order to learn the odor, the brain must alter the strength of synapses. The combinatorial code cannot itself by used to change synaptic strength because all odors use same neurons to form the code, and so all synapses would be changed for any odor and the odors could not be distinguished. In order to learn an odor, the brain must assign a set of neurons - the odor tag - that have the property that these neurons (1) should make use of all of the information available about the odor, and (2) insure that any two tags overlap as little as possible (so one odor does not modify synapses used by other odors). In the talk, I will explain how the olfactory system of both the fruit fly and the mouse produce a tag for each odor that has these two properties. Supported by NSF.

Isolation of Metarhizium anisopliae carboxypeptidase A with native disulfide bonds from the cytosol of Escherichia coli BL21(DE3)

PubMed Central

Austin, Brian P.; Waugh, David S.

2011-01-01

The carboxypeptidase A enzyme from Metarhizium anisopliae (MeCPA) has broader specificity than the mammalian A-type carboxypeptidases, making it a more useful reagent for the removal of short affinity tags and disordered residues from the C-termini of recombinant proteins. When secreted from baculovirus-infected insect cells, the yield of pure MeCPA was 0.25 mg per liter of conditioned medium. Here, we describe a procedure for the production of MeCPA in the cytosol of Escherichia coli that yields approximately 0.5 mg of pure enzyme per liter of cell culture. The bacterial system is much easier to scale up and far less expensive than the insect cell system. The expression strategy entails maintaining the proMeCPA zymogen in a soluble state by fusing it to the C-terminus of maltose-binding protein (MBP) while simultaneously overproducing the protein disulfide isomerase DsbC in the cytosol from a separate plasmid. Unexpectedly, we found that the yield of active and properly oxidized MeCPA was highest when coexpressed with DsbC in BL21(DE3) cells that do not also contain mutations in the trxB and gor genes. Moreover, the formation of active MeCPA was only partially dependent on the disulfide-isomerase activity of DsbC. Intriguingly, we observed that most of the active MeCPA was generated after cell lysis and amylose affinity purification of the MBP-proMeCPA fusion protein, during the time that the partially purified protein was held overnight at 4 °C prior to activation with thermolysin. Following removal of the MBP-propeptide by thermolysin digestion, active MeCPA (with a C-terminal polyhistidine tag) was purified to homogeneity by immobilized metal affinity chromatography (IMAC), ion exchange chromatography and gel filtration. PMID:22197595

Grafting iminodiacetic acid on silica nanoparticles for facilitated refolding of like-charged protein and its metal-chelate affinity purification.

PubMed

Liu, Hu; Dong, Xiaoyan; Sun, Yan

2016-01-15

A series of highly charged nanoscale chelators were fabricated by grafting of poly(glycidyl methacrylate-iminodiacetic acid) (pGI) chains with iminodiacetic acid (IDA) chelating group on silica nanoparticles (SNPs) via atom transfer radical polymerization (ATRP). The nanoscale chelators, denoted as SNPs-pGI, possessed a nickel ion chelating capacity as high as 2800 μmol/g, 50 times higher than the IDA-modified Sepharose FF (IDA-Sepharose) resin reported in literature and offered a high affinity binding capacity for hexahistidine-tagged enhanced green fluorescence protein (6 × His-EGFP) after nickel ion loading. More importantly, the anionic SNPs-pGI of high charge densities displayed much better performance than IDA-Sepharose in facilitating the refolding of like-charged 6 × His-EGFP from inclusion bodies (IBs). For example, for 0.2mg/mL 6 × His-EGFP IB refolding, addition of 6.2 μL/mL SNPs-pGI with the highest charge density led to a refolding yield of 90%, over 43% higher than that obtained with 460 μL/mL IDA-Sepharose. It is notable that the much higher efficiency of the nanoscale chelator was obtained with a chelator consumption corresponding to only 1.4% of IDA-Sepharose. Moreover, the highly charged SNPs-pGI could efficiently facilitate the refolding of 6 × His-EGFP at higher IB concentrations (0.4 and 0.8 mg/mL). After refolding, nickel ions addition led to the recovery of the refolded 6 × His-EGFP with high yield (80%), purity (96%) and enrichment ratio (1.8). All the results suggest that the SNPs-pGI of high charge densities were promising for cost-effective recovery of His-tagged proteins expressed as IBs with the integrative like-charge facilitated refolding and metal-chelate affinity purification strategy. Copyright © 2015 Elsevier B.V. All rights reserved.

Modification degrees at specific sites on heparan sulphate: an approach to measure chemical modifications on biological molecules with stable isotope labelling

PubMed Central

Wu, Zhengliang L.; Lech, Miroslaw

2005-01-01

Chemical modification of biological molecules is a general mechanism for cellular regulation. A quantitative approach has been developed to measure the extent of modification on HS (heparan sulphates). Sulphation on HS by sulphotransferases leads to variable sulphation levels, which allows cells to tune their affinities to various extracellular proteins, including growth factors. With stable isotope labelling and HPLC-coupled MS, modification degrees at various O-sulphation sites could be determined. A bovine kidney HS sample was first saturated in vitro with 34S by an OST (O-sulphotransferase), then digested with nitrous acid and analysed with HPLC-coupled MS. The 34S-labelled oligosaccharides were identified based on their unique isotope clusters. The modification degrees at the sulphotransferase recognition sites were obtained by calculating the intensities of isotopic peaks in the isotope clusters. The modification degrees at 3-OST-1 and 6-OST-1 sites were examined in detail. This approach can also be used to study other types of chemical modifications on biological molecules. PMID:15743272

Using Tidal Fluctuation-Induced Dynamics of Radium Isotopes (224Ra, 223Ra, and 228Ra) to Trace the Hydrodynamics and Geochemical Reactions in a Coastal Groundwater Mixing Zone

NASA Astrophysics Data System (ADS)

Liu, Yi; Jiao, Jiu Jimmy; Liang, Wenzhao; Luo, Xin

2018-04-01

The reactive transport of radium isotopes (224Ra, 223Ra, and 228Ra) in coastal groundwater mixing zones (CGMZs) is sensitive to shifts of redox conditions and geochemical reactions induced by tidal fluctuation. This study presents a spatial distribution and temporal variation of radium isotopes in the CGMZ for the first time. Results show that the activity of radium isotopes in the upper saline plume (USP) is comparatively low due to a short residence time and mixing loss induced by the infiltration of low radium seawater whereas the activity of radium isotopes in the salt wedge (SW) is comparatively high due to a long residence time in the aquifer. The spatial distribution of radium isotopes is determined by the partitioning of radium isotopes, groundwater residence time, and relative ingrowth rates of radium isotopes. In addition, the variation of radium isotopes in the USP lags slightly (˜0 h) whereas the fluctuation of radium isotopes in the SW lags significantly (˜12 h) behind sea level oscillation. Tidal fluctuation affects the partitioning of radium isotopes through controlling seawater infiltration and subsequently influences the dynamics of radium isotopes in the USP. Concurrently, seawater infiltration significantly affects geochemical processes such as the production of nutrients and total alkalinity. Therefore, radium dynamics in the USP have implications for these geochemical processes. The variation of radium isotopes in the USP also has potential implications for transformation of trace metals such as iron and manganese because of the close affinity of radium isotopes to manganese and iron oxides.

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

PubMed Central

Campos, Magnólia de A; Silva, Marilia S; Magalhães, Cláudio P; Ribeiro, Simone G; Sarto, Rafael PD; Vieira, Eduardo A; Grossi de Sá, Maria F

2008-01-01

Background Heterologous protein expression in microorganisms may contribute to identify and demonstrate antifungal activity of novel proteins. The Solanum nigrum osmotin-like protein (SnOLP) gene encodes a member of pathogenesis-related (PR) proteins, from the PR-5 sub-group, the last comprising several proteins with different functions, including antifungal activity. Based on deduced amino acid sequence of SnOLP, computer modeling produced a tertiary structure which is indicative of antifungal activity. Results To validate the potential antifungal activity of SnOLP, a hexahistidine-tagged mature SnOLP form was overexpressed in Escherichia coli M15 strain carried out by a pQE30 vector construction. The urea solubilized His6-tagged mature SnOLP protein was affinity-purified by immobilized-metal (Ni2+) affinity column chromatography. As SnOLP requires the correct formation of eight disulfide bonds, not correctly formed in bacterial cells, we adapted an in vitro method to refold the E. coli expressed SnOLP by using reduced:oxidized gluthatione redox buffer. This method generated biologically active conformations of the recombinant mature SnOLP, which exerted antifungal action towards plant pathogenic fungi (Fusarium solani f. sp.glycines, Colletotrichum spp., Macrophomina phaseolina) and oomycete (Phytophthora nicotiana var. parasitica) under in vitro conditions. Conclusion Since SnOLP displays activity against economically important plant pathogenic fungi and oomycete, it represents a novel PR-5 protein with promising utility for biotechnological applications. PMID:18334031

Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells.

PubMed

Lee, Ji-Hye; Lee, Ji-Eun; Kang, Kyung-Jung; Jang, Young-Joo

2017-07-01

Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H 6 ) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN 6 ) is also efficient for purification as it is easily exposed on the surface of the protein. In this study, four different tagging constructs of hFGF-2 based on tag positions and types (H 6 -FGF2, FGF2-H 6 , HN 6 -FGF2, and FGF2-HN 6 ) were designed and expressed under the inducible T7 expression system in E. coli. The experimental conditions of expression and purification of each recombinant protein were optimized. The effective dosages of the recombinant proteins were determined based on the increase of cell proliferation in human gingival fibroblast. ED50s of H 6 -FGF2, FGF2-H 6 , HN 6 -FGF2, and FGF2-HN 6 were determined (4.42 ng/ml, 3.55 ng/ml, 3.54 ng/ml, and 4.14 ng/ml, respectively) and found to be comparable to commercial FGF-2 (3.67 ng/ml). All the recombinant hFGF-2s inhibit the osteogenic induction and mineralization in human periodontal ligament-derived cells. Our data suggested that biological activities of the recombinant hFGF-2 are irrelevant to types and positions of tags, but may have an influence on the expression efficiency and solubility. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme.

PubMed

Antoniou, Georgia; Papakyriacou, Irineos; Papaneophytou, Christos

2017-10-01

Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.

Novel 6xHis tagged foot-and-mouth disease virus vaccine bound to nanolipoprotein adjuvant via metal ions provides antigenic distinction and effective protective immunity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rai, Devendra K.; Segundo, Fayna Diaz-San; Department of Pathobiology and Veterinary Science, CANR, University of Connecticut, Storrs, CT 06269

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co{sup 2+} affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni{sup 2+}-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDVmore » neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance. - Highlights: • 6xHis-tags in A{sub 24} FMDV enable purification and biding to adjuvants via metal ions. • 6xHis A{sub 24} FMDV:MPLA:NiNLP vaccine enhanced protective immunity against FMDV. • Surface exposed capsid tags allow distinction of infected from vaccinated animals.« less

Escherichia coli DnaA forms helical structures along the longitudinal cell axis distinct from MreB filaments.

PubMed

Boeneman, Kelly; Fossum, Solveig; Yang, Yanhua; Fingland, Nicholas; Skarstad, Kirsten; Crooke, Elliott

2009-05-01

DnaA initiates chromosomal replication in Escherichia coli at a well-regulated time in the cell cycle. To determine how the spatial distribution of DnaA is related to the location of chromosomal replication and other cell cycle events, the localization of DnaA in living cells was visualized by confocal fluorescence microscopy. The gfp gene was randomly inserted into a dnaA-bearing plasmid via in vitro transposition to create a library that included internally GFP-tagged DnaA proteins. The library was screened for the ability to rescue dnaA(ts) mutants, and a candidate gfp-dnaA was used to replace the dnaA gene of wild-type cells. The resulting cells produce close to physiological levels of GFP-DnaA from the endogenous promoter as their only source of DnaA and somewhat under-initiate replication with moderate asynchrony. Visualization of GFP-tagged DnaA in living cells revealed that DnaA adopts a helical pattern that spirals along the long axis of the cell, a pattern also seen in wild-type cells by immunofluorescence with affinity purified anti-DnaA antibody. Although the DnaA helices closely resemble the helices of the actin analogue MreB, co-visualization of GFP-tagged DnaA and RFP-tagged MreB demonstrates that DnaA and MreB adopt discrete helical structures along the length of the longitudinal cell axis.

Thermal ecology of subadult and adult muskellunge in a thermally enriched reservoir

USGS Publications Warehouse

Cole, A. J.; Bettoli, Phillip William

2014-01-01

The movement of adult muskellunge, Esox masquinongy Mitchill, has been investigated in a variety of systems, but temperature selection by muskellunge has not been examined where well-oxygenated waters were available over a range of temperatures for much of the year. Thirty subadult and adult muskellunge tagged internally with temperature-sensing radio tags were tracked from March 2010 to March 2011 in a Tennessee reservoir. Mean tag temperatures were 18.9 °C in spring (March to May), 22.1 °C in summer (June to August), 16.5 °C in autumn and 9.8 °C in winter (December to February). When the greatest range in water temperatures was available (7.1–33.3 °C; May to early August 2010), their realised thermal niche (mean ± 1 SD) was 22.3 °C ± 1.8; the realised thermal niche was affected by fish size (smaller fish selected slightly warmer temperatures) but not sex. An electric generating steam plant discharging warm water resumed operation in January 2011, and most (86%) tagged fish occupied the plume where temperatures were ≈10 °C warmer than ambient water temperatures. No mortalities were observed 15 days later when plant operations ceased. Their affinity for the heated plume prompted concerns that muskellunge will be too easily exploited when the plant operates during winter.

Structural characterization of core-bradavidin in complex with biotin

PubMed Central

Agrawal, Nitin; Määttä, Juha A. E.; Kulomaa, Markku S.; Hytönen, Vesa P.; Johnson, Mark S.; Airenne, Tomi T.

2017-01-01

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin. PMID:28426764

A recombinant estrogen receptor fragment-based homogeneous fluorescent assay for rapid detection of estrogens.

PubMed

Wang, Dan; Xie, Jiangbi; Zhu, Xiaocui; Li, Jinqiu; Zhao, Dongqin; Zhao, Meiping

2014-05-15

In this work, we demonstrate a novel estrogenic receptor fragment-based homogeneous fluorescent assay which enables rapid and sensitive detection of 17β-estradiol (E2) and other highly potent estrogens. A modified human estrogenic receptor fragment (N-His × 6-hER270-595-C-Strep tag II) has been constructed that contains amino acids 270-595 of wild-type human estrogenic receptor α (hER270-595) and two specific tags (6 × His and Strep tag II) fused to the N and C terminus, respectively. The designed receptor protein fragment could be easily produced by prokaryotic expression with high yield and high purity. The obtained protein exhibits high binding affinity to E2 and the two tags greatly facilitate the application of the recombinant protein. Taking advantage of the unique spectroscopic properties of coumestrol (CS), a fluorescent phytoestrogen, a CS/hER270-595-based fluorescent assay has been developed which can sensitively respond to E2 within 1.0 min with a linear working range from 0.1 to 20 ng/mL and a limit of detection of 0.1 ng/mL. The assay was successfully applied for rapid detection of E2 in the culture medium of rat hippocampal neurons. The method also holds great potential for high-throughput monitoring the variation of estrogen levels in complex biological fluids, which is crucial for investigation of the molecular basis of various estrogen-involved processes. Copyright © 2013 Elsevier B.V. All rights reserved.

Spatial and ontogenetic variability in the chemical composition of juvenile common sole ( Solea solea) otoliths

NASA Astrophysics Data System (ADS)

Tanner, S. E.; Vasconcelos, R. P.; Reis-Santos, P.; Cabral, H. N.; Thorrold, S. R.

2011-01-01

A description of variations in the chemical composition of fish otoliths at different spatial scales and life history stages is a prerequisite for their use as natural tags in fish population connectivity and migration studies. Otolith geochemistry of juvenile common sole ( Solea solea), a marine migrant species collected in six Portuguese estuaries was examined. Elemental ratios (Mg:Ca, Mn:Ca, Cu:Ca, Sr:Ca, Ba:Ca, Pb:Ca) were analysed in two zones of the right otolith (corresponding to late larval and juvenile stages) using laser ablation inductively coupled plasma mass spectrometry (ICP-MS). Stable carbon and oxygen isotopes (δ 13C and δ 18O) were determined in left otoliths using isotopic ratio monitoring mass spectrometry (irm-MS). Significant differences in otolith geochemical signatures were found among estuaries, among sites within estuaries and between otolith zones. Several elemental ratios (Mg:Ca, Mn:Ca, Cu:Ca and Sr:Ca) showed consistent patterns between otolith zones and were likely influenced by environmental factors and ontogenetic effects associated with physiological changes during metamorphosis. Assignment of individuals to their collection estuary based on the otolith geochemical signatures was more accurate at the site level (81%) than among estuaries (69%). Site temperature was not correlated with any of the elemental or isotope ratios, but salinity was significantly correlated with Ba:Ca, δ 13C and δ 18O. Observed spatial variations among estuaries and sites within estuaries indicate that geochemical signatures in otoliths are accurate natural tags of estuarine habitat in common sole. Nevertheless, the significant variations observed between otolith zones should be taken into account in the design of population connectivity studies.

Using Satellite Tracking and Isotopic Information to Characterize the Impact of South American Sea Lions on Salmonid Aquaculture in Southern Chile.

PubMed

Sepúlveda, Maritza; Newsome, Seth D; Pavez, Guido; Oliva, Doris; Costa, Daniel P; Hückstädt, Luis A

2015-01-01

Apex marine predators alter their foraging behavior in response to spatial and/or seasonal changes in natural prey distribution and abundance. However, few studies have identified the impacts of aquaculture that represents a spatially and temporally predictable and abundant resource on their foraging behavior. Using satellite telemetry and stable isotope analysis we examined the degree of spatial overlap between the South American sea lion (SASL) and salmon farms, and quantify the amount of native prey versus farmed salmonids in SASL diets. We instrumented eight SASL individuals with SRDL-GPS tags. Vibrissae, hair and skin samples were collected for δ13C and δ15N analyses from five of the tagged individuals and from four males captured in a haul-out located adjacent to salmon farms. Tracking results showed that almost all the foraging areas of SASL are within close proximity to salmon farms. The most important prey for the individuals analyzed was farmed salmonids, with an estimated median (±SD) contribution of 19.7 ± 13.5‰ and 15.3 ± 9.6‰ for hair and skin, respectively. Using vibrissae as a temporal record of diet for each individual, we observed a remarkable switch in diet composition in two SASL, from farmed salmonids to pelagic fishes, which coincided with the decrease of salmon production due to the infectious salmon anemia virus that affected salmon farms in Chile at the end of 2008. Our study demonstrates the usefulness of integrating stable isotope derived dietary data with movement patterns to characterize the impacts of a non-native prey on the foraging ecology of an apex marine predator, providing important applied implications in situations where interactions between aquaculture and wildlife are common.

Using Satellite Tracking and Isotopic Information to Characterize the Impact of South American Sea Lions on Salmonid Aquaculture in Southern Chile

PubMed Central

Sepúlveda, Maritza; Newsome, Seth D.; Pavez, Guido; Oliva, Doris; Costa, Daniel P.; Hückstädt, Luis A.

2015-01-01

Apex marine predators alter their foraging behavior in response to spatial and/or seasonal changes in natural prey distribution and abundance. However, few studies have identified the impacts of aquaculture that represents a spatially and temporally predictable and abundant resource on their foraging behavior. Using satellite telemetry and stable isotope analysis we examined the degree of spatial overlap between the South American sea lion (SASL) and salmon farms, and quantify the amount of native prey versus farmed salmonids in SASL diets. We instrumented eight SASL individuals with SRDL-GPS tags. Vibrissae, hair and skin samples were collected for δ13C and δ15N analyses from five of the tagged individuals and from four males captured in a haul-out located adjacent to salmon farms. Tracking results showed that almost all the foraging areas of SASL are within close proximity to salmon farms. The most important prey for the individuals analyzed was farmed salmonids, with an estimated median (±SD) contribution of 19.7 ± 13.5‰ and 15.3 ± 9.6‰ for hair and skin, respectively. Using vibrissae as a temporal record of diet for each individual, we observed a remarkable switch in diet composition in two SASL, from farmed salmonids to pelagic fishes, which coincided with the decrease of salmon production due to the infectious salmon anemia virus that affected salmon farms in Chile at the end of 2008. Our study demonstrates the usefulness of integrating stable isotope derived dietary data with movement patterns to characterize the impacts of a non-native prey on the foraging ecology of an apex marine predator, providing important applied implications in situations where interactions between aquaculture and wildlife are common. PMID:26309046

Inhibition of ethylene synthesis reduces salt-tolerance in tomato wild relative species Solanum chilense.

PubMed

Gharbi, Emna; Martínez, Juan-Pablo; Benahmed, Hela; Lepoint, Gilles; Vanpee, Brigitte; Quinet, Muriel; Lutts, Stanley

2017-03-01

Exposure to salinity induces a burst in ethylene synthesis in the wild tomato halophyte plant species Solanum chilense. In order to gain information on the role of ethylene in salt adaptation, plants of Solanum chilense (accession LA4107) and of cultivated glycophyte Solanum lycopersicum (cv. Ailsa Craig) were cultivated for 7days in nutrient solution containing 0 or 125mM NaCl in the presence or absence of the inhibitor of ethylene synthesis (aminovinylglycine (AVG) 2μM). Salt-induced ethylene synthesis in S. chilense occurred concomitantly with an increase in stomatal conductance, an efficient osmotic adjustment and the maintenance of carbon isotope discrimination value (Δ 13 C). In contrast, in S. lycopersicum, salt stress decreased stomatal conductance and Δ 13 C values while osmotic potential remained higher than in S. chilense. Inhibition of stress-induced ethylene synthesis by AVG decreased stomatal conductance and Δ 13 C in S. chilense and compromised osmotic adjustment. Solanum chilense behaved as an includer and accumulated high amounts of Na in the shoot but remained able to maintain K nutrition in the presence of NaCl. This species however did not stimulate the expression of genes coding for high-affinity K transport but genes coding for ethylene responsive factor ERF5 and JREF1 were constitutively more expressed in S. chilense than in S. lycopersicum. It is concluded that ethylene plays a key role in salt tolerance of S. chilense. Copyright © 2016. Published by Elsevier GmbH.

Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification.

PubMed

Chen, Yong; Li, Yang; Liu, Peng; Sun, Qun; Liu, Zhu

2014-04-01

A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

PubMed

Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function. Published by Elsevier Masson SAS.

Security Enhancement Mechanism Based on Contextual Authentication and Role Analysis for 2G-RFID Systems

PubMed Central

Tang, Wan; Chen, Min; Ni, Jin; Yang, Ximin

2011-01-01

The traditional Radio Frequency Identification (RFID) system, in which the information maintained in tags is passive and static, has no intelligent decision-making ability to suit application and environment dynamics. The Second-Generation RFID (2G-RFID) system, referred as 2G-RFID-sys, is an evolution of the traditional RFID system to ensure better quality of service in future networks. Due to the openness of the active mobile codes in the 2G-RFID system, the realization of conveying intelligence brings a critical issue: how can we make sure the backend system will interpret and execute mobile codes in the right way without misuse so as to avoid malicious attacks? To address this issue, this paper expands the concept of Role-Based Access Control (RBAC) by introducing context-aware computing, and then designs a secure middleware for backend systems, named Two-Level Security Enhancement Mechanism or 2L-SEM, in order to ensure the usability and validity of the mobile code through contextual authentication and role analysis. According to the given contextual restrictions, 2L-SEM can filtrate the illegal and invalid mobile codes contained in tags. Finally, a reference architecture and its typical application are given to illustrate the implementation of 2L-SEM in a 2G-RFID system, along with the simulation results to evaluate how the proposed mechanism can guarantee secure execution of mobile codes for the system. PMID:22163983

Security enhancement mechanism based on contextual authentication and role analysis for 2G-RFID systems.

PubMed

Tang, Wan; Chen, Min; Ni, Jin; Yang, Ximin

2011-01-01

The traditional Radio Frequency Identification (RFID) system, in which the information maintained in tags is passive and static, has no intelligent decision-making ability to suit application and environment dynamics. The Second-Generation RFID (2G-RFID) system, referred as 2G-RFID-sys, is an evolution of the traditional RFID system to ensure better quality of service in future networks. Due to the openness of the active mobile codes in the 2G-RFID system, the realization of conveying intelligence brings a critical issue: how can we make sure the backend system will interpret and execute mobile codes in the right way without misuse so as to avoid malicious attacks? To address this issue, this paper expands the concept of Role-Based Access Control (RBAC) by introducing context-aware computing, and then designs a secure middleware for backend systems, named Two-Level Security Enhancement Mechanism or 2L-SEM, in order to ensure the usability and validity of the mobile code through contextual authentication and role analysis. According to the given contextual restrictions, 2L-SEM can filtrate the illegal and invalid mobile codes contained in tags. Finally, a reference architecture and its typical application are given to illustrate the implementation of 2L-SEM in a 2G-RFID system, along with the simulation results to evaluate how the proposed mechanism can guarantee secure execution of mobile codes for the system.

Isotopic composition analysis and age dating of uranium samples by high resolution gamma ray spectrometry

NASA Astrophysics Data System (ADS)

Apostol, A. I.; Pantelica, A.; Sima, O.; Fugaru, V.

2016-09-01

Non-destructive methods were applied to determine the isotopic composition and the time elapsed since last chemical purification of nine uranium samples. The applied methods are based on measuring gamma and X radiations of uranium samples by high resolution low energy gamma spectrometric system with planar high purity germanium detector and low background gamma spectrometric system with coaxial high purity germanium detector. The ;Multigroup γ-ray Analysis Method for Uranium; (MGAU) code was used for the precise determination of samples' isotopic composition. The age of the samples was determined from the isotopic ratio 214Bi/234U. This ratio was calculated from the analyzed spectra of each uranium sample, using relative detection efficiency. Special attention is paid to the coincidence summing corrections that have to be taken into account when performing this type of analysis. In addition, an alternative approach for the age determination using full energy peak efficiencies obtained by Monte Carlo simulations with the GESPECOR code is described.

Simulation of multi-photon emission isotopes using time-resolved SimSET multiple photon history generator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiang, Chih-Chieh; Lin, Hsin-Hon; Lin, Chang-Shiun

Abstract-Multiple-photon emitters, such as In-111 or Se-75, have enormous potential in the field of nuclear medicine imaging. For example, Se-75 can be used to investigate the bile acid malabsorption and measure the bile acid pool loss. The simulation system for emission tomography (SimSET) is a well-known Monte Carlo simulation (MCS) code in nuclear medicine for its high computational efficiency. However, current SimSET cannot simulate these isotopes due to the lack of modeling of complex decay scheme and the time-dependent decay process. To extend the versatility of SimSET for simulation of those multi-photon emission isotopes, a time-resolved multiple photon history generatormore » based on SimSET codes is developed in present study. For developing the time-resolved SimSET (trSimSET) with radionuclide decay process, the new MCS model introduce new features, including decay time information and photon time-of-flight information, into this new code. The half-life of energy states were tabulated from the Evaluated Nuclear Structure Data File (ENSDF) database. The MCS results indicate that the overall percent difference is less than 8.5% for all simulation trials as compared to GATE. To sum up, we demonstrated that time-resolved SimSET multiple photon history generator can have comparable accuracy with GATE and keeping better computational efficiency. The new MCS code is very useful to study the multi-photon imaging of novel isotopes that needs the simulation of lifetime and the time-of-fight measurements. (authors)« less

ELROI Extremely Low Resource Optical Identifier. A license plate for your satellite, and more.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palmer, David

ELROI (Extremely Low Resource Optical Identifier) is a license plate for your satellite; a small tag that flashes an optical identification code that can be read by a small telescope on the ground. The final version of the tag will be the size of a thick postage stamp and fully autonomous: you can attach it to everything that goes into space, including small cubesats and inert debris like rocket stages, and it will keep blinking even after the satellite is shut down, reliably identifying the object from launch until re-entry.

Nuclear Forensics and Radiochemistry: Reaction Networks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rundberg, Robert S.

In the intense neutron flux of a nuclear explosion the production of isotopes may occur through successive neutron induced reactions. The pathway to these isotopes illustrates both the complexity of the problem and the need for high quality nuclear data. The growth and decay of radioactive isotopes can follow a similarly complex network. The Bateman equation will be described and modified to apply to the transmutation of isotopes in a high flux reactor. A alternative model of growth and decay, the GD code, that can be applied to fission products will also be described.

Cross sections and differential spectra for reactions of 2-20 MeV neutrons of /sup 27/Al

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blann, M.; Komoto, T.T.

1988-01-01

This report summarizes product yields, secondary n,p and ..cap alpha.. spectra, and ..gamma..-ray spectra calculated for incident neutrons of 2-20 MeV on /sup 27/Al targets. Results are all from the code ALICE, using the version ALISO which does weighting of results for targets which are a mix of isotopes. Where natural isotopic targets are involved, yields and n,p,..cap alpha.. spectra will be reported weighted over isotopic yields. Gamma-ray spectra, however, will be reported for the most abundant isotope.

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

PubMed Central

1991-01-01

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864

Security authentication with a three-dimensional optical phase code using random forest classifier: an overview

NASA Astrophysics Data System (ADS)

Markman, Adam; Carnicer, Artur; Javidi, Bahram

2017-05-01

We overview our recent work [1] on utilizing three-dimensional (3D) optical phase codes for object authentication using the random forest classifier. A simple 3D optical phase code (OPC) is generated by combining multiple diffusers and glass slides. This tag is then placed on a quick-response (QR) code, which is a barcode capable of storing information and can be scanned under non-uniform illumination conditions, rotation, and slight degradation. A coherent light source illuminates the OPC and the transmitted light is captured by a CCD to record the unique signature. Feature extraction on the signature is performed and inputted into a pre-trained random-forest classifier for authentication.

Digital barcodes of suspension array using laser induced breakdown spectroscopy

PubMed Central

He, Qinghua; Liu, Yixi; He, Yonghong; Zhu, Liang; Zhang, Yilong; Shen, Zhiyuan

2016-01-01

We show a coding method of suspension array based on the laser induced breakdown spectroscopy (LIBS), which promotes the barcodes from analog to digital. As the foundation of digital optical barcodes, nanocrystals encoded microspheres are prepared with self-assembly encapsulation method. We confirm that digital multiplexing of LIBS-based coding method becomes feasible since the microsphere can be coded with direct read-out data of wavelengths, and the method can avoid fluorescence signal crosstalk between barcodes and analyte tags, which lead to overall advantages in accuracy and stability to current fluorescent multicolor coding method. This demonstration increases the capability of multiplexed detection and accurate filtrating, expanding more extensive applications of suspension array in life science. PMID:27808270

48 CFR 252.211-7006 - Passive Radio Frequency Identification.

Code of Federal Regulations, 2013 CFR

2013-10-01

... radio frequency identification (RFID) or item unique identification (IUID) information, order... CodeTM (EPC®) means an identification scheme for universally identifying physical objects via RFID tags... passive RFID technology. Exterior container means a MIL-STD-129 defined container, bundle, or assembly...

48 CFR 252.211-7006 - Passive Radio Frequency Identification.

Code of Federal Regulations, 2012 CFR

2012-10-01

... radio frequency identification (RFID) or item unique identification (IUID) information, order... CodeTM (EPC®) means an identification scheme for universally identifying physical objects via RFID tags... passive RFID technology. Exterior container means a MIL-STD-129 defined container, bundle, or assembly...

48 CFR 252.211-7006 - Passive Radio Frequency Identification.

Code of Federal Regulations, 2014 CFR

2014-10-01

... radio frequency identification (RFID) or item unique identification (IUID) information, order... CodeTM (EPC®) means an identification scheme for universally identifying physical objects via RFID tags... passive RFID technology. Exterior container means a MIL-STD-129 defined container, bundle, or assembly...

High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

PubMed Central

2015-01-01

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.

PubMed

Whiteaker, Jeffrey R; Zhao, Lei; Frisch, Christian; Ylera, Francisco; Harth, Stefan; Knappik, Achim; Paulovich, Amanda G

2014-04-04

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli.

PubMed

Lázaro, Beatriz; Villa, Juan A; Santín, Omar; Cabezas, Matilde; Milagre, Cintia D F; de la Cruz, Fernando; Moncalián, Gabriel

2017-01-01

Triglycerides (TAGs), the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT). With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3-fold in the monounsaturated fatty acids (MUFA) occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids.

Bicarbonate Increases Binding Affinity of Vibrio cholerae ToxT to Virulence Gene Promoters

PubMed Central

Thomson, Joshua J.

2014-01-01

The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription. PMID:25182489