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Sample records for affinity purification system

  1. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples. PMID:27498022

  2. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples.

  3. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  4. Dual-tagging system for the affinity purification of mammalian protein complexes

    SciTech Connect

    Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory {Greg} B; Huang, Ying; Wu, Jun; Liu, Yie; Wang, Yisong

    2007-01-01

    Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway{reg_sign}-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations of affinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approximately 16% from as little as 1-7 x 10{sup 7} cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.

  5. Affinity Purification of Antibodies.

    PubMed

    Hnasko, Robert M; McGarvey, Jeffery A

    2015-01-01

    Antibodies are provided in a variety of formats that include antiserum, hybridoma culture supernatant, or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facilitate assay reproducibility, economy, and reduced interference of nonspecific components as well as improved storage, stability, and bio-conjugation. Although not always necessary, the relative simplicity of antibody purification using commercially available protein-A, protein-G, or protein-L resins with basic chromatographic principles warrants purification when antibody source material is available in sufficient quantity. Here, we define three simple methods using immobilized (1) protein-A, (2) protein-G, and (3) protein-L agarose beads to yield highly purified antibody. PMID:26160561

  6. Partial purification of the 5-hydroxytryptophan-reuptake system from human blood platelets using a citalopram-derived affinity resin

    SciTech Connect

    Biessen, E.A.L; Horn, A.S.; Robillard, G.T. )

    1990-04-03

    This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific ({sup 3}H) imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 {mu}M citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after {sup 125}I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of ({sup 3}H) imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and ({sup 3}H)imipramine binding activity.

  7. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

    PubMed

    Carrick, Brian H; Hao, Linxuan; Smaldino, Philip J; Engelke, David R

    2016-03-01

    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  8. Affinity Purification of Protein Complexes Using TAP Tags

    PubMed Central

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities. PMID:26096502

  9. Affinity purification of aprotinin from bovine lung.

    PubMed

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  10. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  11. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  12. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  13. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture. PMID:27473483

  14. Biospecific affinity chromatographic purification of octopine dehydrogenase from molluscs.

    PubMed

    Mulcahy, P; Griffin, T; O'Carra, P

    1997-02-01

    The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata). PMID:9116492

  15. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction.

    PubMed

    Karpol, Alon; Kantorovich, Lia; Demishtein, Alik; Barak, Yoav; Morag, Ely; Lamed, Raphael; Bayer, Edward A

    2009-01-01

    Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module. PMID:18979459

  16. Challenges and recent advances in affinity purification of tag-free proteins.

    PubMed

    Guan, Dongli; Chen, Zhilei

    2014-07-01

    There is currently no generic, simple, lowcost method for affinity chromatographic purification of proteins in which the purified product is free of appended tags. Existing approaches for the purification of tagless proteins fall into two broad categories: (1) direct affinity-based capture of tag-free proteins that utilize affinity ligands specific to the target protein or class of target protein, and (2) removal of an appended affinity tag following tag-mediated protein capture. This paper reviews current state-of-the-art approaches for tagless protein purification in both categories, including specific examples of affinity ligands used for the capture of different classes of proteins and cleavage systems for affinity tag removal following chromatographic capture. A particular focus of this review is on recent developments in affinity tag removal systems utilizing split inteins. PMID:24658742

  17. Dye affinity cryogels for plasmid DNA purification.

    PubMed

    Çimen, Duygu; Yılmaz, Fatma; Perçin, Işık; Türkmen, Deniz; Denizli, Adil

    2015-11-01

    The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9μmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA. PMID:26249596

  18. Affinity chromatography for purification of two urokinases from human urine.

    PubMed

    Takahashi, R; Akiba, K; Koike, M; Noguchi, T; Ezure, Y

    2000-05-26

    A new affinity chromatography (hydrophobic-mediated affinity chromatography), which was characterized by the matrix having both affinity site to urokinase and hydrophobic site, was established for the purification of urokinase from human urine. The hydrophobic affinity matrix (tentatively named PAS in the text) was prepared by immobilizing 6-aminocaproic acid on Sepharose CL-6B, followed by a coupling p-aminobenzamidine to a part of the hydrophobic site on the matrix. The PAS matrix was applied to the purification of urokinase from human urine, and high- and low-molecular weight pure urokinases were efficiently obtained in high yield by the present method. PMID:10892585

  19. Detection of protein-protein interactions using tandem affinity purification.

    PubMed

    Goodfellow, Ian; Bailey, Dalan

    2014-01-01

    Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

  20. 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).

  1. 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

  2. Purification of glycolytic enzymes by using affinity-elution chromatography.

    PubMed Central

    Scopes, R K

    1977-01-01

    1. A systematic procedure for the purification of enzymes by affinity-elution chromatography is described. Enzymes are adsorbed on a cation-exchanger, and eluted with ligands specific for the enzyme concerned. 2. All of the glycolytic and some related enzymes present in rabbit muscle can be purified by the affinity-elution technique. The pH range for adsorption and elution of each enzyme was found, and the effects of minor variations of conditions are described. 3. A description of experimental conditions suitable for affinity elution of each enzyme is given, together with special features relevant to each individual enzyme. 4. Theoretical considerations of affinity elution chromatography are discussed, including its limitations, advantages and disadvantages compared with affinity-adsorption chromatography. Possible developments are suggested to cover enzymes which because of their adsorption characteristics are not at present amenable to affinity-elution procedures. PMID:192194

  3. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  4. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  5. Identification of protein interacting partners using tandem affinity purification.

    PubMed

    Bailey, Dalan; Urena, Luis; Thorne, Lucy; Goodfellow, Ian

    2012-01-01

    A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8). As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10).The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence(8). To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous

  6. Affinity Purification of Sequence-Specific DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Kadonaga, James T.; Tjian, Robert

    1986-08-01

    We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.

  7. Cell-Type-Specific mRNA Purification by Translating Ribosome Affinity Purification (TRAP)

    PubMed Central

    Heiman, Myriam; Kulicke, Ruth; Fenster, Robert J.; Greengard, Paul; Heintz, Nathaniel

    2014-01-01

    Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian central nervous system (CNS) at a molecular level. To address this problem, we recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell population. This methodology, which we termed translating ribosome affinity purification, or TRAP, combines cell-type-specific transgene expression with affinity purification of translating ribosomes. TRAP can be used to study the cell-type-specific mRNA profiles of any genetically defined cell type, and has been successfully used to date in organisms ranging from D. melanogaster to mice and human cultured cells. Unlike other methodologies that rely upon micro-dissection, cell panning, or cell sorting, the TRAP methodology bypasses the need for tissue fixation or single-cell suspensions (and potential artifacts these treatments introduce), and reports on mRNAs in the entire cell body. This protocol provides a step-by-step guide to implementing the TRAP methodology, which takes two days to complete once all materials are in hand. PMID:24810037

  8. Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation

    PubMed Central

    Wang, Jianlong; Cantor, Alan B.; Orkin, Stuart H.

    2009-01-01

    In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed a protein-protein interaction network. This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for in vivo biotinylation system setup in mouse ES cells and affinity purification of multi-protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS-PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification. PMID:19306258

  9. Bimolecular affinity purification: a variation of TAP with multiple applications.

    PubMed

    Starokadomskyy, Petro; Burstein, Ezra

    2014-01-01

    The identification of true interacting partners of any given bait can be plagued by the nonspecific purification of irrelevant proteins. To avoid this problem, Tandem Affinity Purification (TAP) is a widely used procedure in molecular biology as this reduces the chance of nonspecific proteins being present in the final preparation. In this approach, two different affinity tags are fused to the protein bait. Herein, we review in detail a variation on the TAP procedure that we have previously developed, where the affinity moieties are placed on two different proteins that form a complex in vivo. This variation, which we refer to as Bimolecular Affinity Purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known proteins. We have utilized BAP for characterization of molecular complexes and evaluation of proteins interaction. Another application of BAP is the isolation of ubiquitin-like proteins (UBL)-modified fractions of a given protein and characterization of the lysine-acceptor site and structure of UBL-chains. PMID:24943324

  10. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  11. Protein purification by aminosquarylium cyanine dye-affinity chromatography.

    PubMed

    Graça, Vânia C; Sousa, Fani; Santos, Paulo F; Almeida, Paulo S

    2015-01-01

    Affinity chromatography (AC) is one of the most important techniques for the separation and purification of biomolecules, being probably the most selective technique for protein purification. It is based on unique specific reversible interactions between the target molecule and a ligand. In this affinity interaction, the choice of the ligand is extremely important for the success of the purification protocol. The growing interest in AC has motivated an intense research effort toward the development of materials able to overcome the disadvantages of conventional natural ligands, namely their high cost and chemical and biological lability. In this context, synthetic dyes have emerged, in recent decades, as a promising alternative to biological ligands. Herein, detailed protocols for the assembling of a new chromatographic dye-ligand affinity support bearing an immobilized aminosquarylium cyanine dye on an agarose-based matrix (Sepharose CL-6B) and for the separation of a mixture o f three standard proteins: lysozyme, α-chymotrypsin, and trypsin are provided. PMID:25749942

  12. 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.

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

    SciTech Connect

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

    2009-01-01

    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 utilizes 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 have demonstrated 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.

  14. Purification system

    NASA Technical Reports Server (NTRS)

    Flanagan, David T. (Inventor); Gibbons, Randall E. (Inventor)

    1992-01-01

    A system for prolonging the life of a granulated activated charcoal (GAC) water treatment device is disclosed in which an ultraviolet light transparent material is used to constrain water to flow over carbon surfaces. It is configured to receive maximum flux from a UV radiation source for the purpose of preventing microbial proliferation on the carbon surfaces; oxidizing organic contaminants adsorbed from the water onto the carbon surfaces and from biodegradation of adsorbed microbial forms; disinfecting water; and oxidizing organic contaminants in the water.

  15. Affinity-based methodologies and ligands for antibody purification: advances and perspectives.

    PubMed

    Roque, Ana C A; Silva, Cláudia S O; Taipa, M Angela

    2007-08-10

    Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification "paradigm" still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography.

  16. Affinity-based methodologies and ligands for antibody purification: advances and perspectives.

    PubMed

    Roque, Ana C A; Silva, Cláudia S O; Taipa, M Angela

    2007-08-10

    Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification "paradigm" still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography. PMID:17618635

  17. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. PMID:26830537

  18. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented.

  19. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.

  20. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection. PMID:25271333

  1. The Borexino purification system

    NASA Astrophysics Data System (ADS)

    Benziger, Jay

    2014-05-01

    Purification of 278 tons of liquid scintillator and 889 tons of buffer shielding for the Borexino solar neutrino detector is performed with a system of combined distillation, water extraction, gas stripping and filtration. The purification system removed K, U and Th by distillation of the pseudocumene solvent and the PPO fluor. Noble gases, Rn, Kr and Ar were removed by gas stripping. Distillation was also employed to remove optical impurities and reduce the attenuation of scintillation light. The success of the purification system has facilitated the first time real time detection of low energy solar neutrinos.

  2. Affinity purification of a siderophore that exhibits an antagonistic effect against soft rot bacterium.

    PubMed

    Helmy, Mohamed; Baddar, Doa; El'Masry, Mohamed Hisham

    2008-07-01

    Bacterial colonies were isolated from different Egyptian soil samples. From these isolates, one bacterial species was found to produce siderophore. Using classical and biochemical identification methods, the siderophore producing isolate was identified as Pseudomonas fluorescens. Based on the affinity of siderophores for metal ions, an affinity chromatography system was designed for the purification of the siderophore in one step. It was possible to isolate 25 mg siderophore per liter of culture media. The purified siderophore was found to exist in two forms of approximately 30 and 90 kD. They are believed to be polymers of several siderophore molecules. Both forms were found to be active against the pathogen Erwinia carotovora var. carotovora, the causal bacteria of soft rot disease on potato tubers. The advantage of this method over other purification methods is that it uses metal ion so it can be applied for the purification of the known types of siderophores. Moreover, the purification is based on affinity chromatography, so the siderophore purity state permits several biotechnological applications without further treatments. PMID:18707585

  3. Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-05-16

    Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

  4. Purification of muscarinic acetylcholine receptors by affinity chromatography.

    PubMed Central

    André, C; De Backer, J P; Guillet, J C; Vanderheyden, P; Vauquelin, G; Strosberg, A D

    1983-01-01

    Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein. Images Fig. 3. PMID:6605245

  5. Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

    A rapid microscale procedure has been developed for the isolation and purification of yeast alcohol dehydrogenase. Glass beads are used for cytolysis, PEG precipitation for partial purification and a cibacron blue affinity column for the final step. A 27.5 fold purification can be achieved in 2 - 3 hours.

  6. Development of a novel affinity chromatography resin for platform purification of lambda fabs.

    PubMed

    Eifler, Nora; Medaglia, Giovanni; Anderka, Oliver; Laurin, Linus; Hermans, Pim

    2014-01-01

    Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established. PMID:25082738

  7. Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC).

    PubMed

    de Costa, Fernanda; Barber, Carla J S; Pujara, Pareshkumar T; Reed, Darwin W; Covello, Patrick S

    2016-01-01

    Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography. PMID:26843168

  8. Scoring Large Scale Affinity Purification Mass Spectrometry Datasets with MIST

    PubMed Central

    Verschueren, Erik; Von Dollen, John; Cimermancic, Peter; Gulbahce, Natali; Sali, Andrej; Krogan, Nevan

    2015-01-01

    High-throughput Affinity Purification Mass Spectrometry (AP-MS) experiments can identify a large number of protein interactions but only a fraction of these interactions are biologically relevant. Here, we describe a comprehensive computational strategy to process raw AP-MS data, perform quality controls and prioritize biologically relevant bait-prey pairs in a set of replicated AP-MS experiments with Mass spectrometry interaction STatistics (MiST). The MiST score is a linear combination of prey quantity (abundance), abundance invariability across repeated experiments (reproducibility), and prey uniqueness relative to other baits (specificity); We describe how to run the full MiST analysis pipeline in an R environment and discuss a number of configurable options that allow the lay user to convert any large-scale AP-MS data into an interpretable, biologically relevant protein-protein interaction network. PMID:25754993

  9. Water Purification Systems

    NASA Technical Reports Server (NTRS)

    1992-01-01

    A water purification/recycling system developed by Photo-Catalytics, Inc. (PCI) for NASA is commercially available. The system cleanses and recycles water, using a "photo-catalysis" process in which light or radiant energy sparks a chemical reaction. Chemically stable semiconductor powders are added to organically polluted water. The powder absorbs ultraviolet light, and pollutants are oxidized and converted to carbon dioxide. Potential markets for the system include research and pharmaceutical manufacturing applications, as well as microchip manufacture and wastewater cleansing.

  10. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency. PMID:25749943

  11. Magnetic particles as affinity matrix for purification of antithrombin

    NASA Astrophysics Data System (ADS)

    Mercês, A. A. D.; Maciel, J. C.; Carvalho Júnior, L. B.

    2015-11-01

    Immobilization of biomolecules onto insoluble supports is an important tool for the fabrication of a diverse range of functional materials. It provides advantages: enhanced stability and easy separation. In this work two different magnetic composites were synthesized (MAG-PANI-HS and mDAC-HS) to human antithrombin purification. The magnetic particles (MAG) were obtained by co-precipitation method of iron salts II and III and subsequently coated with polyaniline (MAG-PANI particles). Dacron (polyethylene terephthalate) suffered a hydrazinolysis reaction to obtain a powder (Dacron hydrazide) which was subsequently magnetized (mDAC particles) also by co-precipitation method. Heparan sulfate (HS) was immobilized to MAG-PANI and mDAC retained respectively 35μg and 38.6μg per of support. The magnetic composite containing HS immobilized (MAG-PANI-HS and mDAC-HS) was incubated with human blood plasma (1mL) and then washed with NaCl gradients. Electrophoresis of proteins present in eluates showed bands of antithrombin (58kDa). A reduction in the antithrombin activity was detected in plasma that were incubated in the composites magnetic with HS immobilized, suggesting that the antithrombin was removed of the human blood plasma and then purified. Therefore, the above results suggest that both preparations: MAG-PANI-HS and mDAC-HS are able to affinity purify antithrombin, an important component of blood coagulation.

  12. Predicting direct protein interactions from affinity purification mass spectrometry data

    PubMed Central

    2010-01-01

    Background Affinity purification followed by mass spectrometry identification (AP-MS) is an increasingly popular approach to observe protein-protein interactions (PPI) in vivo. One drawback of AP-MS, however, is that it is prone to detecting indirect interactions mixed with direct physical interactions. Therefore, the ability to distinguish direct interactions from indirect ones is of much interest. Results We first propose a simple probabilistic model for the interactions captured by AP-MS experiments, under which the problem of separating direct interactions from indirect ones is formulated. Then, given idealized quantitative AP-MS data, we study the problem of identifying the most likely set of direct interactions that produced the observed data. We address this challenging graph theoretical problem by first characterizing signatures that can identify weakly connected nodes as well as dense regions of the network. The rest of the direct PPI network is then inferred using a genetic algorithm. Our algorithm shows good performance on both simulated and biological networks with very high sensitivity and specificity. Then the algorithm is used to predict direct interactions from a set of AP-MS PPI data from yeast, and its performance is measured against a high-quality interaction dataset. Conclusions As the sensitivity of AP-MS pipeline improves, the fraction of indirect interactions detected will also increase, thereby making the ability to distinguish them even more desirable. Despite the simplicity of our model for indirect interactions, our method provides a good performance on the test networks. PMID:21034440

  13. Production and Purification of Streptokinase by Protected Affinity Chromatography

    PubMed Central

    Babashamsi, Mohammad; Razavian, Mohammad Hossein; Nejadmoghaddam, Mohammad Reza

    2009-01-01

    Streptokinase is an extracellular protein, extracted from certain strains of beta hemolytic streptococcus. It is a non-protease plasminogen activator that activates plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy. The rate of bacterial growth and streptokinase production was studied in condition of excess glucose addition to culture media and its pH maintenance. The streptokinase product of the bacterial culture was preliminary extracted by salt precipitation and then purified by affinity chromatography on plasminogen substituted sepharose-4B in a condition that the plasminogen active site was protected from streptokinase-induced activation. The purity of streptokinase was confirmed by SDS-PAGE and its biological activity determined in a specific streptokinase assay. The results showed that in the fed–batch culture, the rate of streptokinase production increased over two times as compared with the batch culture while at the same time, shortening the streptokinase purification to a single step increased the yield over 95% at the chromatography stage. PMID:23407807

  14. Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2

    PubMed Central

    Locatelli-Hoops, Silvia C.; Yeliseev, Alexei A.

    2016-01-01

    Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor. PMID:24943318

  15. Synthesis and characterization of pseudo-affinity ligand for penicillin acylase purification.

    PubMed

    Keçili, Rüstem; Say, Ridvan; Yavuz, Handan

    2006-11-15

    The aim of this work was to test a chromatographic affinity support containing methacryloyl antipyrine (MAAP) for penicillin acylase (PA) purification by using pure penicillin acylase and crude extract. First, MAAP as a pseudo-specific ligand was synthesized by using methacryloyl chloride and 4-aminoantipyrine. Polymer beads (average size diameter: 40-120 micro m) were prepared by suspension polymerization of ethylene glycol dimethacrylate (EGDMA) and MAAP. This approach for the preparation of adsorbent has several advantages over conventional preparation protocols. An expensive and time consuming step in the preparation of adsorbent is immobilization of a ligand to the adsorption matrix. In this procedure, affinity ligand MAAP acts as comonomer without further modification steps. Poly(EGDMA-MAAP) beads were characterized by FTIR, NMR and screen analysis. Elemental analysis of MAAP for nitrogen was estimated as 89.3 micro mol/g. The prepared adsorbent was then used for the capture of penicillin acylase in batch system. The maximum penicillin acylase adsorption capacity of the poly(EGDMA-MAAP) beads was found to be 82.2 mg/g at pH 5.0. Chromatography with crude feedstock resulted in 23.2-fold purification and 93% recovery with 1.0 M NaOH.

  16. A modular approach to multifunctional polypeptide/ceramic fluorapatite-based self-assembled system in affinity chromatography for the purification of human Immunoglobulin G.

    PubMed

    Islam, Tuhidul; Fernández-Lahore, Marcelo

    2015-03-01

    The multifunctional bone sialoprotein/apatite (AP) self-assembled systems in the mineralized tissues show a pathway for the noncovalent immobilization of ligands on the AP chromatographic matrix. A model approach is presented here regarding the physical immobilization of ligands on the ceramic fluorapatite (CFT) matrix for the purification of human Immunoglobulin G (hIgG). The peptide pIC, HWRGWV-KPRSVSG, composed of a hIgG-specific peptide, HWRGWV (pLI), and a CFT-specific peptide, KPRSVSG (pTC), was synthesized and subjected to physicochemical characterization. A circular dichroism study showed that pIC possesses a flexible structural feature, which is significant in terms of its multifunctional activities. With the current approach, hIgG will be retained selectively by the self-assembled pIC/CFT column, while other biomolecules will pass through the column without being interacted. Therefore, the chromatographic conditions that are the key factors for the successful implementation of this technique were optimized as a function of the composition and pH of the mobile phase. Here, 115 mM sodium chloride (NaCl) in 20 mM sodium phosphate, pH 7.4, was used as the binding buffer, and the elution was performed with 225 mM NaCl in 20 mM sodium phosphate containing 0.3% w/v sodium acetate at pH 6. The binding capacity of the pIC/CFT column was 21.5 mg hIgG/ml matrix with a ligand density of 18.8 µmol/ml, and the binding capacity of the column increased with the increment of ligand density. Afterward, the applicability of a spacer arm between pLI and pTC was also verified. The hIgG-binding capacity of the column decreased with the increment in size of the spacer. In conclusion, the peptide-mediated self-assembled biomimetic system can be used as an alternative to the chemical immobilization of ligands in order to prevent unwanted consequences that result from some of the conventional ligand coupling chemistry.

  17. A modular approach to multifunctional polypeptide/ceramic fluorapatite-based self-assembled system in affinity chromatography for the purification of human Immunoglobulin G.

    PubMed

    Islam, Tuhidul; Fernández-Lahore, Marcelo

    2015-03-01

    The multifunctional bone sialoprotein/apatite (AP) self-assembled systems in the mineralized tissues show a pathway for the noncovalent immobilization of ligands on the AP chromatographic matrix. A model approach is presented here regarding the physical immobilization of ligands on the ceramic fluorapatite (CFT) matrix for the purification of human Immunoglobulin G (hIgG). The peptide pIC, HWRGWV-KPRSVSG, composed of a hIgG-specific peptide, HWRGWV (pLI), and a CFT-specific peptide, KPRSVSG (pTC), was synthesized and subjected to physicochemical characterization. A circular dichroism study showed that pIC possesses a flexible structural feature, which is significant in terms of its multifunctional activities. With the current approach, hIgG will be retained selectively by the self-assembled pIC/CFT column, while other biomolecules will pass through the column without being interacted. Therefore, the chromatographic conditions that are the key factors for the successful implementation of this technique were optimized as a function of the composition and pH of the mobile phase. Here, 115 mM sodium chloride (NaCl) in 20 mM sodium phosphate, pH 7.4, was used as the binding buffer, and the elution was performed with 225 mM NaCl in 20 mM sodium phosphate containing 0.3% w/v sodium acetate at pH 6. The binding capacity of the pIC/CFT column was 21.5 mg hIgG/ml matrix with a ligand density of 18.8 µmol/ml, and the binding capacity of the column increased with the increment of ligand density. Afterward, the applicability of a spacer arm between pLI and pTC was also verified. The hIgG-binding capacity of the column decreased with the increment in size of the spacer. In conclusion, the peptide-mediated self-assembled biomimetic system can be used as an alternative to the chemical immobilization of ligands in order to prevent unwanted consequences that result from some of the conventional ligand coupling chemistry. PMID:25663265

  18. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories. PMID:25749949

  19. Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

    SciTech Connect

    Weslake, R.J.; Chesney, S.L.; Petkau, A.; Friesen, A.D.

    1986-05-15

    Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

  20. Purification of L-( sup 3 H) Nicotine eliminates low affinity binding

    SciTech Connect

    Romm, E.; Marks, M.J.; Collins, A.C. ); Lippiello, P.M. )

    1990-01-01

    Some studies of L-({sup 3}H) nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicated that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-({sup 3}H)nicotine eliminates the low affinity site.

  1. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    PubMed

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

  2. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

    PubMed

    Rimmelzwaan, G F; Groen, J; Juntti, N; Teppema, J S; UytdeHaag, F G; Osterhaus, A D

    1987-03-01

    Immuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infectivity titration system, in an ELISA, in a haemagglutination assay and by negative contrast electron microscopy to quantify CPV or CPV antigen. The degree of purification was further estimated by testing the fractions for total protein content in a colorimetric method, for bovine serum albumin content in an ELISA and by SDS-PAGE. Over 99% of the contaminating proteins proved to be removed, and 20% or 70-90% of infectious CPV or CPV antigen, respectively, was recovered.

  3. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  4. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

  5. Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

    PubMed Central

    Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

    2001-01-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  6. Tandem affinity purification to identify cytosolic and nuclear gβγ-interacting proteins.

    PubMed

    Campden, Rhiannon; Pétrin, Darlaine; Robitaille, Mélanie; Audet, Nicolas; Gora, Sarah; Angers, Stéphane; Hébert, Terence E

    2015-01-01

    It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.

  7. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    PubMed

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.

  8. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    PubMed

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants. PMID:18307162

  9. Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain.

    PubMed

    Mahmood, Rubab

    2016-01-01

    Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)-bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.

  10. Water Purification Systems

    NASA Technical Reports Server (NTRS)

    1994-01-01

    Clearwater Pool Technologies employs NASA-developed silver/copper ionization to purify turtle and dolphin tanks, cooling towers, spas, water recycling systems, etc. The pool purifier consists of a microcomputer to monitor water conditions, a pair of metallic electrodes, and a rheostat controller. Ions are generated by passing a low voltage current through the electrodes; the silver ions kill the bacteria, and the copper ions kill algae. This technology has found broad application because it offers an alternative to chemical disinfectants. It was originally developed to purify water on Apollo spacecraft. Caribbean Clear has been using NASA's silver ionization technology for water purification for more than a decade. Two new products incorporate advancements of the basic technology. One is the AquaKing, a system designed for areas with no source of acceptable drinking water. Another is the Caribbean Clear Controller, designed for commercial pool and water park applications where sanitizing is combined with feedback control of pH and an oxidizer, chlorine or bromine. The technology was originally developed to purify water on Apollo spacecraft.

  11. Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry*

    PubMed Central

    Shen, Zhouxin; Kay, Steve A.

    2016-01-01

    Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling

  12. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  13. Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins.

    PubMed

    Kery, Vladimir; Savage, Justin R; Widjaja, Kartika; Blake, B Kelly; Conklin, David R; Ho, Yew-Seng J; Long, Xinghua; von Rechenberg, Moritz; Zarembinski, Thomas I; Boniface, J Jay

    2003-06-15

    High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.

  14. Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae.

    PubMed

    Trahan, Christian; Aguilar, Lisbeth-Carolina; Oeffinger, Marlene

    2016-01-01

    Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to getting a comprehensive picture of cellular behavior and to understanding biological systems. In the last decade, affinity purification coupled to mass spectrometry has emerged as a powerful tool to comprehensively study interaction networks and their assemblies. However, the study of these interactomes has been hampered by severe methodological limitations. In particular, the affinity purification of intact complexes from cell lysates suffers from protein and RNA degradation, loss of transient interactors, and poor overall yields. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol described here has been optimized for the yeast S. cerevisiae.

  15. An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.

    PubMed

    Bigenzahn, Johannes W; Fauster, Astrid; Rebsamen, Manuele; Kandasamy, Richard K; Scorzoni, Stefania; Vladimer, Gregory I; Müller, André C; Gstaiger, Matthias; Zuber, Johannes; Bennett, Keiryn L; Superti-Furga, Giulio

    2016-03-01

    Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.

  16. The Purification of Natural and Recombinant Peptide Antibodies by Affinity Chromatographic Strategies.

    PubMed

    Ma, Hui; O'Kennedy, Richard

    2015-01-01

    The purification of peptide antibodies (e.g., IgG, IgY, scFv, and Fab) are described in this chapter. Affinity chromatographic purification, a very convenient and effective antibody purification strategy, is used to isolate peptide antibodies based on specific binding, i.e., binding of the antibody to a column on which its specific ligand is immobilized with subsequent elution of the purified antibody. In addition, the application of purification methods based on the use of proteins A, G, and L, each of which bind to specific domains on an antibody/fragment, or the use of specific tags (e.g., histidine and biotin) attached to antibodies or antigens are also described.

  17. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  18. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

  19. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals. PMID:26952369

  20. Designing Chaotic Systems by Piecewise Affine Systems

    NASA Astrophysics Data System (ADS)

    Wu, Tiantian; Li, Qingdu; Yang, Xiao-Song

    Based on mathematical analysis, this paper provides a methodology to ensure the existence of homoclinic orbits in a class of three-dimensional piecewise affine systems. In addition, two chaotic generators are provided to illustrate the effectiveness of the method.

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

    SciTech Connect

    Tykvart, J.; Sacha, P.; Barinka, C.; Knedlik, T.; Starkova, J.; Lubkowski, J.; Konvalinka, J.

    2012-02-07

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

  2. Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme.

    PubMed

    Cass, Brian; Pham, Phuong Lan; Kamen, Amine; Durocher, Yves

    2005-03-01

    Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. PMID:15721774

  3. Single-step purification of native miraculin using immobilized metal-affinity chromatography.

    PubMed

    Duhita, Narendra; Hiwasa-Tanase, Kyoko; Yoshida, Shigeki; Ezura, Hiroshi

    2009-06-24

    Miraculin is a taste-modifying protein that can be isolated from miracle fruit ( Richadella dulcifica ), a shrub native to West Africa. It is able to turn a sour taste into a sweet taste. The commercial exploitation of this sweetness-modifying protein is underway, and a fast and efficient purification method to extract the protein is needed. We succeeded in purifying miraculin from miracle fruit in a single-step purification using immobilized metal-affinity chromatography (IMAC). The purified miraculin exhibited high purity (>95%) in reverse-phase high-performance liquid chromatography. We also demonstrated the necessity of its structure for binding to the nickel-IMAC column. PMID:19469504

  4. NiCoMnO4: A Bifunctional Affinity Probe for His-Tagged Protein Purification and Phosphorylation Sites Recognition.

    PubMed

    Qi, Xiaoyue; Chen, Long; Zhang, Chaoqun; Xu, Xinyuan; Zhang, Yiding; Bai, Yu; Liu, Huwei

    2016-07-27

    A bifunctional affinity probe NiCoMnO4 was designed and prepared with controllable morphology and size using facile methods. It was observed that the probe could be applied in His-tagged proteins purification and phosphopeptides enrichment simply through the buffer modulation. NiCoMnO4 particles showed satisfactory cycling performance for His-tagged proteins purification and broad pH-tolerance of loading buffer for phosphopeptides affinity. Therefore, a high-throughput, cost-effective, and efficient protein/peptide purification method was developed within 10 min based on the novel bifunctional affinity probe. PMID:27381638

  5. A novel affinity disks for bovine serum albumin purification.

    PubMed

    Tuzmen, Nalan; Kalburcu, Tülden; Uygun, Deniz Aktaş; Akgol, Sinan; Denizli, Adil

    2015-01-01

    The adsorption characteristics of bovine serum albumin (BSA) onto the supermacroporous poly(hydroxyethylmethacrylate)-Reactive Green 19 [p(HEMA)-RG] cryogel disks have been investigated in this paper. p(HEMA) cryogel disks were prepared by radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Reactive Green (RG) 19 was covalently attached to the p(HEMA) cryogel disks. These disks were used in BSA adsorption studies to interrogate the effects of pH, initial protein concentration, ionic strength, and temperature. BSA adsorption capacity of the p(HEMA)-RG cryogel disk was significantly improved after the incorporation of RG. Adsorption capacity reached a plateau value at about 0.8 mg/mL at pH 4.0. The amount of adsorbed BSA decreased from 37.7 to 13.9 mg/g with increasing NaCl concentration. The enthalpy of BSA adsorption onto the p(HEMA)-RG cryogel disk was calculated as -58.4 kJ/mol. The adsorption equilibrium isotherm was fitted well by the Freundlich model. BSA was desorbed from cryogel disks (over 90 %) using 0.5 M NaSCN, and the purity of desorbed BSA was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The experimental results showed that the p(HEMA)-RG cryogel disks have potential for the quick protein separation and purification process. PMID:25308615

  6. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants.

    PubMed

    Sainsbury, Frank; Jutras, Philippe V; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

  7. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants

    PubMed Central

    Sainsbury, Frank; Jutras, Philippe V.; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

  8. Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis.

    PubMed

    Ahirwar, Rajesh; Nahar, Pradip

    2015-08-01

    Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A. PMID:26102634

  9. Cyclic peptide ligand with high binding capacity for affinity purification of immunoglobulin G.

    PubMed

    Kang, Hyo Jin; Choe, Weonu; Min, Jeong-Ki; Lee, Young-Mi; Kim, B Moon; Chung, Sang J

    2016-09-30

    The rapidly increasing implementation of antibodies in therapeutic and diagnostic applications has necessitated the development of antibody production and purification technologies for both academic and industrial usage. Bacterial Protein A and Protein G are known to bind antibodies with high affinity and have facilitated the isolation and purification thereof. Recently, small peptide ligands (i.e. IgG Fc domain-binding peptides, FcBP) that specifically bind to the Fc-domain of antibodies were reported. In the present study we describe the development of a reusable high affinity column for antibody purification utilizing immobilized FcBP, comprising 13 amino acids residues, on a sepharose resin. In addition to FcBP, Cys to Ser substituted FcBP (FcBP-Ser), reduced FcBP (FcBP-Red), commercial Protein A and Protein G resins, packed into columns, were evaluated for antibody purification. All these columns except the FcBP-Ser one showed good binding capacity for a humanized IgG (trastuzumab) and a chimeric IgG (cetuximab). The column packed with FcBP-Red allowed antibody purification at a less acidic pH (pH 4.8) than was required for the other ligand affinity columns used in our experiments (i.e., pH 3.2 for Protein G and FcBP columns, and pH 3.5 for Protein A column, respectively). Utilizing the FcBP column, antibodies from swine human sera were isolated with a purity of 95%. Interestingly, the FcBP column could be easily regenerated and operated without loss of efficiency for up to 60 runs, the maximum number of runs performed in the present study.

  10. Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

    PubMed

    Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

    2014-01-01

    Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT).

  11. Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors

    PubMed Central

    Kuester, Miriam; Becker, Gero L.; Hardes, Kornelia; Lindberg, Iris; Steinmetzer, Torsten; Than, Manuel E.

    2013-01-01

    In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied – studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)2-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members. PMID:21875402

  12. Identification of Protein Partners in Mycobacteria Using a Single-Step Affinity Purification Method

    PubMed Central

    Cysewski, Dominik; Stoduś, Krystian; Kowalska, Katarzyna; Dziembowski, Andrzej

    2014-01-01

    Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein–protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein–protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners. PMID:24664103

  13. Identification of protein partners in mycobacteria using a single-step affinity purification method.

    PubMed

    Płociński, Przemysław; Laubitz, Daniel; Cysewski, Dominik; Stoduś, Krystian; Kowalska, Katarzyna; Dziembowski, Andrzej

    2014-01-01

    Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners. PMID:24664103

  14. Affinity purification of egg yolk immunoglobulins (IgY) using a human mycoplasma protein.

    PubMed

    Jiang, Xuemei; Diraviyam, Thirumalai; Zhang, Xiaoying

    2016-02-15

    Egg yolk immunoglobulin (IgY) is a superior functional equivalent to mammalian IgG. However, the preparation of refined and highly purified IgY is still attributed as difficult task. Protein M (a transmembrane protein from human mycoplasma) has been newly demonstrated as an ideal affinity regent for mammalian antibody purification. This study aimed to evaluate the interaction between protein M and IgY. The results showed protein M could be a superior affinity reagent for IgY, scFv as well as IgYΔFc, based on pull down and western blot investigations; in addition, it was found that ∼125 times increase of effective IgY in the elutent was obtained using protein M affinity chromatography column compared with traditional IgY extraction methods. This indicates, the purification strategy of protein M is entirely different to traditional IBPs and the salient purification feature of protein M would be a breakthrough for purifying not only non-mammalian antibodies, but also monoclonal antibodies and engineered antibodies based on variable region.

  15. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-01

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

  16. Design of affinity tags for one-step protein purification from immobilized zinc columns

    SciTech Connect

    Pasquinelli, R.S.; Shepherd, R.E.; Koepsel, R.R.; Zhao, A.; Ataai, M.M.

    2000-02-01

    Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to e superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. for example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper the authors have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

  17. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.

    PubMed

    Coyle, Brandon L; Baneyx, François

    2014-10-01

    We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

  18. Characterization of a dockerin-based affinity tag: application for purification of a broad variety of target proteins.

    PubMed

    Demishtein, Alik; Karpol, Alon; Barak, Yoav; Lamed, Raphael; Bayer, Edward A

    2010-01-01

    Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca(2+)-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K(A)) of 7.7 × 10(8)M(-1), calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum β-glucosidase, purified using this approach, was tested and found to be similar to that of a β-glucosidase preparation (without the ΔDoc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca(2+)-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. PMID:21038354

  19. Yeast 3',5'-bisphosphate nucleotidase: an affinity tag for protein purification.

    PubMed

    Yang, Yang; Ma, Jianhui; Yang, Yilin; Zhang, Xiao; Wang, Yanxing; Yang, Ling; Sun, Meihao

    2014-05-01

    Affinity chromatography is one of the most popular methods for protein purification. Each tag method has its advantages and disadvantages, and combination of different tags and developing of new tags had been proposed and performed. Yeast 3',5'-bisphosphate nucleotidase, also known as HAL2, hydrolyzes 3'-phosphoadenosine 5'-phosphate (PAP) with submicromolar Km, which indicated the tight interactions between HAL2 and PAP. In order to explore the feasibility of HAL2 as a protein purification affinity tag, HAL2 was further characterized with PAP as substrate. Results demonstrated that KmPAP and kcatPAP were ∼0.3μM and ∼11s(-)(1), respectively. Kd for PAP was 0.008μM in the presence of Ca(2+). pH was also found to affect interactions between HAL2 and PAP, with tightest binding (Kd∼8nM) at pH 7.5 and 8. The purification protocol was rationally designed based on nanomolar affinity to PAP agarose in the presence of Ca(2+), which could satisfy the metal requirement for PAP binding, prevent hydrolysis of immobilized PAP and could be chelated by ethylene glycol tetraacetic acid (EGTA) for elution. A series of expression vectors were further constructed and Escherichia coli adenosine 5'-phosphosulfate kinase (APSK) was prokaryotically expressed, purified and characterized. Ready to use expression vector with eight commonly used restriction enzyme recognition sites in multiple cloning site was subsequently constructed. By comparing with current popular tags, HAL2 was found to be an efficient and economical tag for prokaryotic protein expression and purification. PMID:24613729

  20. Short cut of protein purification by integration of cell-disrupture and affinity extraction.

    PubMed

    Schuster, M; Wasserbauer, E; Ortner, C; Graumann, K; Jungbauer, A; Hammerschmid, F; Werner, G

    2000-01-01

    Screening strategies based on functional genomics require the isolation of gene products of several hundred cDNA clones in a fast and versatile manner. Conventional purification strategies will fail to accomplish this goal within a reasonable time frame. In order to short-cut these procedures, we have developed a combination of cell disintegration and affinity technique for rapid isolation and purification. For our purpose, tagged proteins have been produced in yeast by fusing the FLAG-sequence adjacent to the 5' end of cDNAs coding for the respective protein. The example of an over-expressed FLAG-tagged fusion protein, human serum albumin (HSA), was released into the cytoplasm. Detection and purification of the FLAG-fusion protein were carried out by using a mouse monoclonal antibody directed against the FLAG-peptide. For purification purposes, the antibody was immobilized on PROSEP magnetic glass beads. These magnetic glass beads with 500 microns diameter have been investigated for disintegration of yeast and simultaneous capturing of the target protein. After 60 s, 90% of the maximal disintegration level was achieved when a ratio of 20 microliters yeast cell suspension and 100 microliters glass are vortexed. After a wash step, the FLAG-fusion proteins have been eluted with chelating agents such as EDTA. The short-cut procedure has been compared to a conventional purification strategy using an affinity chromatography process. Due to the highly favorable binding characteristics of the applied immunoaffinity sorbent the yield observed in batch operation was 90% and purity in the range of 70-80%.

  1. Optimisation of Downscaled Tandem Affinity Purifications to Identify Core Protein Complexes

    PubMed Central

    Haura, Eric B.; Sacco, Roberto; Li, Jiannong; Müller, André C.; Grebien, Florian; Superti-Furga, Giulio; Bennett, Keiryn L.

    2013-01-01

    In this study we show that via stable, retroviral-expression of tagged EGFR del (L747-S752 deletion mutant) in the PC9 lung cancer cell line and stable doxycycline-inducible expression of tagged Grb2 using a Flp-mediated recombination HEK293 cell system, the SH-TAP can be downscaled to 5 to 12.5 mg total protein input (equivalent to 0.5 - 1 × 15 cm culture plate or 4 - 8 × 106 cells). The major constituents of the EGFR del complex (USB3B, GRB2, ERRFI, HSP7C, GRP78, HSP71) and the Grb2 complex (ARHG5, SOS1, ARG35, CBL, CBLB, PTPRA, SOS2, DYN2, WIPF2, IRS4) were identified. Adjustment of the quantity of digested protein injected into the mass spectrometer reveals that optimisation is required as high quantities of material led to a decrease in protein sequence coverage and the loss of some interacting proteins. This investigation should aid other researchers in performing tandem affinity purifications in general, and in particular, from low quantities of input material. PMID:24077984

  2. Affinity purification of angiotensin converting enzyme inhibitory peptides using immobilized ACE.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, María del Mar; Alaiz, Manuel; Girón-Calle, Julio; Millan, Francisco; Vioque, Javier

    2006-09-20

    A lung extract rich in angiotensin converting enzyme (ACE) and pure ACE were immobilized by reaction with the activated support 4 BCL glyoxyl-agarose. These immobilized ACE derivatives were used for purification of ACE inhibitory peptides by affinity chromatography. The immobilized lung extract was used to purify inhibitory peptides from sunflower and rapeseed protein hydrolysates that had been obtained by treatment of protein isolates with alcalase. The ACE binding peptides that were retained by the derivatives were specifically released by treatment with the ACE inhibitor captopril and further purified by reverse-phase C18 HPLC chromatography. Inhibitory peptides with IC50 50 and 150 times lower than those of the original sunflower and rapeseed hydrolysates, respectively, were obtained. The derivative prepared using pure ACE was used for purification of ACE inhibitory peptides from the same type of sunflower protein hydrolysate. ACE binding peptides were released from the ACE-agarose derivatives by treatment with 1 M NaCl and had an IC50 a little higher than those obtained using immobilized extract and elution with captopril. Affinity chromatography facilitated the purification of ACE inhibitory peptides and potentially other bioactive peptides present in food proteins.

  3. Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel.

    PubMed

    Beyaztaş, Serap; Arslan, Oktay

    2015-06-01

    A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150 kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60 °C and incubated for 60 min. These results indicated that the enzyme was heat stable. PMID:25089709

  4. Affinity chromatography purification of angiotensin II reactor using photoactivable biotinylated probes

    SciTech Connect

    Marie, J.; Seyer, R.; Lombard, C.; Desarnaud, F.; Aumelas, A.; Jard, A.; Bonnafous, J.C. )

    1990-09-25

    The authors have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH{sub 2}){sub 2}SS(CH{sub 2}){sub 2}CO-(Ala{sup 1}, Phe(4N{sub 3}){sup 8})AII, which contains a cleavage disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (K{sub d}{approximately}1 nM), proved to be suitable for indirect affinity chromatography of rate liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

  5. The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions

    PubMed Central

    Di Tomasso, Geneviève; Lampron, Philipe; Dagenais, Pierre; Omichinski, James G.; Legault, Pascale

    2011-01-01

    Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications. PMID:21071425

  6. The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions.

    PubMed

    Di Tomasso, Geneviève; Lampron, Philipe; Dagenais, Pierre; Omichinski, James G; Legault, Pascale

    2011-02-01

    Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications. PMID:21071425

  7. The CRAPome: a Contaminant Repository for Affinity Purification Mass Spectrometry Data

    PubMed Central

    Mellacheruvu, Dattatreya; Wright, Zachary; Couzens, Amber L.; Lambert, Jean-Philippe; St-Denis, Nicole; Li, Tuo; Miteva, Yana V.; Hauri, Simon; Sardiu, Mihaela E.; Low, Teck Yew; Halim, Vincentius A.; Bagshaw, Richard D.; Hubner, Nina C.; al-Hakim, Abdallah; Bouchard, Annie; Faubert, Denis; Fermin, Damian; Dunham, Wade H.; Goudreault, Marilyn; Lin, Zhen-Yuan; Badillo, Beatriz Gonzalez; Pawson, Tony; Durocher, Daniel; Coulombe, Benoit; Aebersold, Ruedi; Superti-Furga, Giulio; Colinge, Jacques; Heck, Albert J. R.; Choi, Hyungwon; Gstaiger, Matthias; Mohammed, Shabaz; Cristea, Ileana M.; Bennett, Keiryn L.; Washburn, Mike P.; Raught, Brian; Ewing, Rob M.; Gingras, Anne-Claude; Nesvizhskii, Alexey I.

    2013-01-01

    Affinity purification coupled with mass spectrometry (AP-MS) is now a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (e.g. proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. While the standard approach is to identify nonspecific interactions using one or more negative controls, most small-scale AP-MS studies do not capture a complete, accurate background protein set. Fortunately, negative controls are largely bait-independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the Contaminant Repository for Affinity Purification (the CRAPome) and describe the use of this resource to score protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely available online at www.crapome.org. PMID:23921808

  8. Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

    PubMed

    DiScipio, Richard G; Liddington, Robert C; Schraufstatter, Ingrid U

    2016-05-01

    A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

  9. Unravelling plant molecular machineries through affinity purification coupled to mass spectrometry.

    PubMed

    Dedecker, Maarten; Van Leene, Jelle; De Jaeger, Geert

    2015-04-01

    Rather than functioning independently, proteins tend to work in concert with each other and with other macromolecules to form macromolecular complexes. Affinity purification coupled to mass spectrometry (AP-MS) can lead to a better understanding of the cellular functions of these complexes. With the development of easy purification protocols and ultra-sensitive MS, AP-MS is currently widely used for screening co-complex membership in plants. Studying complexes in their developmental context through the isolation of specific organs and tissues has now become feasible. Besides, the tagged protein can be employed for probing other interactions like protein-DNA and protein-RNA interactions. With the tools at hand, protein-centred interaction studies will greatly improve our knowledge of how plant cells wire their functional components in relation to their function. PMID:25603557

  10. 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. PMID:26216265

  11. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, H.; Smith, L.M.

    1997-01-07

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support. 3 figs.

  12. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, Huamin; Smith, Lloyd M.

    1997-01-01

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support.

  13. Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps.

    PubMed

    Chandler, D P; Stults, J R; Cebula, S; Schuck, B L; Weaver, D W; Anderson, K K; Egholm, M; Brockman, F J

    2000-08-01

    Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10(-21) M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe

  14. Cell type-specific affinity purification of nuclei for chromatin profiling in whole animals.

    PubMed

    Steiner, Florian A; Henikoff, Steven

    2015-01-01

    Analyzing cell differentiation during development in a complex organism requires the analysis of expression and chromatin profiles in individual cell types. Our laboratory has developed a simple and generally applicable strategy to purify specific cell types from whole organisms for simultaneous analysis of chromatin and expression. The method, termed INTACT for Isolation of Nuclei TAgged in specific Cell Types, depends on the expression of an affinity-tagged nuclear envelope protein in the cell type of interest. These nuclei can be affinity-purified from the total pool of nuclei and used as a source for RNA and chromatin. The method serves as a simple and scalable alternative to FACS sorting or laser capture microscopy to circumvent the need for expensive equipment and specialized skills. This chapter provides detailed protocols for the cell-type specific purification of nuclei from Caenorhabditis elegans.

  15. Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.

    PubMed

    Babu, Mohan; Kagan, Olga; Guo, Hongbo; Greenblatt, Jack; Emili, Andrew

    2012-01-01

    Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems(1, 2). Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria(1-6). In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes(1, 2, 7). Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast(8, 9), and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system(10). Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large

  16. Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry

    PubMed Central

    2010-01-01

    Background Poly(ADP-ribose) polymerases (PARPs) catalyze the formation of poly(ADP-ribose) (pADPr), a post-translational modification involved in several important biological processes, namely surveillance of genome integrity, cell cycle progression, initiation of the DNA damage response, apoptosis, and regulation of transcription. Poly(ADP-ribose) glycohydrolase (PARG), on the other hand, catabolizes pADPr and thereby accounts for the transient nature of poly(ADP-ribosyl)ation. Our investigation of the interactomes of PARP-1, PARP-2, and PARG by affinity-purification mass spectrometry (AP-MS) aimed, on the one hand, to confirm current knowledge on these interactomes and, on the other hand, to discover new protein partners which could offer insights into PARPs and PARG functions. Results PARP-1, PARP-2, and PARG were immunoprecipitated from human cells, and pulled-down proteins were separated by gel electrophoresis prior to in-gel trypsin digestion. Peptides were identified by tandem mass spectrometry. Our AP-MS experiments resulted in the identifications of 179 interactions, 139 of which are novel interactions. Gene Ontology analysis of the identified protein interactors points to five biological processes in which PARP-1, PARP-2 and PARG may be involved: RNA metabolism for PARP-1, PARP-2 and PARG; DNA repair and apoptosis for PARP-1 and PARP-2; and glycolysis and cell cycle for PARP-1. Conclusions This study reveals several novel protein partners for PARP-1, PARP-2 and PARG. It provides a global view of the interactomes of these proteins as well as a roadmap to establish the systems biology of poly(ADP-ribose) metabolism. PMID:20388209

  17. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  18. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.

    PubMed

    Banks, Charles A S; Kong, Stephanie E; Washburn, Michael P

    2012-12-01

    Characterizing protein complexes and identifying their subunits promote our understanding of the machinery involved in many in vivo processes. Proteomic studies can identify a protein's binding partners, and this can provide insight into how protein complexes function and how they are regulated. In addition, the composition of a protein complex within an organism can be investigated as a function of time, as a function of location, or during the response of an organism to a change in environment. There are many ways to isolate a complex and identify its constituents. This review will focus on complex isolation using affinity purification and will address issues that biochemists should bear in mind as they isolate protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT)(1). Protein complex analysis by mass spectrometry frequently involves the collaborative efforts of biochemists or biologists who purify protein complexes and proteomic specialists who analyze the samples - for fruitful collaborations it can be helpful for these specialized groups to be acquainted with basic principles of their collaborator's discipline. With this in mind, we first review the variety of affinity purification methods which might be considered for preparing complexes for analysis, and then provide brief primers on the principles of MudPIT mass spectrometry and data analysis. From this foundation, we then discuss how these techniques are integrated and optimized and suggest salient points to consider when preparing purified samples for protein identification, performing mass spectrometry runs, and analyzing the resulting data.

  19. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies.

    PubMed

    Boulet-Audet, Maxime; Kazarian, Sergei G; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  20. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

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

    PubMed

    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

  2. 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

  3. Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography.

    PubMed

    Kuhar, Kalika; Mittal, Anuradha; Kansal, Rekha; Gupta, Vijay Kumar

    2014-02-01

    Plant protease inhibitors (PIs) are generally small proteins which play key roles in regulation of endogenous proteases and may exhibit antifeedant, antifungal, antitumor and cytokine inducing activities. Dolichos biflorus (horse gram) is an unexploited legume, which is rich in nutrients and also has therapeutic importance. It contains a double-headed PI, which is an anti-nutritional factor. As there is no report available on its simultaneous removal and purification in single step, in this study, a double-headed PI active against both trypsin and chymotrypsin was purified from Dolichos biflorus to -14-fold with -84% recovery using an immobilized metal affinity chromatography (IMAC) medium consisting of Zn-alginate beads. The method was single-step, fast, simple, reliable and economical. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 16 kDa and was stable over a pH range of 2.0-12.0 and up to a temperature of 100 degrees C for 20 min. The optimum temperature for trypsin and chymotrypsin inhibitor was observed to be 50 degrees C and 37 degrees C, respectively and pH optimum was pH 7.0 and 8.0, respectively. Thus, IMAC using Zn-alginate beads was useful in simultaneous purification and removal of an anti-nutritional factor from horse gram flour in single step. This procedure may also be employed for purification of other plant PIs in one step.

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

    PubMed

    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.

  5. DNA purification by triplex-affinity capture and affinity capture electrophoresis

    DOEpatents

    Cantor, Charles R.; Ito, Takashi; Smith, Cassandra L.

    1996-01-01

    The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel.

  6. DNA purification by triplex-affinity capture and affinity capture electrophoresis

    DOEpatents

    Cantor, C.R.; Ito, Takashi; Smith, C.L.

    1996-01-09

    The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel. 6 figs.

  7. Affinity tag for protein purification and detection based on the disulfide-linked complex of InaD and NorpA.

    PubMed

    Kimple, Michelle E; Sondek, John

    2002-09-01

    Affinity tags are not only used for the expression and purification of recombinant proteins but also for the detection of protein-protein interactions. Common problems with many affinity tags are excessive length, which may interfere with the structure and function of tagged proteins, and low affinity and/or specificity for primary detection and purification agents. Preliminary results suggest that the C-terminalfive residues of the Drosophila protein NorpA, based on the short, covalent interaction they make with the N-terminal PDZ domain (PDZI) of InaD, are useful as a general affinity tag. First, a PDZI-alkaline phosphatase fusion protein specifically detects both its physiological ligand and a heterologous protein expressing the NorpA C-terminal five residues. The interaction of PDZI with a NorpA-tagged protein is reversible by a reducing agent, which allows nitrocellulose membranes to be stripped completely and reused. In addition, a NorpA-tagged protein can specifically bind to immobilized PDZI resin, while other cellular proteins are washed through. After washing, the NorpA-tagged protein is eluted by a reducing buffer. The NorpA tag's short length makes it the smallest affinity tag available, and its specific and high-affinity interaction with PDZI could yield a powerful system that improves on currently available technology.

  8. Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling

    SciTech Connect

    Katoh, M.; Djiane, J.; Kelly, P.A.

    1985-06-01

    The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with (/sup 125/I)ovine PRL ((/sup 125/I)oPRL). PRL receptors were solubilized from mammary microsomes with 3-((3-cholamidopropyl)dimethylammonio)1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with /sup 125/I using the chloramine-T method. Covalent labeling of PRL receptors with (/sup 125/I)oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of (/sup 125/I)oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five (/sup 125/I)oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit.

  9. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  10. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  11. One-step purification of lactoperoxidase from bovine milk by affinity chromatography.

    PubMed

    Atasever, Ali; Ozdemir, Hasan; Gulcin, Ilhami; Irfan Kufrevioglu, O

    2013-01-15

    Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC(50) of 0.848.10(-5)M. The K(i) for sulphanilamide was determined to be 3.57.10(-5)M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-L-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and L-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS-PAGE and its R(z) (A(412)/A(280)) value. The R(z) value for the purified LPO was found to be 0.7. Maximum binding was achieved and K(m) and V(max) values were determined.

  12. Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Bering, C. Larry; Kuhns, Jennifer J.; Rowlett, Roger

    1998-08-01

    We have developed a rapid and inexpensive experiment utilizing affinity chromatography to isolate carbonic anhydrase (CA) from bovine blood. The more specific an affinity gel is the better the purification, but the greater the cost. Some costs would be prohibitive in the undergraduate biochemistry laboratory. Less specific resins may be more affordable but may bind a number of closely related proteins. One alternative would be to couple a specific ligand to an inexpensive resin such as an ion exchanger. We describe a simple procedure for preparing a sulfonamide-coupled resin which specifically binds CA from a blood hemolysate. The CA is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that only a single band of 31 kD was obtained. The instructor can readily prepare the affinity gel prior to the lab, and the students, beginning with packed red blood cells can carry out the lysis, binding to the gel, elution, enzymatic assays, and electrophoresis.

  13. Reverse osmosis water purification system

    NASA Technical Reports Server (NTRS)

    Ahlstrom, H. G.; Hames, P. S.; Menninger, F. J.

    1986-01-01

    A reverse osmosis water purification system, which uses a programmable controller (PC) as the control system, was designed and built to maintain the cleanliness and level of water for various systems of a 64-m antenna. The installation operates with other equipment of the antenna at the Goldstone Deep Space Communication Complex. The reverse osmosis system was designed to be fully automatic; with the PC, many complex sequential and timed logic networks were easily implemented and are modified. The PC monitors water levels, pressures, flows, control panel requests, and set points on analog meters; with this information various processes are initiated, monitored, modified, halted, or eliminated as required by the equipment being supplied pure water.

  14. 3'-Amino thymidine affinity matrix for the purification of herpes simplex virus thymidine kinase.

    PubMed Central

    Tung, P. P.; Respass, J.; Summers, W. C.

    1996-01-01

    A simple procedure for preparation of an affinity resin with 3'-amino thymidine linked to the carboxyl residues on 6-amino-hexanoic agarose is described. We have used this column for a rapid and simple purification of the thymidine kinase encoded by the herpes simplex virus type 1 genome. This resin has two major advantages over the most widely use used resin made with thymidine-p-nitrophenyl phosphate: first it is easily obtainable, and second, it is not subject to destruction by phosphodiesterases. The two resins are very similar in behavior and the resin made with amino thymidine has allowed us to prepare large quantities of highly purified HSV TK for crystallization studies. Images Figure 3 Figure 4 PMID:9436293

  15. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    PubMed

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

  16. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 2: Experimental Study

    PubMed Central

    2015-01-01

    We present an experimental study of coupling of isotachophoresis (ITP) and affinity chromatography (AC) to effect rapid, selective purification with high column utilization and high resolution. We provide a detailed protocol for performing ITP-AC and describe the design of a buffer system to perform sequence specific separation of nucleic acids. We describe the synthesis and functionalization of our affinity substrate, poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith (GMA-EDMA PPM). This substrate allows easy immobilization of affinity probes, is nonsieving (even to macromolecules), and exhibits negligible nonspecific binding. We demonstrate ITP-AC with 25 nt, Cy5 labeled DNA target and a DNA probe and study the spatiotemporal dynamics using epifluorescence imaging. We make qualitative and quantitative comparisons between these data and the model presented in the first part of this two-paper series. We vary the target concentration from 1 pg μL–1 to 100 pg μL–1 and ITP velocity over the range of 10–50 μm s–1, and thereby explore over 4 orders of magnitude of scaled target amount. We observe very good agreement between predictions and experimental data for the spatiotemporal behavior of the coupled ITP and affinity process, and for key figures of merit, including scaled capture length and maximum capture efficiency. Lastly, we demonstrate that the resolution of ITP-AC increases linearly with time and purify 25 nt target DNA from 10 000-fold higher abundance background (contaminating) genomic fish sperm DNA. We perform this capture from 200 μL of sample in under 1 mm column length and within <10 min. PMID:24937777

  17. Affinity purification of in vitro transcribed RNA with homogeneous ends using a 3'-ARiBo tag.

    PubMed

    Di Tomasso, Geneviève; Salvail-Lacoste, Alix; Bouvette, Jonathan; Omichinski, James G; Legault, Pascale

    2014-01-01

    Common approaches for purification of RNAs synthesized in vitro by the T7 RNA polymerase often denature the RNA and produce RNAs with chemically heterogeneous 5'- and 3'-ends. Thus, native affinity purification strategies that incorporate 5' and 3' trimming technologies provide a solution to two main disadvantages that arise from standard approaches for RNA purification. This chapter describes procedures for nondenaturing affinity purification of in vitro transcribed RNA using a 3'-ARiBo tag, which yield RNAs with a homogeneous 3'-end. The applicability of the method to RNAs of different sequences, secondary structures, and sizes (29-614 nucleotides) is described, including suggestions for troubleshooting common problems. In addition, this chapter presents three complementary approaches to producing 5'-homogeneity of the affinity-purified RNA: (1) selection of the starting sequence; (2) Cse3 endoribonuclease cleavage of a 5'-CRISPR tag; or (3) self-cleavage of a 5'-hammerhead ribozyme tag. The additional steps to express and purify the Cse3 endonuclease are detailed. In light of recent results, the advantages and limitations of current approaches to achieve 5'-homogeneity of affinity-purified RNA are discussed, such that one can select a suitable strategy to purify the RNA of interest. PMID:25432744

  18. Ligand affinity chromatography, an indispensable method for the purification of soluble cytokine receptors and binding proteins.

    PubMed

    Novick, Daniela; Rubinstein, Menachem

    2012-01-01

    Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity. PMID:22131033

  19. Purification of infective bluetongue virus particles by immuno-affinity chromatography using anti-core antibody.

    PubMed

    Chand, Karam; Biswas, Sanchay K; Mondal, Bimalendu

    2016-03-01

    An immuno-affinity chromatography technique for purification of infective bluetongue virus (BTV) has been descried using anti-core antibodies. BTV anti-core antibodies (prepared in guinea pig) were mixed with cell culture-grown BTV-1 and then the mixture was added to the cyanogens bromide-activated protein-A Sepharose column. Protein A binds to the antibody which in turn binds to the antigen (i.e. BTV). After thorough washing, antigen-antibody and antibody-protein A couplings were dissociated with 4M MgCl2, pH6.5. Antibody molecules were removed by dialysis and virus particles were concentrated by spin column ultrafiltration. Dialyzed and concentrated material was tested positive for BTV antigen by a sandwich ELISA and the infectivity of the chromatography-purified virus was demonstrated in cell culture. This method was applied for selective capture of BTV from a mixture of other viruses. As group-specific antibodies (against BTV core) were used to capture the virus, it is expected that virus of all BTV serotypes could be purified by this method. This method will be helpful for selective capture and enrichment of BTV from concurrently infected blood or tissue samples for efficient isolation in cell culture. Further, this method can be used for small scale purification of BTV avoiding ultracentrifugation. PMID:26925450

  20. Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics.

    PubMed Central

    Wright, C L; Warsy, A S; Holroyde, M J; Trayer, I P

    1978-01-01

    The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity

  1. Metal chelate affinity precipitation: purification of BSA using poly(N-vinylcaprolactam-co-methacrylic acid) copolymers.

    PubMed

    Ling, Yuan-Qing; Nie, Hua-Li; Brandford-White, Christopher; Williams, Gareth R; Zhu, Li-Min

    2012-06-01

    This investigation involves the metal chelate affinity precipitation of bovine serum albumin (BSA) using a copper ion loaded thermo-sensitive copolymer. The copolymer of N-vinylcaprolactam with methacrylic acid PNVCL-co-MAA was synthesized by free radical polymerization in aqueous solution, and Cu(II) ions were attached to provide affinity properties for BSA. A maximum loading of 48.1mg Cu(2+) per gram of polymer was attained. The influence of pH, temperature, BSA and NaCl concentrations on BSA precipitation and of pH, ethylenediaminetetraacetic acid (EDTA) and NaCl concentrations on elution were systematically probed. The optimum conditions for BSA precipitation occurred when pH, temperature and BSA concentration were 6.0, 10°C and 1.0 mg/ml, respectively and the most favorable elution conditions were at pH 4.0, with 0.2M NaCl and 0.06 M EDTA. The maximum amounts of BSA precipitation and elution were 37.5 and 33.7 mg BSA/g polymer, respectively. It proved possible to perform multiple precipitation/elution cycles with a minimal loss of polymer efficacy. The results show that PNVCL-co-MAA is a suitable matrix for the purification of target proteins from unfractionated materials.

  2. p53-Encoding pDNA Purification by Affinity Chromatography for Cancer Therapy.

    PubMed

    Sousa, Ângela; Queiroz, João A; Sousa, Fani

    2015-01-01

    The gene therapy approach based on reestablishment of p53 tumor suppressor, which acts as a prevailing guardian against malignant cell transformation, is raising new prospects on the outcome of an effective anticancer treatment. It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Therefore, several downstream methods have been proposed to achieve high quantities of supercoiled plasmid DNA with pharmaceutical grade purity. Affinity chromatography with amino acids as ligands has recently yielded interesting results because these ligands take advantage of their biological function or chemical structure to promote specific interactions with different nucleic acids. Here, we describe detailed procedures for the preparation and purification of supercoiled plasmid DNA, with the purity degree required by regulatory agencies, by using arginine affinity chromatography. With this methodology pure pDNA is obtained, efficient on eukaryotic cell transfection and biologically active, resulting in the reestablishment of the p53 protein levels in cancer cell lines. PMID:26072404

  3. Purification of beta-glucuronidase and structural assessment of the carbohydrate chains by lectin affinity immunoelectrophoresis.

    PubMed

    Wójczyk, B; Hoja, D; Lityńska, A

    1991-08-01

    The purification of rat liver beta-glucuronidase from a lysosomal fraction by methods including affinity chromatography, chromatofocusing and preparative PAGE steps is described. Molecular weights of 300,000 and 150,000 were estimated by two dimensional gradient PAGE/immunoelectrophoresis of the lysosomal extract. Isoelectrofocusing in agarose gel followed by immunoelectrophoresis in the second dimension revealed the presence of at least five maxima in the range pH 4.3-7.4. The structural assessment of the carbohydrate chains of lysosomal and microsomal beta-glucuronidase was performed by lectin affinity immunoelectrophoresis. Reaction with Concanavalin A indicated the presence of bi-antennary complex, oligomannosidic and hybrid type structures, whereas the absence of tri- and tetra-antennary complex type structures was deduced from the lack of interaction with phytohemagglutinin-L. The reaction with Lens culinaris agglutinin, Pisum sativum agglutinin and Lotus tetragonolobus lectin revealed that part of the glycans contained a fucose alpha(1-6)-linked to the N-acetylglucosamine attached to asparagine. The presence of terminal beta(1-4)-galactose residues was detected with Ricinus communis agglutinin I. PMID:1841676

  4. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    PubMed

    Salehi, Nasrin; Peng, Ching-An

    2016-07-01

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. PMID:27110670

  5. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

    PubMed Central

    Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T

    2009-01-01

    The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766

  6. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    PubMed

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d.

  7. Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

    PubMed

    Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

    2015-01-01

    A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation.

  8. High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification.

    PubMed

    Nie, Minghua; Vashisht, Ajay A; Wohlschlegel, James A; Boddy, Michael N

    2015-09-25

    Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO "cloud" phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function.

  9. Novel cross-linked alcohol-insoluble solid (CL-AIS) affinity gel from pea pod for pectinesterase purification.

    PubMed

    Wu, Ming-Chang; Lin, Guan-Hui; Wang, Yuh-Tai; Jiang, Chii-Ming; Chang, Hung-Min

    2005-10-01

    Alcohol-insoluble solids (AIS) from pea pod were cross-linked (CL-AIS) and used as an affinity gel matrix to isolate pectin esterases (PEs) from tendril shoots of chayote (TSC) and jelly fig achenes (JFA), and the results were compared with those isolated by ion-exchange chromatography with a commercial resin. CL-AIS gel matrix in a column displayed poor absorption and purification fold of PE; however, highly methoxylated CL-AIS (HM-CL-AIS), by exposing CL-AIS to methanolic sulfuric acid to increase the degree of esterification (DE) to 92%, facilitated the enzyme purification. The purified TSC PE and JFA PE by the HM-CL-AIS column were proofed as a single band on an SDS-PAGE gel, showing that the HM-CL-AIS column was a good matrix for purification of PE, either with alkaline isoelectric point (pI) (TSC PE) or with acidic pI (JFA PE).

  10. Purification of Hemoglobin from Red Blood Cells using Tangential Flow Filtration and Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Elmer, Jacob; Harris, David; Palmer, Andre F.

    2011-01-01

    Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are examined and compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter. PMID:21195679

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

    PubMed

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

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. PMID:26657801

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

    PubMed

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

    2015-10-30

    Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin. PMID:26427325

  13. Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum.

    PubMed

    Kollmar, Martin

    2006-08-15

    The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species. PMID:16516959

  14. Monosize poly(glycidyl methacrylate) beads for dye-affinity purification of lysozyme.

    PubMed

    Altintaş, Evrim Banu; Denizli, Adil

    2006-03-30

    Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for purification of lysozyme from chicken egg white. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by a dispersion polymerization technique. The content of epoxy groups on the surface of the poly(GMA) sample determined by the HCl-pyridine method (3.8 mmol/g). Cibacron Blue F3GA loading was 1.73 mmol/g. The monosize beads were characterized by elemental analysis, FTIR and SEM. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration, temperature and ionic strength). Maximum lysozyme adsorption amount of poly(GMA) and poly(GMA)-Cibacron Blue F3GA beads were 1.6 and 591.7 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, poly(GMA)-Cibacron Blue F3GA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg-white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the eluted lysozyme was analyzed by SDS-PAGE and found to be 88% with recovery about 79%. The specific activity of the eluted lysozyme was high as 43,600 U/mg.

  15. Using ProHits to store, annotate and analyze affinity purification - mass spectrometry (AP-MS) data

    PubMed Central

    Liu, Guomin; Zhang, Jianping; Choi, Hyungwon; Lambert, Jean-Philippe; Srikumar, Tharan; Larsen, Brett; Nesvizhskii, Alexey I.; Raught, Brian; Tyers, Mike; Gingras, Anne-Claude

    2012-01-01

    Affinity purification coupled with mass spectrometry (AP-MS) is a robust technique used to identify protein-protein interactions. With recent improvements in sample preparation, and dramatic advances in MS instrumentation speed and sensitivity, this technique is becoming more widely used throughout the scientific community. To meet the needs of research groups both large and small, we have developed software solutions for tracking, scoring and analyzing AP-MS data. Here, we provide details for the installation and utilization of ProHits, a Laboratory Information Management System designed specifically for AP-MS interaction proteomics. This protocol explains: (i) how to install the complete ProHits system, including modules for the management of mass spectrometry files and the analysis of interaction data, and (ii) alternative options for the use of pre-existing search results in simpler versions of ProHits, including a virtual machine implementation of our ProHits Lite software. We also describe how to use the main features of the software to analyze AP-MS data. PMID:22948730

  16. Evaluation system of negative electron affinity photocathode

    NASA Astrophysics Data System (ADS)

    Fu, Rongguo; Chang, Benkang; Qian, Yunsheng; Wang, Guihua; Zong, Zhiyuan

    2001-10-01

    This article first describes the background of the research and manufacture of evaluation system of Negative Electron Affinity photocathode. This article designs a set of super high vacuum system for activating NEA photocathode on the base of activation theory, the process of design and debugging is given. The system is composed of three parts: super high vacuum system for GaAs material activation, multi-meter testing system, surface analysis system. The system is used for on-line evaluation of activating of NEA photocathode. The technical parameters and structure of the evaluation system of NEA photocathode are given in the paper. The system is finished and experiments are made. At last the picture of the system is given.

  17. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

    PubMed Central

    Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Xu, Zhikai; Lei, Yingfeng

    2015-01-01

    Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions. PMID:26389898

  18. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1.

    PubMed

    Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Xu, Zhikai; Lei, Yingfeng

    2015-09-16

    Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3' end of dengue virus (DENV) 5'-3' UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.

  19. Affinity Purification of a Recombinant Protein Expressed as a Fusion with the Maltose-Binding Protein (MBP) Tag

    PubMed Central

    Duong-Ly, Krisna C.; Gabelli, Sandra B.

    2015-01-01

    Expression of fusion proteins such as MBP fusions can be used as a way to improve the solubility of the expressed protein in E. coli (Fox and Waugh, 2003; Nallamsetty et al., 2005; Nallamsetty and Waugh, 2006) and as a way to introduce an affinity purification tag. The protocol that follows was designed by the authors as a first step in the purification of a recombinant protein fused with MBP, using fast protein liquid chromatography (FPLC). Cells should have been thawed, resuspended in binding buffer, and lysed by sonication or microfluidization before mixing with the amylose resin or loading on the column. Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment. PMID:26096500

  20. Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags

    PubMed Central

    Salvail-Lacoste, Alix; Di Tomasso, Geneviève; Piette, Benjamin L.; Legault, Pascale

    2013-01-01

    Affinity purification of RNA using the ARiBo tag technology currently provides an ideal approach to quickly prepare RNA with 3′ homogeneity. Here, we explored strategies to also ensure 5′ homogeneity of affinity-purified RNAs. First, we systematically investigated the effect of starting nucleotides on the 5′ heterogeneity of a small SLI RNA substrate from the Neurospora VS ribozyme purified from an SLI-ARiBo precursor. A series of 32 SLI RNA sequences with variations in the +1 to +3 region was produced from two T7 promoters (class III consensus and class II ϕ2.5) using either the wild-type T7 RNA polymerase or the P266L mutant. Although the P266L mutant helps decrease the levels of 5′-sequence heterogeneity in several cases, significant levels of 5′ heterogeneity (≥1.5%) remain for transcripts starting with GGG, GAG, GCG, GGC, AGG, AGA, AAA, ACA, AUA, AAC, ACC, AUC, and AAU. To provide a more general approach to purifying RNA with 5′ homogeneity, we tested the suitability of using a small CRISPR RNA stem–loop at the 5′ end of the SLI-ARiBo RNA. Interestingly, we found that complete cleavage of the 5′-CRISPR tag with the Cse3 endoribonuclease can be achieved quickly from CRISPR–SLI-ARiBo transcripts. With this procedure, it is possible to generate SLI-ARiBo RNAs starting with any of the four standard nucleotides (G, C, A, or U) involved in either a single- or a double-stranded structure. Moreover, the 5′-CRISPR-based strategy can be combined with affinity purification using the 3′-ARiBo tag for quick purification of RNA with both 5′ and 3′ homogeneity. PMID:23657939

  1. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    SciTech Connect

    Lee, Byung-Kwon; Jung, Kyung-Sik; Son, Cagdas D; Kim, Heejung; Verberkmoes, Nathan C; Arshava, Boris; Naider, Fred; Becker, Jeffrey Marvin

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  2. Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

    PubMed

    Maltezos, Anastasios; Platis, Dimitris; Vlachakis, Dimitrios; Kossida, Sophia; Marinou, Marigianna; Labrou, Nikolaos E

    2014-01-01

    The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

  3. Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

    PubMed

    Maltezos, Anastasios; Platis, Dimitris; Vlachakis, Dimitrios; Kossida, Sophia; Marinou, Marigianna; Labrou, Nikolaos E

    2014-01-01

    The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. PMID:24375581

  4. Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

    PubMed Central

    Lackmann, M; Bucci, T; Mann, R J; Kravets, L A; Viney, E; Smith, F; Moritz, R L; Carter, W; Simpson, R J; Nicola, N A; Mackwell, K; Nice, E C; Wilks, A F; Boyd, A W

    1996-01-01

    Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor. Images Fig. 2 Fig. 3 PMID:8637907

  5. A simple one pot purification of bacterial amylase from fermented broth based on affinity toward starch-functionalized magnetic nanoparticle.

    PubMed

    Paul, Tanima; Chatterjee, Saptarshi; Bandyopadhyay, Arghya; Chattopadhyay, Dwiptirtha; Basu, Semanti; Sarkar, Keka

    2015-08-18

    Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet-visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION.

  6. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 1: Theory

    PubMed Central

    2015-01-01

    We present a novel technique that couples isotachophoresis (ITP) with affinity chromatography (AC) to achieve rapid, selective purification with high column utilization. ITP simultaneously preconcentrates an analyte and purifies it, based on differences in mobility of sample components, excluding species that may foul or compete with the target at the affinity substrate. ITP preconcentration accelerates the affinity reaction, reducing assay time, improving column utilization, and allowing for capture of targets with higher dissociation constants. Furthermore, ITP-AC separates the target and contaminants into nondiffusing zones, thus achieving high resolution in a short distance and time. We present an analytical model for spatiotemporal dynamics of ITP-AC. We identify and explore the effect of key process parameters, including target distribution width and height, ITP zone velocity, forward and reverse reaction constants, and probe concentration on necessary affinity region length, assay time, and capture efficiency. Our analytical approach shows collapse of these variables to three nondimensional parameters. The analysis yields simple analytical relations for capture length and capture time in relevant ITP-AC regimes, and it demonstrates how ITP greatly reduces assay time and improves column utilization. In the second part of this two-part series, we will present experimental validation of our model and demonstrate ITP-AC separation of the target from 10,000-fold more-abundant contaminants. PMID:24937679

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

    PubMed

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

    2014-04-25

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

  8. Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers.

    PubMed

    Croucher, David R; Iconomou, Mary; Hastings, Jordan F; Kennedy, Sean P; Han, Jeremy Z R; Shearer, Robert F; McKenna, Jessie; Wan, Adrian; Lau, Joseph; Aparicio, Samuel; Saunders, Darren N

    2016-01-01

    The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal-regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells. PMID:27405979

  9. Engineering foot-and-mouth disease virus serotype O IND R2/1975 for one-step purification by immobilized metal affinity chromatography.

    PubMed

    Biswal, Jitendra K; Bisht, Punam; Subramaniam, Saravanan; Ranjan, Rajeev; Sharma, Gaurav K; Pattnaik, Bramhadev

    2015-09-01

    Immobilized metal affinity chromatography (IMAC) allows for the efficient protein purification via metal affinity tag such as hexa-histidine (His6) sequence. To develop a new chromatography strategy for the purification and concentration of foot-and-mouth disease virus (FMDV) particles, we inserted the His6-tag at the earlier reported site in the VP1 G-H loop of the FMD virus serotype O vaccine strain IND R2/1975. Display of the His6-tag on the capsid surface, endowed the virus with an increased affinity for immobilized nickel ions. We demonstrated that the His6-tagged FMDV could be produced to high titre and purified from the infected BHK-21 cell lysates by IMAC efficiently. Further, a 1150-fold reduction in protein contaminant level and an 8400-fold reduction in DNA contaminant level were achieved in the IMAC purification of His6-tagged FMDV. Through various functional assays it has been found that the tagged virus retains its functionality and infectivity similar to the non-tagged virus. The affinity purification of the His6-tagged FMDV may offer a feasible, alternative approach to the current methods of FMDV antigen purification, concentration and process scalability. PMID:26123433

  10. Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

    PubMed

    Shiomi, K; Yamaguchi, S; Shimakura, K; Nagashima, Y; Yamamori, K; Matsui, T

    1993-12-01

    A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

  11. Diesel exhaust-gas purification system

    SciTech Connect

    Doherty, B.J.

    1982-07-01

    The design of a diesel exhaust gas purification system is presented. It will provide 2000 scfm of dry, anerobic gas (essentially nitrogen) for use in air drilling operations where drill pipe corrosion is a problem, such as geothermal applications. The system is operable in the field and may be transported via highways. It will operate at ambient temperatures up to 110/sup 0/F and requires no water - diesel fuel is used to combust excess oxygen and to generate electricity for the system. Gas production costs, including capital amortization, operations, fuel and maintenance (for reasonable utilization) are about $1.50/1000 scf.

  12. Purification of phosphinothricin acetyltransferase using Reactive brown 10 affinity in a single chromatography step.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2013-08-01

    The expression of phosphinothricin N-acetyltransferase (PAT) protein in transgenic plants confers tolerance to the herbicide glufosinate. To enable the characterization of PAT protein expressed in plants, it is necessary to obtain high purity PAT protein from the transgenic grain. Because transgenically expressed proteins are typical present at very low levels (i.e. 0.1-50 μg protein/g grain), a highly specific and efficient purification protocol is required to purify them. Based on the physicochemical properties of PAT, we developed a novel purification method that is simple, time-saving, inexpensive and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein was purified to homogeneity from cottonseed with high recovery efficiency. As expected, the Reactive brown 10-produced PAT was enzymatically active. Other applications of the method on protein expression and purification, and development of PAT enzymatic inhibitors were also discussed. PMID:23748142

  13. Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Voráčková, Irena; Suchanová, Šárka; Ulbrich, Pavel; Diehl, William E.; Ruml, Tomáš

    2011-01-01

    Heterologous proteins are frequently purified by Immobilized Metal Ion Affinity Chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e. CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state. PMID:21600288

  14. L-histidine functionalized multi-walled carbon nanotubes for on-line affinity separation and purification of immunoglobulin G in serum.

    PubMed

    Du, Zhuo; Zhang, Suling; Zhou, Chanyuan; Liu, Miao; Li, Gongke

    2012-09-15

    In this work, the multi-walled carbon nanotubes were covalently functionalized with L-histidine (His-MWNTs) as online pseudospecific affinity adsorbent for immunoglobulin G (IgG) separation and purification with a simple surface modification method, using 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimde (NHS). The affinity of the His-MWNTs toward IgG was investigated in a microcolumn incorporated into a sequential injection system, which also involves an UV spectrometer with a flow cell for online real-time detection. The incorporation of histidine as affinity groups noticeably increased the selectivity and binding capacity of MWNTs for IgG and the His-MWNTs exhibited high retention and recovery rate of nearly 100% under optimized conditions. This separation and enrichment process made it possible to determine a lower concentration range of IgG in serum from 1.0-33 μg/mL with a detection limit of 0.3 μg/mL with a sampling volume of 4.0 mL. The static and dynamic adsorption capacities obtained were 267 mg of IgG/g His-MWNTs and 35 mg/g in aqueous solution, respectively, which are among the highest reported results in literatures employing affinity separation methods. Desorption of IgG from His-MWNTs could be accomplished by lowering the pH to 1.5 with glycine-HCl buffer. The practical application of His-MWNTs for separation of IgG in serum was evaluated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis which confirmed that the purity of recovered IgG from human serum was over 85% and better than a commercial product.

  15. Affinity Purification of O-Acetylserine(thiol)lyase from Chlorella sorokiniana by Recombinant Proteins from Arabidopsis thaliana.

    PubMed

    Salbitani, Giovanna; Wirtz, Markus; Hell, Rüdiger; Carfagna, Simona

    2014-01-01

    In the unicellular green alga Chlorella sorokiniana (211/8 k), the protein O-acetylserine(thiol)lyase (OASTL), representing the key-enzyme in the biosynthetic cysteine pathway, was isolated and purified to apparent homogeneity. The purification was carried out in cells grown in the presence of all nutrients or in sulphate (S) deprived cells. After 24 h of S-starvation, a 17-fold increase in the specific activity of OASTL was measured. In order to enable the identification of OASTL proteins from non-model organisms such as C. sorokiniana, the recombinant his-tagged SAT5 protein from Arabidopsis thaliana was immobilized by metal chelate chromatography. OASTL proteins from C. sorokiniana were affinity purified in one step and activities were enhanced 29- and 41-fold, from S-sufficient and S-starved (24 h) cells, respectively. The successful application of SAT/OASTL interaction for purification confirms for the first time the existence of the cysteine synthase complexes in microalgae. The purified proteins have apparent molecular masses between 32-34 kDa and are thus slightly larger compared to those found in Arabidopsis thaliana and other vascular plants. The enhanced OASTL activity in S-starved cells can be attributed to increased amounts of plastidic and the emergence of cytosolic OASTL isoforms. The results provide proof-of-concept for the biochemical analysis of the cysteine synthase complex in diverse microalgal species. PMID:25093930

  16. Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

    SciTech Connect

    James, W.M.; Emerick, M.C.; Agnew, W.S. )

    1989-07-11

    The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

  17. Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography.

    PubMed

    Tan, Lik Chern Melvin; Chua, Anthony Jin Shun; Goh, Li Shan Liza; Pua, Shu Min; Cheong, Yuen Kuen; Ng, Mah Lee

    2010-11-01

    Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) protein is highly immunogenic and capable of inducing neutralizing antibodies against wild-type virus, it is both a potential protein subunit vaccine candidate and a suitable diagnostic reagent. Here, we describe the use of metal affinity membrane chromatography as a rapid and improved alternative for the purification of recombinant DIII (rDIII) antigens from DENV serotypes 1-4 and WNV - New York, Sarafend, Wengler and Kunjin strains. Optimum conditions for the expression, solubilization, renaturation and purification of these proteins were established. The purified proteins were confirmed by MALDI-TOF mass spectrometry and ELISA using antibodies raised against the respective viruses. Biological function of the purified rDIII proteins was confirmed by their ability to generate DIII-specific antibodies in mice that could neutralize the virus.

  18. Automated Water-Purification System

    NASA Technical Reports Server (NTRS)

    Ahlstrom, Harlow G.; Hames, Peter S.; Menninger, Fredrick J.

    1988-01-01

    Reverse-osmosis system operates and maintains itself with minimal human attention, using programmable controller. In purifier, membranes surround hollow cores through which clean product water flows out of reverse-osmosis unit. No chemical reactions or phase changes involved. Reject water, in which dissolved solids concentrated, emerges from outer membrane material on same side water entered. Flow controls maintain ratio of 50 percent product water and 50 percent reject water. Membranes expected to last from 3 to 15 years.

  19. Affinity purifications of aldose reductase and xylitol dehydrogenase from the xylose-fermenting yeast Pachysolen tannophilus

    SciTech Connect

    Bolen, P.L.; Roth, K.A.; Freer, S.N.

    1986-10-01

    Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.

  20. Exploration of cone cyclic nucleotide-gated channel-interacting proteins using affinity purification and mass spectrometry.

    PubMed

    Ding, Xi-Qin; Matveev, Alexander; Singh, Anil; Komori, Naoka; Matsumoto, Hiroyuki

    2014-01-01

    Photopic (cone) vision essential for color sensation, central vision, and visual acuity is mediated by the activation of photoreceptor cyclic nucleotide-gated (CNG) channels. Naturally occurring mutations in the cone channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. This work investigated the functional modulation of cone CNG channel by exploring the channel-interacting proteins. Retinal protein extracts prepared from cone-dominant Nrl (- / -) mice were used in CNGA3 antibody affinity purification, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. The peptide mass fingerprinting of the tryptic digests and database search identified a number of proteins including spectrin alpha-2, ATPase (Na(+)/K(+) transporting) alpha-3, alpha and beta subunits of ATP synthase (H(+) transporting, mitochondrial F1 complex), and alpha-2 subunit of the guanine nucleotide-binding protein. In addition, the affinity-binding assays demonstrated an interaction between cone CNG channel and calmodulin but not cone Na(+)/Ca(2+)-K(+) exchanger in the mouse retina. Results of this study provide insight into our understanding of cone CNG channel-interacting proteins and the functional modulations.

  1. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

    PubMed

    Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Vercruysse, Leen; Dedecker, Maarten; Verkest, Aurine; Vandepoele, Klaas; Martens, Lennart; Witters, Erwin; Gevaert, Kris; De Jaeger, Geert

    2015-01-01

    Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

  2. The Plasma Membrane Ca(2+) ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein.

    PubMed

    Niggli, Verena; Carafoli, Ernesto

    2016-01-01

    Plasma membrane Ca(2+) ATPases (PMCA pumps) are key regulators of cytosolic Ca(2+) in eukaryotes. They extrude Ca(2+) from the cytosol, using the energy of ATP hydrolysis and operate as Ca(2+)-H(+) exchangers. They are activated by the Ca(2+)-binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylation. Isolation of the PMCA in pure and active form is essential for the analysis of its structure and function. In this chapter, the purification of the pump, as first achieved from erythrocyte plasma membranes by calmodulin-affinity chromatography, is described in detail. The reversible, high-affinity, Ca(2+)-dependent interaction of the pump with calmodulin is the basis of the procedure. Either phospholipids or glycerol have to be present in the isolation buffers to keep the pump active during the isolation procedure. After the isolation of the PMCA pump from human erythrocytes the pump was purified from other cell types, e.g., heart sarcolemma, plant microsomal fractions, and cells that express it ectopically. The reconstitution of the purified pump into phospholipid vesicles using the cholate dialysis method will also be described. It allows studies of transport mechanism and of regulation of pump activity. The purified pump can be stored in the reconstituted form for several days at 4 °C with little loss of activity, but it rapidly loses activity when stored in the detergent-solubilized form. PMID:26695022

  3. One-pot preparation of a sulfamethoxazole functionalized affinity monolithic column for selective isolation and purification of trypsin.

    PubMed

    Xiao, Yuan; Guo, Jialiang; Ran, Danni; Duan, Qianqian; Crommen, Jacques; Jiang, Zhengjin

    2015-06-26

    A facile and efficient "one-pot" copolymerization strategy was used for the preparation of sulfonamide drug (SA) functionalized monolithic columns. Two novel SA-immobilized methacrylate monolithic columns, i.e. poly(GMA-SMX-co-EDMA) and poly(GMA-SAA-co-EDMA) were prepared by one-pot in situ copolymerization of the drug ligand (sulfamethoxazole (SMX) or sulfanilamide (SAA)), the monomer (glycidyl methacrylate, GMA) and the cross-linker (ethylene dimethacrylate, EDMA) within 100 μm i.d. capillaries under optimized polymerization conditions. The physicochemical properties and column performance of the fabricated monolithic columns were characterized by elemental analysis, scanning electron microscopy and micro-HPLC. Satisfactory column permeability, efficiency and separation performance were obtained on the optimized poly(GMA-SMX-co-EDMA) monolithic column for small molecules, such as a standard test mixture and eight aromatic ketones. Notably, it was found that the poly(GMA-SMX-co-EDMA) monolith showed a selective affinity to trypsin, while the poly(GMA-SAA-co-EDMA) monolith containing sulfanilamide did not exhibit such affinity at all. This research not only provides a novel monolith for the selective isolation and purification of trypsin, but it also offers the possibility to easily prepare novel drug functionalized methacrylate monoliths through a one-pot copolymerization strategy.

  4. Spotlite: web application and augmented algorithms for predicting co-complexed proteins from affinity purification--mass spectrometry data.

    PubMed

    Goldfarb, Dennis; Hast, Bridgid E; Wang, Wei; Major, Michael B

    2014-12-01

    Protein-protein interactions defined by affinity purification and mass spectrometry (APMS) suffer from high false discovery rates. Consequently, lists of potential interactions must be pruned of contaminants before network construction and interpretation, historically an expensive, time-intensive, and error-prone task. In recent years, numerous computational methods were developed to identify genuine interactions from the hundreds of candidates. Here, comparative analysis of three popular algorithms, HGSCore, CompPASS, and SAINT, revealed complementarity in their classification accuracies, which is supported by their divergent scoring strategies. We improved each algorithm by an average area under a receiver operating characteristics curve increase of 16% by integrating a variety of indirect data known to correlate with established protein-protein interactions, including mRNA coexpression, gene ontologies, domain-domain binding affinities, and homologous protein interactions. Each APMS scoring approach was incorporated into a separate logistic regression model along with the indirect features; the resulting three classifiers demonstrate improved performance on five diverse APMS data sets. To facilitate APMS data scoring within the scientific community, we created Spotlite, a user-friendly and fast web application. Within Spotlite, data can be scored with the augmented classifiers, annotated, and visualized ( http://cancer.unc.edu/majorlab/software.php ). The utility of the Spotlite platform to reveal physical, functional, and disease-relevant characteristics within APMS data is established through a focused analysis of the KEAP1 E3 ubiquitin ligase.

  5. A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids.

    PubMed

    Maeda, Kenji; Poletto, Mattia; Chiapparino, Antonella; Gavin, Anne-Claude

    2014-09-01

    Interactions between lipids and proteins in the aqueous phases of cells contribute to many aspects of cell physiology. Here we describe a detailed protocol to systematically characterize in vivo-assembled complexes of soluble proteins and lipids. Saccharomyces cerevisiae strains expressing physiological amounts of a protein of interest fused to the tandem-affinity purification (TAP) tag are first lysed in the absence of detergent to capture intact protein-lipid complexes. The affinity-purified complexes (typically 30-50 kDa) are subjected to analytical size-exclusion chromatography (SEC) to remove contaminating lipids that elute at the void volume (>600 kDa), in order to achieve sufficient signal-to-background lipid ratios. Proteins in the SEC fractions are then analyzed by denaturing gel electrophoresis. Lipidomics techniques such as high-performance thin-layer chromatography or gas or liquid chromatography-mass spectrometry can then be applied to measure the elution profiles of lipids and to pinpoint the true interactors co-eluting with the TAP fusions. The procedure (starting from cell lysis) requires 2 d, and it can easily be adapted to other organisms.

  6. Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p-aminobenzamidine affinity chromatography.

    PubMed

    Bansal, Vibha; Roychoudhury, Pradip K; Mattiasson, Bo; Kumar, Ashok

    2006-01-01

    An integrated product recovery system was developed to separate urokinase from the cell culture broth of human kidney cells HT1080. Supermacroporous monolithic cryogels provided ideal matrices with respect to surface and flow properties for use as cell culture scaffold as well as for affinity chromatographic capture step of the enzyme in the integrated system. The urokinase was produced continuously in the reactor running for 4 weeks with continuous circulation of 500 ml of culture medium. The enzyme activity in the culture medium reached to 280 Plough units (PU)/mg protein. Cu(II)-iminodiacetic acid (IDA)-polyacrylamide (pAAm) cryogel column was used to capture urokinase by integrating with the gelatin-coupled pAAm-cryogel bioreactor for HT1080 cell culture. After removing the urokinase capture column from the integrated system the bound protein was eluted. The metal affinity capture step gave 4.5-fold purification of the enzyme thus achieving a specific activity of 1300 PU/mg protein. The enzyme eluate from Cu(II)-IDA-pAAm cryogel capture column was further purified on benzamidine-Sepharose affinity column. This step finally led to a homogeneous preparation of different forms of urokinase in two different elution peaks with a best urokinase activity of 13 550 PU/mg of protein. As compared to initial activity in the cell culture broth, about 26.2- and 48.4-fold increase in specific activity was achieved with enzyme yields corresponding to 32% and 35% in two different peak fractions, respectively. Native electrophoresis and SDS-PAGE showed multiple protein bands corresponding to different forms of the urokinase, which were confirmed by Western blotting and zymography. PMID:16761300

  7. Aryl thioglycoside-based affinity purification of exo-acting cellulases.

    PubMed

    Piyachomkwan, K; Penner, M H

    1998-01-15

    The influence of ligand-coupling chemistry and mobile-phase composition on the interaction of exo-acting cellulases with an immobilized complementary ligand was investigated. p-Aminophenyl 1-thio-beta-D-cellobioside (APTC) was used as a representative affinity ligand to which exo-acting cellulases (cellobiohydrolases, CBHs) preferentially bind. A "crude" cellulase preparation from the fungus Trichoderma reesei served as an enzyme source. The adsorption properties of the two principal exo-acting CBHs in this preparation, CBH I and CBH II, are shown to be distinctly different under several scenarios. Their relative affinities, based on column elution behavior and partition equilibrium experiments, are shown to be highly dependent on the functional groups employed for ligand coupling, the extent of functional group hydrolysis, the composition of the mobile phase, and the inherent nature of the enzymes. The dependency on the chemistry of the supporting matrix was illustrated using agarose supports containing cyanate ester, N-hydroxy-succinimide, and epoxy functional groups. When compared under apparent optimal conditions, the affinity of CBH II for immobilized APTC was approximately 10-fold that of CBH I. However, selective adsorption of CBH I or CBH II can be achieved by adjusting experimental parameters. PMID:9451508

  8. Metal chelate affinity precipitation of RNA and purification of plasmid DNA

    NASA Technical Reports Server (NTRS)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

    2003-01-01

    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

  9. Rapid purification of the gastric H+/K(+)-ATPase complex by tomato-lectin affinity chromatography.

    PubMed Central

    Callaghan, J M; Toh, B H; Simpson, R J; Baldwin, G S; Gleeson, P A

    1992-01-01

    We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the beta-subunit of the gastric H+/K(+)-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris & Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Mortiz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis & Baldwin (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K(+)-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1% Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein beta-subunit. The two components were identified as the 95 kDa alpha-subunit and the 60-90 kDa beta-subunit of the gastric H+/K(+)-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The beta-subunit was present in approximately equimolar amounts to the catalytic alpha-subunit. Whereas the gastric H+/K(+)-ATPase was not active after solubilization in 1% Triton X-100, solubilization of density-gradient-purified membranes in the non-ionic detergent, C12E8, followed by chromatography of the extract on tomato lectin-Sepharose 4B, resulted in the purification of the gastric H+/K(+)-ATPase complex which exhibited K(+)-dependent phosphatase activity. This is the first report of a rapid purification of a partially active solubilized

  10. Affinity purification of antibodies using immobilized FB domain of protein A.

    PubMed

    Solomon, B; Raviv, O; Leibman, E; Fleminger, G

    1992-04-24

    A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A. PMID:1517325

  11. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Water purification system for hemodialysis. 876.5665 Section 876.5665 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665 Water purification system for hemodialysis....

  12. Purification of subunit B of Shiga toxin using a synthetic trisaccharide-based affinity matrix.

    PubMed

    Pozsgay, V; Trinh, L; Shiloach, J; Robbins, J B; Donohue-Rolfe, A; Calderwood, S B

    1996-01-01

    The blood group P1 antigenic trisaccharide (3), which is the receptor-binding ligand of Shiga-like toxins, is synthesized in a spacer-equipped form (32) from 2-(trimethylsilyl)ethyl glucoside 5 and the 1-thiogalactoside building blocks 10 and 22 in a stereocontrolled, stepwise fashion. Covalent attachment of 32 to hydrazine group-containing agarose gel by reductive amination provided the P1 trisaccharide-containing affinity sorbent which was used for preparative scale isolation of subunit B of Shiga toxin. PMID:8741990

  13. A tandem laboratory scale protein purification process using Protein A affinity and anion exchange chromatography operated in a weak partitioning mode.

    PubMed

    Shamashkin, Michael; Godavarti, Ranga; Iskra, Timothy; Coffman, Jon

    2013-10-01

    A significant consequence of scaling up production of high titer monoclonal antibody (mAb) processes in existing facilities is the generation of in-process pools that exceed the capacity of storage vessels. A semi-continuous downstream process where columns and filters are linked and operated in tandem would eliminate the need for intermediate holding tanks. This study is a bench-scale demonstration of the feasibility of a tandem process for the purification of mAbs employing an affinity Protein A capture step, followed by a flow-through anion-exchange (AEX) step with the possibility of adding an in-line virus filtration step (VF). All three steps were linked sequentially and operated as one continuous process using an ÄKTA FPLC equipped with two pumps and a system of valves and bypasses that allowed the components to be engaged at different stages of the process. The AEX column was operated in a weak partitioning (WP) mode enabled by a precise in-line titration of Protein A effluent. In order to avoid complex control schemes and facilitate validation, quality and robustness were built into the system through selection of buffers based on thermodynamic and empirical models. The tandem system utilized the simplest possible combination of valves, pumps, controls, and automation, so that it could easily be implemented in a clinical or commercial production facility. Linking the purification steps in a tandem process is expected to generate savings in time and production costs and also reduce the size of quality systems due to reduced documentation requirements, microbial sampling, and elimination of hold time validation. PMID:23633385

  14. Purification of peroxidase from red cabbage (Brassica oleracea var. capitata f. rubra) by affinity chromatography.

    PubMed

    Somtürk, Burcu; Kalın, Ramazan; Özdemir, Nalan

    2014-08-01

    Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9% from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K m and V max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC50 and K i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702±0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.

  15. IgA-affinity purification and characterization of the lectin jacalin.

    PubMed

    Roque-Barreira, M C; Praz, F; Halbwachs-Mecarelli, L; Greene, L J; Campos-Neto, A

    1986-01-01

    We describe the use of IgA-Sepharose 4B affinity chromatography to purify the lectin jacalin from saline extracts of Artocarpus integrifolia L. seeds. Elution with 0.8 M D-galactose provides 10-15 mg lectin/50 mg seed protein. Jacalin behaved like a single component on immunoelectrophoresis and a single, somewhat diffuse band was obtained by polyacrylamide gel electrophoresis (PAGE) at pH 4.5. A single peak corresponding to an apparent molecular weight of 43 kDa was obtained by gel filtration on Sephadex G-75 (10 mM phosphate buffered saline (PBS), pH 7.4). On SDS-PAGE +/- 2-mercaptoethanol two bands of apparent molecular weights 11.8 and 14.7 kDa were detected. Jacalin behaved like a protein of apparent molecular weight of 13-14 kDa on Sephadex G-50 eluted with PBS containing 0.2% SDS. These data indicate that the jacalin molecule consists of 3-4 non-identical polypeptide subunits not connected by disulfide bridges. The amino acid composition of IgA affinity-purified jacalin (mol/405 mol amino acids) is Lys (24), His (5), Arg (4), Trp (6), Asx (36), Thr (35), Ser (48), Glx (31), Pro (18), Gly (53), Ala (13), Val (25), Met (3), Ile (23), Leu (25), Tyr (30), Phe (26), which corresponds to a molecular weight of 44.163 kDa.

  16. Necator americanus secretory acetylcholinesterase and its purification from excretory-secretory products by affinity chromatography.

    PubMed

    Pritchard, D I; Leggett, K V; Rogan, M T; McKean, P G; Brown, A

    1991-03-01

    Acetylcholinesterase (AChE) secretion by adult N. americanus was enhanced in vitro by incorporating insoluble collagen rafts into culture dishes. Enzyme produced in this way had preferential substrate specificity for acetylthiocholine iodide (ATC), and its activity was inhibited by eserine (1.1 x 10(-8) M). Ancylostoma ceylanicum, another hookworm species, failed to produce comparable amounts of AChE in culture. AChE was efficiently purified from culture medium by affinity chromatography on edrophonium sepharose; 81% of the AChE activity was retained by the affinity matrix, although this fraction contained only 4.3% of the protein loaded. Antisera raised against purified AChE in rabbits immunohistochemically stained the oesophageal glands of the parasite, and reacted with molecules of 32, 60, 80, 140 and 220 kDa in reduced adult ES products on Western blotting, although differential activity was observed against worm homogenates and earlier developmental stages. On IEF, purified AChE resolved predominantly with a pl of 3.55; proteins with a similar pl were recognized by rabbit anti-AChE. IgG preparations of this antiserum inhibited AChE activity in ES products, and inhibited AChE secretion by adult worms in culture. The availability of this immunological probe will allow definitive experiments to be conducted on the role of this enigmatic enzyme in the host-parasite relationship. PMID:2052405

  17. Submersible purification system for radioactive water

    DOEpatents

    Abbott, Michael L.; Lewis, Donald R.

    1989-01-01

    A portable, submersible water purification system for use in a pool of water containing radioactive contamination includes a prefilter for filtering particulates from the water. A resin bed is then provided for removal of remaining dissolved, particulate, organic, and colloidal impurities from the prefiltered water. A sterilizer then sterilizes the water. The prefilter and resin bed are suitably contained and are submerged in the pool. The sterilizer is water tight and located at the surface of the pool. The water is circulated from the pool through the prefilter, resin bed, and sterilizer by suitable pump or the like. In the preferred embodiment, the resin bed is contained within a tank which stands on the bottom of the pool and to which a base mounting the prefilter and pump is attached. An inlet for the pump is provided adjacent the bottom of the pool, while the sterilizer and outlet for the system is located adjacent the top of the pool.

  18. Transient conformational modification of immunoglobulin G during purification by protein A affinity chromatography.

    PubMed

    Gagnon, Pete; Nian, Rui; Leong, Denise; Hoi, Aina

    2015-05-22

    Exposure of three native IgG1 monoclonal antibodies to 100mM acetate, pH 3.5 had no significant effect on their hydrodynamic size (11.5±0.5nm), while elution from protein A with the same buffer created a conformation of 5.5±1.0nm. Formation of the reduced-size conformation was preceded by the known destabilization of the second constant domain of the heavy chain (Cγ2) by contact with protein A, then compounded by exposure to low pH, creating extended flexibility in the hinge-Cγ2 region and allowing the Fab region to fold over the Fc region. The reduced-size conformation was necessary for complete elution. It persisted unchanged for at least 7 days under elution conditions. Physiological conditions restored native size, and it was maintained on re-exposure to 100mM acetate, pH 3.5. Protein A-mediated destabilization and subsequent restoration of native size did not create aggregates, but the reduced-size conformation was more susceptible to aggregation by secondary stress than native antibody. Protein A-mediated formation of the reduced-size conformation is probably universal during purification of human IgG1 antibodies, and may occur with other subclasses and IgG from other species, as well as Fc-fusion proteins. PMID:25882588

  19. Purification of rat liver arylsulfatase A and its microheterogeneity assayed by crossed affinity-immunoelectrophoresis.

    PubMed

    Wójczyk, B; Hoja, D; Lityńska, A

    1992-01-01

    Arylsulfatase A (arylsulfatase sulfohydrolase) EC 3.1.6.1 was purified from rat liver by a procedure consisting of differential centrifugation, Con A-Sepharose and Blue Sepharose chromatography, PBE 94 chromatofocusing, DEAE-cellulose and gel filtration chromatography followed by preparative electrophoresis. A molecular mass of 132,000 was estimated by gradient PAGE. Particular proteins were detected by immunoelectrophoresis. Isoelectric focusing combined with immunoelectrophoresis gave two peaks of arylsulfatase A, with isoelectric points of pH 3.9 and 4.5. Microheterogeneity of rat liver arylsulfatase A was studied by affinity immunoelectrophoresis with 9 different lectins. The presence of concanavalin A-, Lens culinaris agglutinin-, Lotus tetragonolobus agglutinin- and wheat germ agglutinin-reactive forms permitted assessment of the types of carbohydrate moieties in arylsulfatase A.

  20. Affinity purification of proteins binding to kinase inhibitors immobilized on self-assembling monolayers.

    PubMed

    Bantscheff, Marcus; Hobson, Scott; Kuster, Bernhard

    2012-01-01

    Kinase inhibitors represent a relatively new class of drugs that offer novel therapies targeting specific -malfunctioning kinase-mediated signaling pathways in oncology and potentially inflammation. As the ATP binding sites of the ∼500 human kinases are structurally conserved and because most current drugs target the ATP binding site, there is a need to profile all the kinases that a drug may bind and/or inhibit. We have developed a chemical proteomics method that affinity purifies kinases from cell or tissue lysates using kinase inhibitors immobilized on self-assembling monolayers. The method can be applied to assess the selectivity of a given kinase inhibitor and thus to guide its preclinical or clinical development.

  1. Purification of rat liver arylsulfatase A and its microheterogeneity assayed by crossed affinity-immunoelectrophoresis.

    PubMed

    Wójczyk, B; Hoja, D; Lityńska, A

    1992-01-01

    Arylsulfatase A (arylsulfatase sulfohydrolase) EC 3.1.6.1 was purified from rat liver by a procedure consisting of differential centrifugation, Con A-Sepharose and Blue Sepharose chromatography, PBE 94 chromatofocusing, DEAE-cellulose and gel filtration chromatography followed by preparative electrophoresis. A molecular mass of 132,000 was estimated by gradient PAGE. Particular proteins were detected by immunoelectrophoresis. Isoelectric focusing combined with immunoelectrophoresis gave two peaks of arylsulfatase A, with isoelectric points of pH 3.9 and 4.5. Microheterogeneity of rat liver arylsulfatase A was studied by affinity immunoelectrophoresis with 9 different lectins. The presence of concanavalin A-, Lens culinaris agglutinin-, Lotus tetragonolobus agglutinin- and wheat germ agglutinin-reactive forms permitted assessment of the types of carbohydrate moieties in arylsulfatase A. PMID:1363453

  2. A large set of estrogen receptor β-interacting proteins identified by tandem affinity purification in hormone-responsive human breast cancer cell nuclei.

    PubMed

    Nassa, Giovanni; Tarallo, Roberta; Ambrosino, Concetta; Bamundo, Angela; Ferraro, Lorenzo; Paris, Ornella; Ravo, Maria; Guzzi, Pietro H; Cannataro, Mario; Baumann, Marc; Nyman, Tuula A; Nola, Ernesto; Weisz, Alessandro

    2011-01-01

    Estrogen receptors α (ER-α) and β (ER-β) play distinct biological roles in onset and progression of hormone-responsive breast cancer, with ER-β exerting a modulatory activity on ER-α-mediated estrogen signaling and stimulation of cell proliferation by mechanisms still not fully understood. We stably expressed human ER-β fused to a tandem affinity purification-tag in estrogen-responsive MCF-7 cells and applied tandem affinity purification and nanoLC-MS/MS to identify the ER-β interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co-purify with ER-β from nuclear extracts identify several new molecular partners of this receptor subtype that represents nodal points of a large protein network controlling multiple processes and functions in breast cancer cells. PMID:21182203

  3. From pathways to networks: connecting dots by establishing protein-protein interaction networks in signaling pathways using affinity purification and mass spectrometry

    PubMed Central

    Li, Xu; Wang, Wenqi; Chen, Junjie

    2015-01-01

    Signal transductions are the basis of biological activities in all living organisms. Studying the signaling pathways, especially under physiological conditions, has become one of the most important facets of modern biological research. During the last decade, mass spectrometry has been used extensively in biological research and is proven to be effective in addressing important biological questions. Here, we review the current progress in the understanding of signaling networks using mass spectrometry approaches. We will focus on studies of protein-protein interactions that use affinity purification followed by mass spectrometry approach. We discuss obstacles to affinity purification, data processing, functional validation, and identification of transient interactions and provide potential solutions for pathway-specific proteomics analysis, which we hope one day will lead to a comprehensive understanding of signaling networks in humans. PMID:25137225

  4. From pathways to networks: connecting dots by establishing protein-protein interaction networks in signaling pathways using affinity purification and mass spectrometry.

    PubMed

    Li, Xu; Wang, Wenqi; Chen, Junjie

    2015-01-01

    Signal transductions are the basis of biological activities in all living organisms. Studying the signaling pathways, especially under physiological conditions, has become one of the most important facets of modern biological research. During the last decade, MS has been used extensively in biological research and is proven to be effective in addressing important biological questions. Here, we review the current progress in the understanding of signaling networks using MS approaches. We will focus on studies of protein-protein interactions that use affinity purification followed by MS approach. We discuss obstacles to affinity purification, data processing, functional validation, and identification of transient interactions and provide potential solutions for pathway-specific proteomics analysis, which we hope one day will lead to a comprehensive understanding of signaling networks in humans. PMID:25137225

  5. Identification of BZR1-interacting Proteins as Potential Components of the Brassinosteroid Signaling Pathway in Arabidopsis Through Tandem Affinity Purification*

    PubMed Central

    Wang, Chunming; Shang, Jian-Xiu; Chen, Qi-Xiu; Oses-Prieto, Juan A.; Bai, Ming-Yi; Yang, Yihong; Yuan, Min; Zhang, Yu-Lan; Mu, Cong-Cong; Deng, Zhiping; Wei, Chuang-Qi; Burlingame, Alma L.; Wang, Zhi-Yong; Sun, Ying

    2013-01-01

    Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14–3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response. PMID:24019147

  6. PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

    PubMed Central

    Schildbach, Stefan; Blumert, Conny; Horn, Friedemann; von Bergen, Martin; Labudde, Dirk

    2016-01-01

    The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6. PMID:26966684

  7. Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

    PubMed Central

    Brehm, R D; Tranter, H S; Hambleton, P; Melling, J

    1990-01-01

    A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production. Images PMID:2339869

  8. Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography.

    PubMed

    Efremenko, E; Votchitseva, Y; Plieva, F; Galaev, I; Mattiasson, B

    2006-05-01

    Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.

  9. Identification of BZR1-interacting proteins as potential components of the brassinosteroid signaling pathway in Arabidopsis through tandem affinity purification.

    PubMed

    Wang, Chunming; Shang, Jian-Xiu; Chen, Qi-Xiu; Oses-Prieto, Juan A; Bai, Ming-Yi; Yang, Yihong; Yuan, Min; Zhang, Yu-Lan; Mu, Cong-Cong; Deng, Zhiping; Wei, Chuang-Qi; Burlingame, Alma L; Wang, Zhi-Yong; Sun, Ying

    2013-12-01

    Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response. PMID:24019147

  10. Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

    PubMed

    Hellstern, Simon; Mutzel, Rupert

    2016-06-01

    We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established. PMID:26892535

  11. Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography.

    PubMed

    Efremenko, E; Votchitseva, Y; Plieva, F; Galaev, I; Mattiasson, B

    2006-05-01

    Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration. PMID:16088350

  12. Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin.

    PubMed

    Narahari, Akkaladevi; Swamy, Musti J

    2010-04-21

    The chito-oligosaccharide-specific lectin from pumpkin (Cucurbita maxima) phloem exudate has been purified to homogeneity by affinity chromatography on chitin. After SDS/PAGE in the presence of 2-mercaptoethanol, the pumpkin phloem lectin yielded a single band corresponding to a molecular mass of 23.7 kDa, whereas ESI-MS (electrospray ionization MS) gave the molecular masses of the subunit as 24645 Da. Analysis of the CD spectrum of the protein indicated that the secondary structure of the lectin consists of 9.7% alpha-helix, 35.8% beta-sheet, 22.5% beta-turn and 32.3% unordered structure. Saccharide binding did not significantly affect the secondary and tertiary structures of the protein. The haemagglutinating activity of pumpkin phloem lectin was mostly unaffected in the temperature range 4-70 degrees C, but a sharp decrease was seen between 75 and 85 degrees C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 degrees C, indicating that it is a relatively stable protein.

  13. Data on the identification of protein interactors with the Evening Complex and PCH1 in Arabidopsis using tandem affinity purification and mass spectrometry (TAP-MS).

    PubMed

    Huang, He; Alvarez, Sophie; Nusinow, Dmitri A

    2016-09-01

    Tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis is a powerful biochemical approach to identify protein-protein associations. Here we describe two datasets generated by a series of TAP-MS analyses to co-purify proteins associated with either ELF3 or ELF4 of the Evening Complex (EC) ("Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry" (Huang et al., 2016a) [1]) or proteins associated with PCH1, which is a newly identified output of the circadian clock to regulate photoperiodic growth in Arabidopsis thaliana ("PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis" (Huang et al. 2016b) [2]). We used either ELF3, ELF4 or PCH1 fused to a C-terminal tandem affinity tag (6xHis-3xFLAG) as baits and conducted purifications in various genetic mutant backgrounds. These data are discussed in recent publications [1,2], and are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002606 (for EC) and PRIDE: PXD003352 (for PCH1).

  14. Data on the identification of protein interactors with the Evening Complex and PCH1 in Arabidopsis using tandem affinity purification and mass spectrometry (TAP-MS).

    PubMed

    Huang, He; Alvarez, Sophie; Nusinow, Dmitri A

    2016-09-01

    Tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis is a powerful biochemical approach to identify protein-protein associations. Here we describe two datasets generated by a series of TAP-MS analyses to co-purify proteins associated with either ELF3 or ELF4 of the Evening Complex (EC) ("Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry" (Huang et al., 2016a) [1]) or proteins associated with PCH1, which is a newly identified output of the circadian clock to regulate photoperiodic growth in Arabidopsis thaliana ("PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis" (Huang et al. 2016b) [2]). We used either ELF3, ELF4 or PCH1 fused to a C-terminal tandem affinity tag (6xHis-3xFLAG) as baits and conducted purifications in various genetic mutant backgrounds. These data are discussed in recent publications [1,2], and are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002606 (for EC) and PRIDE: PXD003352 (for PCH1). PMID:27274533

  15. Affinity column for purification of the human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor

    SciTech Connect

    Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

    1987-05-01

    The TXA/sub 2//PGH/sub 2/ receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific /sup 3/H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA/sub 2//PGH/sub 2/ receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor.

  16. Recombinant expression and affinity purification of snake venom gland parvalbumin in Escherichia coli.

    PubMed

    Jia, Ying; Pérez, John C

    2009-07-01

    Parvalbumins (PV) are small, acidic, water soluble and calcium-binding proteins generally present in muscular and nervous tissues. In the present study, we identified and characterized a cDNA clone encoding PV, named AplPV, from a snake (Agkistrodon piscivorus leucostoma) venom gland cDNA library. AplPV belongs to EF-hand proteins with six alpha-helices constituting three EF-hand domains. The deduced amino acid sequence of AplPV is 91% and 68% identical to the previously characterized PVs of Boa constrictor and Cyprinus carpio, respectively. The full-length cDNA was subcloned into the expression vector pGEX and transformed into Escherichia coli (E.coli) to produce recombinant protein. The bacterially expressed GST-AplPV fusion protein was highly expressed, and effectively purified by Glutathione-Sepharose affinity chromatography. A high concentration of thrombin protease specifically cleaved and removed the GST tag from fusion protein, and further purified by Benzamidine column for removal of thrombin protease. As a result, the 12 kDa AplPV recombinant protein alone was purified. To investigate the tissue-specific biological occurrence of AplPV, a polyclonal antibody (anti-AplPV-antibody) was raised against GST-AplPV fusion protein in rabbit. Western blot analysis revealed that immunoreactive bands were exhibited in both recombinant protein and samples of venom glands, but not in any crude venom. This specific occurrence indicates a specialized function of AplPV in snake venom glands.

  17. Identification of Novel Surface-Exposed Proteins of Rickettsia rickettsii by Affinity Purification and Proteomics

    PubMed Central

    Gong, Wenping; Xiong, Xiaolu; Qi, Yong; Jiao, Jun; Duan, Changsong; Wen, Bohai

    2014-01-01

    Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens. PMID:24950252

  18. Challenges and opportunities in the purification of recombinant tagged proteins.

    PubMed

    Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

    2014-01-01

    The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. PMID:24334194

  19. Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

    PubMed

    O'Shaughnessy, Colette M; Micoli, Francesca; Gavini, Massimiliano; Goodall, Margaret; Cobbold, Mark; Saul, Allan; Maclennan, Calman A

    2013-01-31

    Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella

  20. The Amicon Pro system--a centrifugal device capable of performing all steps in the protein purification workflow.

    PubMed

    Cappione, Amedeo; Mabuchi, Masaharu; Suhrawardy, Saosan; Briggs, David; Nadler, Timothy

    2013-01-01

    raditional protein purification is a long process with many steps utilizing multiple devices, often resulting in protein degradation and loss. The Amicon Pro device streamlines the affinity purification process by providing a single adaptable centrifugation unit capable of performing all steps in the affinity purification process. The device combines affinity-based spin column purification with downstream sample concentration and buffer exchange, eliminating the need for multiple sample transfers, thereby minimizing protein loss. The results presented in this work indicate that purification of His-tagged protein using the Amicon Pro device is faster, easier, and provides better yields than other traditional methods (eg. spin-column and slurry method). PMID:24364216

  1. 9. Water Purification System and Instrument Air Receiver Tank, view ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. Water Purification System and Instrument Air Receiver Tank, view to the south. The water purification system is visible in the right foreground of the photograph and the instrument air receiver tank is visible in the right background of the photograph. - Washington Water Power Clark Fork River Cabinet Gorge Hydroelectric Development, Powerhouse, North Bank of Clark Fork River at Cabinet Gorge, Cabinet, Bonner County, ID

  2. Dynamic output feedback H ∞ control for affine fuzzy systems

    NASA Astrophysics Data System (ADS)

    Wang, Huimin; Yang, Guang-Hong

    2013-06-01

    This article investigates the problem of designing H ∞ dynamic output feedback controllers for nonlinear systems, which are described by affine fuzzy models. The system outputs have been chosen as premise variables, which can guarantee that the plant and the controller always switch to the same region. By using a piecewise Lyapunov function and adding slack matrix variables, a piecewise-affine dynamic output feedback controller design method is obtained in the formulation of linear matrix inequalities (LMIs), which can be efficiently solved numerically. In contrast to the existing work, the proposed approach needs less LMI constraints and leads to less conservatism. Finally, numerical examples illustrate the effectiveness of the new result.

  3. Purification of human immunoglobulins A, G and M from Cohn fraction II/III by small peptide affinity chromatography.

    PubMed

    Liu, Zhuo; Gurgel, Patrick V; Carbonell, Ruben G

    2012-11-01

    This work describes attempts to purify human IgG, IgA and IgM from Cohn fraction II/III using HWRGWV affinity peptide resin. The effects of peptide density and different elution additives on recovery of the three antibodies were investigated. At low peptide density, salting-in salts such as magnesium chloride and calcium chloride facilitated antibody elution. Ethylene glycol, urea and arginine also facilitated elution because of their ability to decrease hydrophobic interactions, hydrogen bonding and electrostatic interactions. However, at high peptide density, no recovery improvements were observed because of increased non-specific hydrophobic interactions. The final elution conditions for each antibody were chosen based on the resulting yields and purities when a 10:2:1mg/mL mixture of human IgG, IgA and IgM was used as starting material. Different pretreatment methods were employed in order to improve the purity of antibodies from Cohn fraction II/III. After pretreatment with caprylic acid precipitation or combination of caprylic acid and polyethylene glycol precipitation, purities over 95% and yields of about 60% were obtained for hIgG, which are comparable to current chromatographic purification methods involving two chromatography steps when hIgG is isolated from plasma fractions. A hIgA-enriched fraction with 42% hIgA and 56% hIgG, as well as a hIgM enriched fraction with 46% hIgM, 28% hIgA and 24% hIgG, were obtained as the by-products. PMID:23026261

  4. Engineered protein A ligands, derived from a histidine-scanning library, facilitate the affinity purification of IgG under mild acidic conditions

    PubMed Central

    2014-01-01

    Background In antibody purification processes, the acidic buffer commonly used to elute the bound antibodies during conventional affinity chromatograph, can damage the antibody. Herein we describe the development of several types of affinity ligands which enable the purification of antibodies under much milder conditions. Results Staphylococcal protein A variants were engineered by using both structure-based design and combinatorial screening methods. The frequency of amino acid residue substitutions was statistically analyzed using the sequences isolated from a histidine-scanning library screening. The positions where the frequency of occurrence of a histidine residue was more than 70% were thought to be effective histidine-mutation sites. Consequently, we identified PAB variants with a D36H mutation whose binding of IgG was highly sensitive to pH change. Conclusion The affinity column elution chromatograms demonstrated that antibodies could be eluted at a higher pH (∆pH**≧2.0) than ever reported (∆pH = 1.4) when the Staphylococcal protein A variants developed in this study were used as affinity ligands. The interactions between Staphylococcal protein A and IgG-Fab were shown to be important for the behavior of IgG bound on a SpA affinity column, and alterations in the affinity of the ligands for IgG-Fab clearly affected the conditions for eluting the bound IgG. Thus, a histidine-scanning library combined with a structure-based design was shown to be effective in engineering novel pH-sensitive proteins. PMID:25057290

  5. 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. PMID:26616099

  6. Proteome-wide identification of novel binding partners to the oncogenic fusion gene protein, NPM-ALK, using tandem affinity purification and mass spectrometry.

    PubMed

    Wu, Fang; Wang, Peng; Young, Leah C; Lai, Raymond; Li, Liang

    2009-02-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion gene protein that is characteristically found in a subset of anaplastic large cell lymphomas, promotes tumorigenesis through its functional and physical interactions with various biologically important proteins. The identification of these interacting proteins has proven to be useful to further our understanding of NPM-ALK-mediated tumorigenesis. For the first time, we performed a proteome-wide identification of NPM-ALK-binding proteins using tandem affinity purification and a highly sensitive mass spectrometric technique. Tandem affinity purification is a recently developed method that carries a lower background and higher sensitivity compared with the conventional immunoprecipitation-based protein purification protocols. The NPM-ALK gene was cloned into an HB-tagged vector and expressed in GP293 cells. Three independent experiments were performed and the reproducibility of the data was 68%. The vast majority of the previously reported NPM-ALK-binding proteins were detected. We also identified proteins that are involved in various cellular processes that were not previously described in association with NPM-ALK, such as MCM6 and MSH2 (DNA repair), Nup98 and importin 8 (subcellular protein transport), Stim1 (calcium signaling), 82Fip (RNA regulation), and BAG2 (proteosome degradation). We believe that these data highlight the functional diversity of NPM-ALK and provide new research directions for the study of the biology of this oncoprotein.

  7. 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.

  8. Human IgA-binding peptides selected from random peptide libraries: affinity maturation and application in IgA purification.

    PubMed

    Hatanaka, Takaaki; Ohzono, Shinji; Park, Mirae; Sakamoto, Kotaro; Tsukamoto, Shogo; Sugita, Ryohei; Ishitobi, Hiroyuki; Mori, Toshiyuki; Ito, Osamu; Sorajo, Koichi; Sugimura, Kazuhisa; Ham, Sihyun; Ito, Yuji

    2012-12-14

    Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1-A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (K(d) = 1.3 μm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (K(d) = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.

  9. Affinity purification of antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

  10. Integrative refolding and purification of histidine-tagged protein by like-charge facilitated refolding and metal-chelate affinity adsorption.

    PubMed

    Liu, Hu; Du, Wen-Jie; Dong, Xiao-Yan; Sun, Yan

    2014-05-30

    This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification.

  11. Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.

    PubMed

    Arimitsu, Hideyuki; Sasaki, Keiko; Kojima, Hiroe; Yanaka, Tadashi; Tsuji, Takao

    2013-01-01

    Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease. PMID:24340102

  12. Simple Method for Shiga Toxin 2e Purification by Affinity Chromatography via Binding to the Divinyl Sulfone Group

    PubMed Central

    Arimitsu, Hideyuki; Sasaki, Keiko; Kojima, Hiroe; Yanaka, Tadashi; Tsuji, Takao

    2013-01-01

    Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease. PMID:24340102

  13. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    PubMed

    Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

  14. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis

    PubMed Central

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  15. The purification of human enterokinase by affinity chromatography and immunoadsorption. Some observations on its molecular characteristics and comparisons with the pig enzyme.

    PubMed Central

    Grant, D A; Hermon-Taylor, J

    1976-01-01

    A method is described for the purification of human enterokinase from accumulated duodenal fluid by affinity chromatography using p-aminobenzamidine as the ligand. Resolution was greatest when glycylglycine was substituted as the spacer arm. Purification was not a one-step procedure, and some contamination, principally by the alpha-glucosidases, remained. Their removal was completed by immunoadsorption using antisera raised to enterokinase-free material containing these enzymes, prepared as a by-product of the purification procedure. The final preparation had an activity of 4260 nmol of trypsin/min per mg and was free of other enzymic activity tested. Amino acid and sugar analyses of the highly purified enzyme indicated an acidic glycoprotein containing 57% sugar (neutral sugars 47%, amino sugars 10%). The apparent mol.wts. and Stokes radii of human and pig enterokinase were 296 000 and 316 000, and 5.65 and 5.78 nm respectively. Two isoenzymes were identified for human enterokinase and three for the pig enzyme. Human enterokinase demonstrated a resistance to reduction of disulphide linkages and to sodium dodecyl sulphate binding, which may be related to the need for it to retain its integrity in the digestive environment of the upper small intestine. Antisera to highly purified pig and human enterokinases specifically inhibited enterokinase activity. Immuno-inhibition of intestinal aminopeptidase, maltase and glucoamylase by homologous antisera was not observed. Images PLATE 1 PMID:945736

  16. Preliminary Hazards Assessment: Iron disulfide purification system

    SciTech Connect

    1991-07-30

    A process for the purification (washing) of iron disulfide (FeS{sub 2}) powder is conducted in the Northeast corner (Area 353) of the main plant building (Building 100). This location is about 130 feet from the fenced boundary of the Partnership School/Child Development Center. In the first steps of the process, raw iron disulfide powder is ground and separated by particle size. The ground and sized powder is then purified in a three-step acid washing process using both hydrochloric acid (HCI) and hydrofluoric (HF) acid. The iron disulfide process is an intermittent batch process conducted four to eight times a year. This study is a Preliminary Hazards Assessment (PHA) to assess the hazards associated with the iron disulfide process. This is a preliminary study and will be used to determine if additional safety analysis is necessary. The scope of the PHA includes assessment of the process steps of grinding, size classification, and purification. The purpose is to identify major hazards and determine if the current and newly added safeguards are adequate for operation. The PHA also lists recommendations for additional safety features that should be added to reduce the risks of operation.

  17. Immobilized iminodiacetic acid (IDA)-type Cu2+ -chelating membrane affinity chromatography for purification of bovine liver catalase.

    PubMed

    Yang, L; Jia, L; Zou, H; Zhang, Y

    1999-05-01

    A metal ion chelating membrane medium based on iminodiacetate-substituted modified short cotton cellulose was examined for the purification of bovine liver catalase (BLC). The effect of buffer pH, chelator surface density, initial concentration of crude enzyme and flow rate on BLC binding efficiency to the copper ion chelating membrane adsorbent were examined. Under the chromatographic conditions chosen, 67.7% recovery of BLC was attained with an overall 4.2-fold increase in specific activity in a single step. After performance of BLC purification, the chelating membrane adsorbent can be easily regenerated by imidazole or EDTA buffer with higher reviving effectiveness with the latter. PMID:10375124

  18. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  19. FYWHCLDE-based affinity chromatography of IgG: effect of ligand density and purifications of human IgG and monoclonal antibody.

    PubMed

    Zhao, Wei-Wei; Shi, Qing-Hong; Sun, Yan

    2014-08-15

    This work reports the development of an octapeptide-based affinity adsorbent for the purification of human IgG (hIgG) and monoclonal antibody (mAb). The octapeptide was FYWHCLDE selected earlier by the biomimetic design of affinity peptide ligands for hIgG. The ligand was coupled to Sepharose gel at four densities from 10.4 to 31.0μmol/mL, and the effect of peptide density on the adsorption of hIgG and bovine serum albumin (BSA) was first investigated. The binding capacity of hIgG increased from 104.2 to 176.4mg/mL within the ligand density range, and the binding affinity (dissociation constant) kept at 2.4-3.7μM. Batch adsorption revealed that the selectivity of FYWHCLDE-Sepharose for IgG was 30-40 times over BSA. The effective pore diffusivity of IgG decreased somewhat with increasing ligand density, but the dynamic binding capacity at 10% breakthrough, measured by using 10-fold diluted human serum as feedstock, doubled with increasing ligand density from 10.4 to 31.0μmol/mL due to the remarkable increase of static binding capacity. By using the affinity column with a ligand density of 23.9μmol/mL, hIgG and humanized mAb purifications from human serum and cell culture supernatant, respectively, were achieved at high purities and recovery yields. Finally, the robustness of the peptide gel was demonstrated by recycled use of the affinity column in 20 breakthrough cycles. PMID:24947889

  20. Optimiziing the laboratory monitoring of biological wastewater-purification systems

    SciTech Connect

    S.V. Gerasimov

    2009-05-15

    Optimization of the laboratory monitoring of biochemical wastewater-treatment systems at coke plants is considered, for the example of OAO Koks. By adopting a methodological approach to determine the necessary data from chemical analysis, it is possible to reduce the time, labor, and materials required for monitoring, without impairing the purification process or compromising the plant's environmental policies.

  1. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Water purification system for hemodialysis. 876.5665 Section 876.5665 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665...

  2. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Water purification system for hemodialysis. 876.5665 Section 876.5665 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665...

  3. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Water purification system for hemodialysis. 876.5665 Section 876.5665 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665...

  4. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Water purification system for hemodialysis. 876.5665 Section 876.5665 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665...

  5. Systems, compositions, and methods for fluid purification

    DOEpatents

    Ho, W.S. Winston; Verweij, Hendrik; Shqau, Krenar; Ramasubranian, Kartik

    2015-12-22

    Disclosed herein are membranes comprising a substrate, a support layer, and a selective layer. In some embodiments the membrane may further comprise a permeable layer. Methods of forming membranes are also disclosed comprising forming a support layer on a substrate, removing adsorbed species from the support layer, preparing a solution containing inorganic materials of a selective layer, contacting the support layer with the solution, drying the membrane, and exposing the membrane to rapid thermal processing. Also disclosed are methods of fluid purification comprising providing a membrane having a feed side and a permeable side, passing a fluid mixture across the feed side of the membrane, providing a driving force for transmembrane permeation, removing from the permeate side a permeate stream enriched in a purified fluid, and withdrawing from the feed side a fluid that is depleted in a purified fluid.

  6. A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme.

    PubMed

    Kaya, Mustafa Oguzhan; Arslan, Oktay; Guler, Ozen Ozensoy

    2015-01-01

    In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. L-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-L-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-L-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants K(M) and V(Max) for BTH were determined by using hyaluronic acid as a substrate. K(M) and V(Max) values obtained from the Lineweaver-Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, β-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH. PMID:25373501

  7. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high...

  8. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high...

  9. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high...

  10. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high...

  11. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high...

  12. Applications of novel affinity cassette methods: use of peptide fusion handles for the purification of recombinant proteins.

    PubMed

    Hearn, M T; Acosta, D

    2001-01-01

    In this article, recent progress related to the use of different types of polypeptide fusion handles or 'tags' for the purification of recombinant proteins are critically discussed. In addition, novel aspects of the molecular cassette concept are elaborated, together with areas of potential application of these fundamental principles in molecular recognition. As evident from this review, the use of these concepts provides a powerful strategy for the high throughput isolation and purification of recombinant proteins and their derived domains, generated from functional genomic or zeomic studies, as part of the bioprocess technology leading to their commercial development, and in the study of molecular recognition phenomena per se. In addition, similar concepts can be exploited for high sensitivity analysis and detection, for the characterisation of protein bait/prey interactions at the molecular level, and for the immobilisation and directed orientation of proteins for use as biocatalysts/biosensors.

  13. Identification of proteins associated with ligand-activated estrogen receptor α in human breast cancer cell nuclei by tandem affinity purification and nano LC-MS/MS.

    PubMed

    Tarallo, Roberta; Bamundo, Angela; Nassa, Giovanni; Nola, Ernesto; Paris, Ornella; Ambrosino, Concetta; Facchiano, Angelo; Baumann, Marc; Nyman, Tuula A; Weisz, Alessandro

    2011-01-01

    Estrogen receptor α (ER-α) is a key mediator of estrogen actions in breast cancer (BC) cells. Understanding the effects of ligand-activated ER-α in target cells requires identification of the molecular partners acting in concert with this nuclear receptor to transduce the hormonal signal. We applied tandem affinity purification (TAP), glycerol gradient centrifugation and MS analysis to isolate and identify proteins interacting with ligand-activated ER-α in MCF-7 cell nuclei. This led to the identification of 264 ER-associated proteins, whose functions highlight the hinge role of ER-α in the coordination of multiple hormone-regulated nuclear processes in BC cells. PMID:21182205

  14. Influence of affinity on antibody determination in microtiter ELISA systems

    SciTech Connect

    Peterman, J.H.; Voss, E.W. Jr.; Butler, J.E.

    1986-03-01

    Theoretically, all immunoassays are affinity (Ka) dependent when the product of the antibody (Ab) Ka and the free epitope concentration is less than 10. Thus, the degree of dependence on Ka depends on the concentration of available antigen in the system. The authors examined the binding of /sup 125/I-anti-fluorescein (a-FLU) monoclonal antibodies of different affinities to FLU-gelatin adsorbed on Immunlon 2 microtiter plates. Data obtained were in general agreement with our theoretical predictions; the percent of /sup 125/I-a-FLU which bound correlated with Ka, as did the shape of the titration curves. Measurement of 5 a-FLU monoclonals by the ELISA showed that the determination of Ab concentrations depends on the FLU-gelatin concentration, epitope density, and on the relationship between the Kas of test samples and the reference standard Ab preparation. Thus the ELISA is Ka dependent and should not be used routinely to estimate the absolute amount to Ab in unknown samples. However, the Ka dependency of the ELISA might provide a convenient assay for the estimation of the relative functional Ka (rfKa) of antibody preparations.

  15. 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…

  16. A scintillator purification plant and fluid handling system for SNO+

    NASA Astrophysics Data System (ADS)

    Ford, Richard J.

    2015-08-01

    A large capacity purification plant and fluid handling system has been constructed for the SNO+ neutrino and double-beta decay experiment, located 6800 feet underground at SNOLAB, Canada. SNO+ is a refurbishment of the SNO detector to fill the acrylic vessel with liquid scintillator based on Linear Alkylbenzene (LAB) and 2 g/L PPO, and also has a phase to load natural tellurium into the scintillator for a double-beta decay experiment with 130Te. The plant includes processes multi-stage dual-stream distillation, column water extraction, steam stripping, and functionalized silica gel adsorption columns. The plant also includes systems for preparing the scintillator with PPO and metal-loading the scintillator for double-beta decay exposure. We review the basis of design, the purification principles, specifications for the plant, and the construction and installations. The construction and commissioning status is updated.

  17. A scintillator purification plant and fluid handling system for SNO+

    SciTech Connect

    Ford, Richard J.

    2015-08-17

    A large capacity purification plant and fluid handling system has been constructed for the SNO+ neutrino and double-beta decay experiment, located 6800 feet underground at SNOLAB, Canada. SNO+ is a refurbishment of the SNO detector to fill the acrylic vessel with liquid scintillator based on Linear Alkylbenzene (LAB) and 2 g/L PPO, and also has a phase to load natural tellurium into the scintillator for a double-beta decay experiment with {sup 130}Te. The plant includes processes multi-stage dual-stream distillation, column water extraction, steam stripping, and functionalized silica gel adsorption columns. The plant also includes systems for preparing the scintillator with PPO and metal-loading the scintillator for double-beta decay exposure. We review the basis of design, the purification principles, specifications for the plant, and the construction and installations. The construction and commissioning status is updated.

  18. Exhaust gas purification system for lean burn engine

    DOEpatents

    Haines, Leland Milburn

    2002-02-19

    An exhaust gas purification system for a lean burn engine includes a thermal mass unit and a NO.sub.x conversion catalyst unit downstream of the thermal mass unit. The NO.sub.x conversion catalyst unit includes at least one catalyst section. Each catalyst section includes a catalytic layer for converting NO.sub.x coupled to a heat exchanger. The heat exchanger portion of the catalyst section acts to maintain the catalytic layer substantially at a desired temperature and cools the exhaust gas flowing from the catalytic layer into the next catalytic section in the series. In a further aspect of the invention, the exhaust gas purification system includes a dual length exhaust pipe upstream of the NO.sub.x conversion catalyst unit. The dual length exhaust pipe includes a second heat exchanger which functions to maintain the temperature of the exhaust gas flowing into the thermal mass downstream near a desired average temperature.

  19. ( sup 3 H)phenamil binding protein of the renal epithelium Na+ channel. Purification, affinity labeling, and functional reconstitution

    SciTech Connect

    Barbry, P.; Chassande, O.; Marsault, R.; Lazdunski, M.; Frelin, C. )

    1990-01-30

    This paper describes a large-scale purification procedure of the amiloride binding component of the epithelium Na+ channel. (3H)Phenamil was used as a labeled ligand to follow the purification. The first two steps are identical with those previously described. A third step was a hydroxyapatite column. The purified material consisted of a homodimer of two 88-kDa proteins that migrated anomalously in SDS-PAGE to give an apparent Mr of 105,000. Deglycosylation by treatment with neuraminidase and endoglycosidase F or with neuraminidase and glycopeptidase F indicated that less than 5% of the mass of the native receptor was carbohydrate. Sedimentation analysis of the purified Na+ channel in H2O and D2O sucrose gradients and gel filtration experiments led to an estimated molecular weight of the (3H)phenamil receptor protein-detergent-phospholipid complex of 288,000 and of the native (3H)phenamil receptor protein of 158,000. (3H)Br-benzamil is another labeled derivative of amiloride that recognized binding sites that had the same pharmacological properties as (3H)phenamil binding sites and that copurified with them. Upon irradiation of kidney membranes, (3H)Br-benzamil incorporated specifically into a 185-kDa polypeptide chain under nonreducing electrophoretic conditions and a 105-kDa protein under reducing conditions. The same labeling pattern was observed at the different steps of the purification. Reconstitution of the purified phenamil receptor into large unilamellar vesicles was carried out. A low but significant phenamil- and amiloride-sensitive electrogenic Na+ transport was observed.

  20. Anacardium occidentale bark lectin: purification, immobilization as an affinity model and influence in the uptake of technetium-99M by rat adipocytes.

    PubMed

    Maciel, Maria Inês Sucupira; de Mendonça Cavalcanti, Maria do Socorro; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes; de Almeida Catanho, Maria Teresa Jansem; Coelho, Luana Cassandra Breitenbach Barroso

    2012-10-01

    Lectins, proteins that recognize carbohydrates, have been immobilized on inert supports and used in the screening or purification of glycoproteins. Anacardium occidentale bark infusion has been used as a hypoglycemic agent in Brazil. The toxicity of natural products may be evaluated determining their capability to alter the biodistribution of technetium-99M ((99m)Tc). This work reports the isolation and characterization of a lectin from A. occidentale bark (AnocBL), its evaluation as an affinity support for glycoprotein isolation and lectin effect on the uptake of (99m)Tc by rat adipocytes. AnocBL was isolated from 80 % ammonium sulphate supernatant by affinity chromatography on fetuin-agarose. SDS-PAGE showed a single protein band of 47 kDa. The monossacharide L-arabinose and the glycoproteins fetuin, asialofetuin, ovomucoid, casein, thyroglobulin, peroxidase, fetal bovine serum and IgG inhibited the activity. The lectin activity was stable until 70 °C and at a pH range of 3.0-7.5. AnocBL-Sepharose column bound fetuin indicating that the lectin matrix may be used to obtain glycoconjugates of biotechnological interest. In vitro assay revealed that glucose and insulin increase (99m)Tc uptake by rat adipocytes. AnocBL decreases (99m)Tc uptake, and this effect was not detected in the presence of glucose. Fetuin inhibited AnocBL effect in all insulin concentrations.

  1. An optimized system for expression and purification of secreted bacterial proteins.

    PubMed

    Geisbrecht, Brian V; Bouyain, Samuel; Pop, Mihai

    2006-03-01

    In this report, we describe an optimized system for the efficient overexpression, purification, and refolding of secreted bacterial proteins. Candidate secreted proteins were produced recombinantly in Escherichia coli as Tobacco Etch Virus protease-cleavable hexahistidine-c-myc eptiope fusion proteins. Without regard to their initial solubility, recombinant fusion proteins were extracted from whole cells with guanidium chloride, purified under denaturing conditions by immobilized metal affinity chromatography, and refolded by rapid dilution into a solution containing only Tris buffer and sodium chloride. Following concentration on the same resin under native conditions, each protein was eluted for further purification and/or characterization. Preliminary studies on a test set of 12 secreted proteins ranging in size from 13 to 130 kDa yielded between 10 and 50 mg of fusion protein per liter of induced culture at greater than 90% purity, as judged by Coomassie-stained SDS-PAGE. Of the nine proteins further purified, analytical gel filtration chromatography indicated that each was a monomer in solution and circular dichroism spectroscopy revealed that each had adopted a well-defined secondary structure. While there are many potential applications for this system, the results presented here suggest that it will be particularly useful for investigators employing structural approaches to understand protein function, as attested to by the crystal structures of three proteins purified using this methodology (B.V. Geisbrecht, B.Y. Hamaoka, B. Perman, A. Zemla, D.J. Leahy, J. Biol. Chem. 280 (2005) 17243-17250). PMID:16260150

  2. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    SciTech Connect

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F. )

    1990-11-15

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of {sup 125}I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-{sup 125}I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.

  3. Robust adaptive control for a class of uncertain non-affine nonlinear systems using affine-type neural networks

    NASA Astrophysics Data System (ADS)

    Zhao, Shitie; Gao, Xianwen

    2016-08-01

    A robust adaptive control is proposed for a class of single-input single-output non-affine nonlinear systems. In order to approximate the unknown nonlinear function, a novel affine-type neural network is used, and then to compensate the approximation error and external disturbance a robust control term is employed. By Lyapunov stability analysis for the closed-loop system, it is proved that tracking errors asymptotically converge to zero. Moreover, an observer is designed to estimate the system states because all the states may not be available for measurements. Furthermore, the adaptation laws of neural networks and the robust controller are given out based on the Lyapunov stability theory. Finally, two simulation examples are presented to demonstrate the effectiveness of the proposed control method.

  4. Resonant purification of mixed states for closed and open quantum systems

    SciTech Connect

    Romano, Raffaele

    2007-02-15

    Pure states are fundamental for the implementation of quantum technologies, and several methods for the purification of the state of a quantum system S have been developed in the past years. In this work we describe a mechanism leading to purification of mixed states, based on the interaction of S with an auxiliary system P. Considering two-level systems and assuming a particular interaction between them, we study how the dynamical parameters of the system P affect the purification of S. By using analytical and numerical tools, we show that the purification process exhibits a resonant behavior in both the cases of system isolated from the external environment or not.

  5. An affinity-directed protein missile system for targeted proteolysis

    PubMed Central

    Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

    2016-01-01

    The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

  6. Method of gas purification and system therefor

    DOEpatents

    Szwarc, Raphael

    1985-04-23

    A method and device for conducting gettering. The gettering is conducted with one of an LiB, LiSi or LiAl system. Preferably the LiB system is of the formula Li.sub.x B.sub.1-x wherein 0

  7. Method of gas purification and system therefor

    DOEpatents

    Szwarc, R.

    1983-12-29

    A method and device are disclosed for conducting gettering. The gettering is conducted with one of an LiB, LiSi or LiAl system. Preferably the LiB system is of the formula Li/sub x/B/sub 1-x/ wherein 0 < x < 1 with gettering conducted at room or slightly elevated temperature of about 100 to 200/sup 0/C.

  8. Affinity purification and characterisation of zinc chelating peptides from rapeseed protein hydrolysates: possible contribution of characteristic amino acid residues.

    PubMed

    Xie, Ningning; Huang, Jingjing; Li, Bo; Cheng, Jianghua; Wang, Zhuochen; Yin, Junfeng; Yan, Xiaoming

    2015-04-15

    Zinc is an essential trace element for human growth and development. In this work, zinc-chelating peptides from rapeseed protein hydrolysates produced with alcalase were investigated by affinity chromatography with immobilized zinc and Sephadex G-25 gel filtration. Four small peptides, namely, Ala-Arg, Asn-Ser-Met (NSM), Gly-Lys-Arg, and Glu-Pro-Ser-His, were obtained and identified by reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The zinc-chelating ability of the four peptides was further validated by inductively coupled plasma atomic emission spectrometry (ICP-AES). NSM was found to exhibit the highest zinc-chelating rate, which was better than that of reduced glutathione. We speculated that the Asn residue at the amino-terminus might facilitate this zinc-chelating ability. Therefore, utilizing small peptides from rapeseed protein as novel carriers for zinc supplement was feasible.

  9. cDNA cloning, bacterial expression, in vitro renaturation and affinity purification of the zinc endopeptidase astacin.

    PubMed Central

    Reyda, S; Jacob, E; Zwilling, R; Stöcker, W

    1999-01-01

    Astacin (EC 3.4.24.21) from the freshwater crayfish (Astacus astacus) is a prototype for the metzincin superfamily and for the astacin family of zinc peptidases, enzymes which are involved in hatching processes, embryonic patterning and tissue remodelling. Here we report on the cloning and overexpression in Escherichia coli of an astacin cDNA which was reverse-transcribed from crayfish midgut-gland mRNA. A cDNA construct based on this clone was generated which comprised the nucleotide sequence encoding mature astacin devoid of the signal and propeptide. This construct was cloned into the pET3a vector and used to transform E. coli BL21(DE3) cells. Recombinant astacin was purified from inclusion bodies and dissolved under reducing conditions. For folding, the protein was diluted into neutral buffer containing l-arginine, GSH and EDTA. Eventually, Zn(2+) was added by dialysis and the fraction of active enzyme was affinity-purified on immobilized Pro-Leu-Gly hydroxamate. As shown by superimposition of the corresponding three-dimensional structures, this inhibitor binds to a region of the active-site cleft that is conserved in most metzincins. Therefore this principle behind this affinity technique, originally introduced for fibroblast collagenase by Moore and Spilburg [Biochemistry (1986) 25, 5189-5195], is applicable throughout the metzincin superfamily of metalloproteases, despite their otherwise differing cleavage specificities. Recombinant astacin is active on gelatine zymograms and in a quenched fluorescence assay, yielding kinetic parameters comparable with those of wild-type astacin purified from crayfish stomach. PMID:10585873

  10. Mathematical model accurately predicts protein release from an affinity-based delivery system.

    PubMed

    Vulic, Katarina; Pakulska, Malgosia M; Sonthalia, Rohit; Ramachandran, Arun; Shoichet, Molly S

    2015-01-10

    Affinity-based controlled release modulates the delivery of protein or small molecule therapeutics through transient dissociation/association. To understand which parameters can be used to tune release, we used a mathematical model based on simple binding kinetics. A comprehensive asymptotic analysis revealed three characteristic regimes for therapeutic release from affinity-based systems. These regimes can be controlled by diffusion or unbinding kinetics, and can exhibit release over either a single stage or two stages. This analysis fundamentally changes the way we think of controlling release from affinity-based systems and thereby explains some of the discrepancies in the literature on which parameters influence affinity-based release. The rate of protein release from affinity-based systems is determined by the balance of diffusion of the therapeutic agent through the hydrogel and the dissociation kinetics of the affinity pair. Equations for tuning protein release rate by altering the strength (KD) of the affinity interaction, the concentration of binding ligand in the system, the rate of dissociation (koff) of the complex, and the hydrogel size and geometry, are provided. We validated our model by collapsing the model simulations and the experimental data from a recently described affinity release system, to a single master curve. Importantly, this mathematical analysis can be applied to any single species affinity-based system to determine the parameters required for a desired release profile. PMID:25449806

  11. 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.

  12. New research on bioregenerative air/water purification systems

    NASA Technical Reports Server (NTRS)

    Johnson, Anne H.; Ellender, R. D.; Watkins, Paul J.

    1991-01-01

    For the past several years, air and water purification systems have been developed and used. This technology is based on the combined activities of plants and microorganisms as they function in a natural environment. More recently, researchers have begun to address the problems associated with indoor air pollution. Various common houseplants are currently being evaluated for their abilities to reduce concentrations of volatile organic compounds (VOCS) such as formaldehyde and benzene. With development of the Space Exploration Initiative, missions will increase in duration, and problems with resupply necessitates implementation of regenerative technology. Aspects of bioregenerative technology have been included in a habitat known as the BioHome. The ultimate goal is to use this technology in conjunction with physicochemical systems for air and water purification within closed systems. This study continued the risk assessment of bioregenerative technology with emphasis on biological hazards. In an effort to evaluate the risk for human infection, analyses were directed at enumeration of fecal streptococci and enteric viruses with the BioHome waste water treatment system.

  13. Air Purification in Closed Environments: An Overview of Spacecraft Systems

    NASA Technical Reports Server (NTRS)

    Perry, Jay L.; LeVan, Douglas; Crumbley, Robert (Technical Monitor)

    2002-01-01

    The primary goal for a collective protection system and a spacecraft environmental control and life support system (ECLSS) are strikingly similar. Essentially both function to provide the occupants of a building or vehicle with a safe, habitable environment. The collective protection system shields military and civilian personnel from short-term exposure to external threats presented by toxic agents and industrial chemicals while an ECLSS sustains astronauts for extended periods within the hostile environment of space. Both have air quality control similarities with various aircraft and 'tight' buildings. This paper reviews basic similarities between air purification system requirements for collective protection and an ECLSS that define surprisingly common technological challenges and solutions. Systems developed for air revitalization on board spacecraft are discussed along with some history on their early development as well as a view of future needs. Emphasis is placed upon two systems implemented by the National Aeronautics and Space Administration (NASA) onboard the International Space Station (ISS): the trace contaminant control system (TCCS) and the molecular sieve-based carbon dioxide removal assembly (CDRA). Over its history, the NASA has developed and implemented many life support systems for astronauts. As the duration, complexity, and crew size of manned missions increased from minutes or hours for a single astronaut during Project Mercury to days and ultimately months for crews of 3 or more during the Apollo, Skylab, Shuttle, and ISS programs, these systems have become more sophisticated. Systems aboard spacecraft such as the ISS have been designed to provide long-term environmental control and life support. Challenges facing the NASA's efforts include minimizing mass, volume, and power for such systems, while maximizing their safety, reliability, and performance. This paper will highlight similarities and differences among air purification systems

  14. Functional characterization of the kinase activation loop in nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) using tandem affinity purification and liquid chromatography-mass spectrometry.

    PubMed

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of >or=1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.

  15. Studies on ram acrosin. Activation of proacrosin accompanying the isolation of acrosin from spermatozoa, and purification of the enzyme by affinity chromatography.

    PubMed Central

    Brown, C R; Hartree, E F

    1978-01-01

    1. A previously described, freeze-dried, partially purified ram acrosin preparation was fractionated on a column of Sepharose linked to the acrosin inhibitor p-(p'-aminophenoxypropoxy)benzamidine. Two acrosin fractions were obtained. 2. beta-Acrosin was homogeneous, quite stable at low pH and very stable when freeze-dried. Its molecular weight is about 38000, and it contains about six sugar residues per molecule, but no sialic acid. psi-Acrosin consisted of at least three unstable forms of acrosin. 3. When the entire purification process, starting from collection of semen, was carried out as rapidly as possible, the yield of beta-acrosin was increased and very little psi-acrosin was obtained. 4. In fresh ram semen the acrosin is present as the intra-acrosomal zymogen, proacrosin. After its extraction from spermatozoa autoproteolytic reactions convert proacrosin into beta-acrosin; psi-acrosin appears to be breakdown products of beta-acrosin. 5. When beta-acrosin was passed through a column of Sepharose linked to the non-inhibitory deamidinated analogue of the inhibitor it behaved as a hydrophobic protein. This is consistent with our view that acrosin (as zymogen) occurs in spermatozoa as a membrane-bound protein. 6. Success in the isolation of pure acrosin in high yield calls for an affinity adsorbent with the appropriate subsidiary hydrophobic properties. PMID:736895

  16. Raw data for the identification of SUMOylated proteins in S. cerevisiae subjected to two types of osmotic shock, using affinity purification coupled with mass spectrometry

    PubMed Central

    Srikumar, Tharan; Lewicki, Megan C.; Raught, Brian

    2014-01-01

    The small ubiquitin-related modifier (SUMO) “stress response” (SSR) is a poorly understood evolutionarily conserved phenomenon in which steady-state SUMO conjugate levels are dramatically increased in response to environmental stresses. Here we describe the data acquired using affinity-purification coupled with mass spectrometry to identify proteins that are SUMOylated in response to two different types of osmotic stress, 1 M sorbitol and 1 M KCl. The mass spectrometry dataset described here has been uploaded to the MassIVE repository with ID: MSV000078739, and consists of 32 raw MS files acquired in data-dependent mode on a Thermo Q-Exactive instrument. iProphet-processed MS/MS search results and associated SAINT scores are also included as a reference. These data are discussed and interpreted in “The S. cerevisiae SUMO stress response is a conjugation–deconjugation cycle that targets the transcription machinery”, by Lewicki et al. in the Journal of Proteomics, 2014 [1]. PMID:26217701

  17. Atom transfer radical polymerization to fabricate monodisperse poly[glycidyl methacrylate-co-poly (ethylene glycol) methacrylate] microspheres and its application for protein affinity purification.

    PubMed

    Yu, Ling; Shi, Zhuan Zhuan; Li, Chang Ming

    2015-09-01

    Poly[glycidyl methacrylate-co-poly (ethylene glycol) methacrylate] microspheres for the first time were successfully synthesized by atom transfer radical polymerization (ATRP) method at room temperature. The co-polymerization approach was investigated to delicately control the microsphere morphology and size-distribution by reaction conditions including solvent percentage, monomer loading and rotation speed. The results show that the average size of the microspheres is ∼5.7 μm with coexistence of epoxy, hydroxyl and ether groups, which provide plentiful functional sites for protein anchoring. The mechanism of the microsphere formation is proposed. The microsphere successfully demonstrates its unique application for affinity purification of proteins, in which the functional epoxy group facilitates a simple and efficient protein covalent immobilization to purify immunoglobulin G on the microspheres, while the hydrophilic poly (ethylene glycol) motif can repulse nonspecific protein adsorption for good specificity. This microspheres can be used in broad protein biosensors due to their abundant functional groups and high surface to volume ratio.

  18. Purification by cobalamin-Sepharose affinity chromatography and intrinsic factor-binding activity of an extramembrane proteolytic product from pig ileal mucosa.

    PubMed Central

    Yerima, A; Safi, A; Gastin, I; Michalski, J C; Saunier, M; Gueant, J L

    1996-01-01

    We have purified a cobalamin-binding protein obtained by papain digestion of pig intestine by cobalamin-AH-Sepharose affinity chromatography, with a purification factor of 17,300, a yield of 63% and a cobalamin-binding activity of 11,260 pmol/mg of protein. The protein contained 3.8% carbohydrate and was O- and N-glycosylated. Its molecular mass was 69 kDa on SDS/PAGE and its isoelectric point was 5.1. It had a binding activity for both [57Co]cobalamin and [57Co]cobalamin-intrinsic factor in native PAGE autoradiography and it inhibited the binding of intrinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein shared a binding activity for both cobalamin and intrinsic factor-cobalamin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor. PMID:8573109

  19. Molecular insight in the purification of immunoglobulin by pseudobiospecific ligand l-histidine and histidyl moieties in histidine ligand affinity chromatography (HLAC) by molecular docking.

    PubMed

    Savane, Tushar S; Kumar, Sanjit; Janakiraman, Vignesh Narasimhan; Kamalanathan, Agamudi S; Vijayalakshmi, Mookambeswaran A

    2016-05-15

    Pseudobiospecific ligand l-histidine is an inexpensive, highly stable, non-toxic ligand explored successfully over the last twenty years for the purification of immunoglobulins in immobilised histidine ligand affinity chromatography. It is of great interest to know the molecular recognition sites of IgG to immobilized l-histidine. Here, we have used an in silico approach to explore the molecular recognition of l-histidine by IgG. We have assessed the feasible binding modes of histidine and its moieties at different sites of IgG and considered only those binding conformations which are exhibited via the imidazole ring NH group or any other OH donating group apart from the ones which are terminally conjugated with the support matrix. We categorised binding site into two categories; category I: inner binding groove and category II: surface binding groove and observed that the hinge region of IgG has most favourable binding pocket for l-histidine and histidyl moieties. Ser and Tyr residues on the hinge region make several significant interactions with l-histidine and histidyl moieties. In case of Fc region of IgG, l-histidine and histidyl moieties closely resemble the binding modes of Protein A, biomimetic ligand 22/8 and B domain of SpA to IgG. In addition to these we have also observed a significant binding site for l-histidine and histidyl moieties at Fab region of IgG. PMID:26476866

  20. Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma.

    PubMed

    Huangfu, Chaoji; Ma, Yuyuan; Lv, Maomin; Jia, Junting; Zhao, Xiong; Zhang, Jingang

    2016-07-01

    As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource. PMID:27214605

  1. Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry*

    PubMed Central

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C.

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of ≥1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK. PMID:19887368

  2. Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis.

    PubMed

    Suzuki, Ken; Sakamoto, Hironori; Shinozaki, Yukiko; Tabata, Jun; Watanabe, Takashi; Mochizuki, Atsushi; Koitabashi, Motoo; Fujii, Takeshi; Tsushima, Seiya; Kitamoto, Hiroko K

    2013-09-01

    Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca(2+) or Mg(2+) at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).

  3. Identification of mRNA-Interacting Factors by MS2-TRAP (MS2-Tagged RNA Affinity Purification).

    PubMed

    Yoon, Je-Hyun; Gorospe, Myriam

    2016-01-01

    Posttranscriptional gene expression is governed by the interaction of mRNAs with vast families of RNA-binding proteins (RBPs) and noncoding (nc)RNAs. RBPs and ncRNAs jointly influence all aspects of posttranscriptional metabolism, including pre-mRNA splicing and maturation, mRNA transport, editing, stability, and translation. Given the impact of mRNA-interacting molecules on gene expression, there is great interest in identifying mRNA-binding factors comprehensively. Here, we provide a detailed protocol to tag mRNAs with MS2 hairpins and then affinity-purify trans-binding factors (RBPs, ncRNAs) associated with the MS2-tagged mRNA. This method, termed MS2-TRAP, permits the systematic characterization of ribonucleoprotein (RNP) complexes formed on a given mRNA of interest. We describe how to prepare the mRNA-MS2 expression vector, purify the MS2-tagged RNP complexes, and detect bound RNAs and RBPs, as well as variations of this methodology to address related questions of RNP biology. PMID:26965253

  4. Circulation and Purification in the LUX-ZEPLIN System Test

    NASA Astrophysics Data System (ADS)

    Alsum, Shaun; Lz Collaboration

    2016-03-01

    LZ is a dark-matter direct detection experiment whose detector is a two-phase TPC using approximately seven tons of active xenon as its scintillator. The xenon must have few electronegative impurities to ensure sufficient electron transport through the drift region. The LZ purification system is being prototyped in the LZ system test, a test platform located at SLAC using about 100kg of Xenon, which consists of gas circulation through a SAES getter. We utilize a dual-phase and a gas-phase heat exchanger to reduce needed cooling power. To achieve this circulation we employ an all metal seal triple diaphragm pump, also prototyped in the System Test. This talk will present early results from the system test as well as some baseline LZ designs. The LUX-ZEPLIN dark matter direct detection experiment.

  5. Calculation of antibody affinity in homogeneous and heterogeneous systems.

    PubMed

    Chalquest, R R

    1988-12-01

    Antibody affinity is an important determinant of all antibody-antigen reactions. A new computer program, AFCRV, was developed to calculate binding constants with data from a radioimmunoassay on most microcomputers in the laboratory by using constant-ratio dilution curves. Evaluation of a homogeneous or heterogeneous antibody in the presence of a single antigen can be accomplished.

  6. Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

    PubMed Central

    Gounaris, A D; Brown, M A; Barrett, A J

    1984-01-01

    Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated. Images Fig. 2. Fig. 3. Fig. 4. PMID:6548132

  7. A Temperature-driven Liquid Xenon Recirculation and Purification System

    NASA Astrophysics Data System (ADS)

    Benitez-Medina, Julio Cesar; Hall, Kendy

    2006-10-01

    We have built a liquid xenon recirculation and purification system in order to address the problem of inconsistencies in our Ba^+ fluorescence spectra. In our previous work our liquid xenon purity system did not include recirculation, and the liquid xenon contained ppm of electronegative impurities. By continuous recirculation through a getter purifier, ppb purity is expected. Our recirculation system is driven thermally, by applying heat to the evaporation region, instead of by the pump method used by others. The advantage of thermal driven recirculation is that there are no pressure surges. Therefore, the liquid is calm as it evaporates and condenses. This gives excellent optical quality for Ba^+ spectroscopy in liquid xenon. The goal of this work is to detect fluorescence from single Ba^+ daughter ions in the Enriched Xenon Observatory (EXO) double beta decay experiment.

  8. DEVELOPMENT OF A NON-NOBLE METAL HYDROGEN PURIFICATION SYSTEM

    SciTech Connect

    Korinko, P; Kyle Brinkman, K; Thad Adams, T; George Rawls, G

    2008-11-25

    Development of advanced hydrogen separation membranes in support of hydrogen production processes such as coal gasification and as front end gas purifiers for fuel cell based system is paramount to the successful implementation of a national hydrogen economy. Current generation metallic hydrogen separation membranes are based on Pd-alloys. Although the technology has proven successful, at issue is the high cost of palladium. Evaluation of non-noble metal based dense metallic separation membranes is currently receiving national and international attention. The focus of the reported work was to develop a scaled reactor with a VNi-Ti alloy membrane to replace a production Pd-alloy tube-type purification/diffuser system.

  9. Regenerable Air Purification System for Gas-Phase Contaminant Control

    NASA Technical Reports Server (NTRS)

    Constantinescu, Ileana C.; Qi, Nan; LeVan, M. Douglas; Finn, Cory K.; Finn, John E.; Luna, Bernadette (Technical Monitor)

    2000-01-01

    A regenerable air purification system (RAPS) that uses water vapor to displace adsorbed contaminants from an. adsorbent column into a closed oxidation loop is under development through cooperative R&D between Vanderbilt University and NASA Ames Research Center. A unit based on this design can be used for removing trace gas-phase contaminants from spacecraft cabin air or from polluted process streams including incinerator exhaust. Recent work has focused on fabrication and operation of a RAPS breadboard at NASA Ames, and on measurement of adsorption isotherm data for several important organic compounds at Vanderbilt. These activities support the use and validation of RAPS modeling software also under development at Vanderbilt, which will in turn be used to construct a prototype system later in the project.

  10. Negative homotropic cooperativity and affinity heterogeneity: preparation of yeast glyceraldehyde-3-phosphate dehydrogenase with maximal affinity homogeneity.

    PubMed Central

    Gennis, L S

    1976-01-01

    A three-step procedure including affinity chromatography on NAD+-azobenzamidopropyl-Sepharose has been designed for the purification of yeast glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] with maximized specific activity and maximized homogeneity with respect to affinity for the coenzyme, NAD+.Binding isotherms allow the analysis of cooperativity patterns that disclose both the average ligand affinity in the system and the distribution of ligands among the sites, only for systems with complete affinity homogeneity. The presence of affinity heterogeneity, resulting from multiple oligomeric species differing only in their affinity for coenzyme, gives rise to isotherms which falsely manifest apparent negative cooperativity. A method for distinguishing negative homotropic cooperativity from affinity heterogeneity is suggested. PMID:186779

  11. Revealing novel telomere proteins using in vivo cross-linking, tandem affinity purification, and label-free quantitative LC-FTICR-MS.

    PubMed

    Nittis, Thalia; Guittat, Lionel; LeDuc, Richard D; Dao, Ben; Duxin, Julien P; Rohrs, Henry; Townsend, R Reid; Stewart, Sheila A

    2010-06-01

    Telomeres are DNA-protein structures that protect chromosome ends from the actions of the DNA repair machinery. When telomeric integrity is compromised, genomic instability ensues. Considerable effort has focused on identification of telomere-binding proteins and elucidation of their functions. To date, protein identification has relied on classical immunoprecipitation and mass spectrometric approaches, primarily under conditions that favor isolation of proteins with strong or long lived interactions that are present at sufficient quantities to visualize by SDS-PAGE. To facilitate identification of low abundance and transiently associated telomere-binding proteins, we developed a novel approach that combines in vivo protein-protein cross-linking, tandem affinity purification, and stringent sequential endoprotease digestion. Peptides were identified by label-free comparative nano-LC-FTICR-MS. Here, we expressed an epitope-tagged telomere-binding protein and utilized a modified chromatin immunoprecipitation approach to cross-link associated proteins. The resulting immunoprecipitant contained telomeric DNA, establishing that this approach captures bona fide telomere binding complexes. To identify proteins present in the immunocaptured complexes, samples were reduced, alkylated, and digested with sequential endoprotease treatment. The resulting peptides were purified using a microscale porous graphite stationary phase and analyzed using nano-LC-FTICR-MS. Proteins enriched in cells expressing HA-FLAG-TIN2 were identified by label-free quantitative analysis of the FTICR mass spectra from different samples and ion trap tandem mass spectrometry followed by database searching. We identified all of the proteins that constitute the telomeric shelterin complex, thus validating the robustness of this approach. We also identified 62 novel telomere-binding proteins. These results demonstrate that DNA-bound protein complexes, including those present at low molar ratios, can be

  12. Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification.

    PubMed

    Alekseyenko, Artyom A; Gorchakov, Andrey A; Kharchenko, Peter V; Kuroda, Mitzi I

    2014-02-18

    Understanding the composition of epigenetic regulators remains an important challenge in chromatin biology. Traditional biochemical analysis of chromatin-associated complexes requires their release from DNA under conditions that can also disrupt key interactions. Here we develop a complementary approach (BioTAP-XL), in which cross-linking (XL) enhances the preservation of protein interactions and also allows the analysis of DNA targets under the same tandem affinity purification (BioTAP) regimen. We demonstrate the power of BioTAP-XL through analysis of human EZH2, a core subunit of polycomb repressive complex 2 (PRC2). We identify and validate two strong interactors, C10orf12 and C17orf96, which display enrichment with EZH2-BioTAP at levels similar to canonical PRC2 components (SUZ12, EED, MTF2, JARID2, PHF1, and AEBP2). ChIP-seq analysis of BioTAP-tagged C10orf12 or C17orf96 revealed the similarity of each binding pattern with the location of EZH2 and the H3K27me3-silencing mark, validating their physical interaction with PRC2 components. Interestingly, analysis by mass spectrometry of C10orf12 and C17orf96 interactions revealed that these proteins may be mutually exclusive PRC2 subunits that fail to interact with each other or with JARID2 and AEBP2. C10orf12, in addition, shows a strong and unexpected association with components of the EHMT1/2 complex, thus potentially connecting PRC2 to another histone methyltransferase. Similarly, results from CBX4-BioTAP protein pulldowns are consistent with reports of a diversity of PRC1 complexes. Our results highlight the importance of reciprocal analyses of multiple subunits and suggest that iterative use of BioTAP-XL has strong potential to reveal networks of chromatin-based interactions in higher organisms.

  13. Mathematical modeling of the low and high affinity arabinose transport systems in Escherichia coli.

    PubMed

    Yildirim, Necmettin

    2012-04-01

    A mathematical model was developed for the low and high affinity arabinose transport systems in E. coli. The model is a system of three ordinary differential equations and takes the dynamics of mRNAs for the araE and araFGH proteins and the internal arabinose into account. Special attention was paid to estimate the model parameters from the literature. Our analysis and simulations suggest that the high affinity transport system helps the low affinity transport system to respond to high concentration of extracellular arabinose faster, whereas the high affinity transport system responds to a small amount of extracellular arabinose. Steady state analysis of the model also predicts that there is a regime for the extracellular concentration of arabinose where the arabinose system can show bistable behavior.

  14. Regenerable Air Purification System for Gas-Phase Contaminant Control

    NASA Technical Reports Server (NTRS)

    Constantinescu, Ileana C.; Finn, John E.; LeVan, M. Douglas; Lung, Bernadette (Technical Monitor)

    2000-01-01

    Tests of a pre-prototype regenerable air purification system (RAPS) that uses water vapor to displace adsorbed contaminants from an adsorbent column have been performed at NASA Ames Research Center. A unit based on this design can be used for removing trace gas-phase contaminants from spacecraft cabin air or from polluted process streams including incinerator exhaust. During the normal operation mode, contaminants are removed from the air on the column. Regeneration of the column is performed on-line. During regeneration, contaminants are displaced and destroyed inside the closed oxidation loop. In this presentation we discuss initial experimental results for the performance of RAPS in the removal and treatment of several important spacecraft contaminant species from air.

  15. A versatile multidimensional protein purification system with full internet remote control based on a standard HPLC system.

    PubMed

    Riek, Uwe; Ramirez, Sacnicte; Wallimann, Theo; Schlattner, Uwe

    2009-05-01

    The standard Akta Explorer high-performance liquid chromatography (HPLC) system has limitations for the automation of multidimensional protein purification. Here, we describe simple modifications that allow for automated multidimensional purification protocols to extend the possibilities of the Akta three-dimensional purification kit in terms of column number, flexibility of volumes stocked for re-injection of samples, and available choice of buffers. These modifications do not preclude the use of standard one-dimensional purification protocols. Additionally, we demonstrate a technology for encrypted full remote control of the machine over the Internet by cost-effective use of standard asymmetric digital subscriber line (ADSL) that enables direct remote interaction with the machine without preventing local control. A 4-column purification scheme, including equilibration and cleaning in place (CIP) procedures, was implemented on such a system. It significantly increased reproducibility and shortened processing time by 85%, as compared with manual operation, thus allowing for automated protein purification overnight.

  16. Synthesis of nonlinear discrete control systems via time-delay affine Takagi-Sugeno fuzzy models.

    PubMed

    Chang, Wen-Jer; Chang, Wei

    2005-04-01

    The affine Takagi-Sugeno (TS) fuzzy model played a more important role in nonlinear control because it can be used to approximate the nonlinear systems more than the homogeneous TS fuzzy models. Besides, it is known that the time delays exist in physical systems and the previous works did not consider the time delay effects in the analysis of affine TS fuzzy models. Hence a parallel distributed compensation based fuzzy controller design issue for discrete time-delay affine TS fuzzy models is considered in this paper. The time-delay effect is considered in the discrete affine TS fuzzy models and the stabilization issue is developed for the nonlinear time-delay systems. Finally, a numerical simulation for a time-delayed nonlinear truck-trailer system is given to show the applications of the present approach.

  17. Construction of a Class of Four-Dimensional Piecewise Affine Systems with Homoclinic Orbits

    NASA Astrophysics Data System (ADS)

    Wu, Tiantian; Yang, Xiao-Song

    2016-06-01

    Based on mathematical analysis, this paper provides a methodology to ensure the existence of homoclinic orbits in a class of four-dimensional piecewise affine systems. In addition, an example is provided to illustrate the effectiveness of the method.

  18. Best approximation of functions in L{sub p} by polynomials on affine system

    SciTech Connect

    Terekhin, Pavel A

    2011-02-28

    Estimates of the best L{sub p}-approximation of functions by polynomials in an affine system (system of dilations and translations), which are similar to well-known estimates due to Ul'yanov and Golubov for approximations in the Haar system, are obtained. An analogue of A.F. Timan and M.F. Timan's inequality is shown to hold under certain conditions on the generating function of the affine system; this analogue fails for the Haar system for 1

  19. Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes.

    PubMed

    Umesh Hebbar, H; Sumana, B; Raghavarao, K S M S

    2008-07-01

    Reverse micellar systems of CTAB/isooctane/hexanol/butanol and AOT/isooctane are used for the extraction and primary purification of bromelain from crude aqueous extract of pineapple wastes (core, peel, crown and extended stem). The effect of forward as well as back extraction process parameters on the extraction efficiency, activity recovery and purification fold is studied in detail for the pineapple core extract. The optimized conditions for the extraction from core resulted in forward and back extraction efficiencies of 45% and 62%, respectively, using reverse micellar system of cationic surfactant CTAB. A fairly good activity recovery (106%) and purification (5.2-fold) of bromelain is obtained under these conditions. Reverse micellar extraction from peel, extended stem and crown using CTAB system resulted in purification folds of 2.1, 3.5, and 1.7, respectively. Extraction from extended stem using anionic surfactant AOT in isooctane did not yield good results under the operating conditions employed.

  20. Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes.

    PubMed

    Umesh Hebbar, H; Sumana, B; Raghavarao, K S M S

    2008-07-01

    Reverse micellar systems of CTAB/isooctane/hexanol/butanol and AOT/isooctane are used for the extraction and primary purification of bromelain from crude aqueous extract of pineapple wastes (core, peel, crown and extended stem). The effect of forward as well as back extraction process parameters on the extraction efficiency, activity recovery and purification fold is studied in detail for the pineapple core extract. The optimized conditions for the extraction from core resulted in forward and back extraction efficiencies of 45% and 62%, respectively, using reverse micellar system of cationic surfactant CTAB. A fairly good activity recovery (106%) and purification (5.2-fold) of bromelain is obtained under these conditions. Reverse micellar extraction from peel, extended stem and crown using CTAB system resulted in purification folds of 2.1, 3.5, and 1.7, respectively. Extraction from extended stem using anionic surfactant AOT in isooctane did not yield good results under the operating conditions employed. PMID:17964777

  1. Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

    PubMed

    Matheson, Nicholas J; Peden, Andrew A; Lehner, Paul J

    2014-01-01

    Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.

  2. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Stellwagen, Earle

    1981-04-01

    Neutral salts exhibit very marked differences as eluants of proteins from affinity columns. We observe: (i) that the relative potencies of neutral salts as eluants are independent of the protein or the affinity ligand in the systems studied, (ii) that the absolute salt concentration necessary to elute any given protein bound to the affinity matrix is proportional to the algebraic sum of a set of elution coefficients defined herein for the separate ions present in the solution, and (iii) that the proportionality between elution potency and elution coefficient is a function of the affinity of the protein for the immobilized ligand. Given the concentration of one neutral salt required for elution of a protein of interest from an affinity column, the elution capability of any neutral salt at any temperature can be quantitatively predicted for that protein. Accordingly, application and elution protocols for affinity chromatography can be designed to optimize the yield and fold purification of proteins.

  3. Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension.

    PubMed

    Malm, Magdalena; Bass, Tarek; Gudmundsdotter, Lindvi; Lord, Martin; Frejd, Fredrik Y; Ståhl, Stefan; Löfblom, John

    2014-09-01

    Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.

  4. Purification of liquid metal systems with sodium coolant from oxygen using getters

    NASA Astrophysics Data System (ADS)

    Kozlov, F. A.; Konovalov, M. A.; Sorokin, A. P.

    2016-05-01

    For increasing the safety and economic parameters of nuclear power stations (NPSs) with sodium coolant, it was decided to install all systems contacting radioactive sodium, including purification systems of circuit I, in the reactor vessel. The performance and capacity of cold traps (CTs) (conventional element of coolant purification systems) in these conditions are limited by their volume. It was proposed to use hot traps (HTs) in circuit I for coolant purification from oxygen. It was demonstrated that, at rated parameters of the installation when the temperature of the coolant streamlining the getter (gas absorber) is equal to 550°C, the hot trap can provide the required coolant purity. In shutdown modes at 250-300°C, the performance of the hot trap is reduced by four orders of magnitude. Possible HT operation regimes for shutdown modes and while reaching rated parameters were proposed and analyzed. Basic attention was paid to purification modes at power rise after commissioning and accidental contamination of the coolant when the initial oxygen concentration in it reached 25 mln-1. It was demonstrated that the efficiency of purification systems can be increased using HTs with the getter in the form of a foil or granules. The possibility of implementing the "fast purification" mode in which the coolant is purified simultaneously with passing over from the shutdown mode to the rated parameters was substantiated.

  5. Application of the Protein Maker as a platform purification system for therapeutic antibody research and development.

    PubMed

    Hélie, Geneviève; Parat, Marie; Massé, Frédéric; Gerdts, Cory J; Loisel, Thomas P; Matte, Allan

    2016-01-01

    Within the research and development environment, higher throughput, parallelized protein purification is required for numerous activities, from small scale purification of monoclonal antibodies (mAbs) and antibody fragments for in vitro and in vivo assays to process development and optimization for manufacturing. Here, we describe specific applications and associated workflows of the Protein Maker liquid handling system utilized in both of these contexts. To meet the requirements for various in vitro assays, for the identification and validation of new therapeutic targets, small quantities of large numbers of purified antibodies or antibody fragments are often required. Reducing host cell proteins (HCP) levels following capture with Protein A by evaluating various wash buffers is an example of how parallelized protein purification can be leveraged to improve a process development outcome. Stability testing under various conditions of in-process intermediates, as an example, the mAb product from a clarified harvest, requires parallelized protein purification to generate concurrent samples for downstream assays. We have found that the Protein Maker can be successfully utilized for small-to-mid scale platform purification or for process development applications to generate the necessary purified protein samples. The ability to purify and buffer exchange up to 24 samples in parallel offers a significant reduction in time and cost per sample compared to serial purification using a traditional FPLC system. By combining the Protein Maker purification system with a TECAN Freedom EVO liquid handler for automated buffer exchange we have created a new, integrated platform for a variety of protein purification and process development applications. PMID:27418955

  6. A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri.

    PubMed

    Dantas, Giordanni C; Martins, Paula M M; Martins, Daniela A B; Gomes, Eleni; Ferreira, Henrique

    2016-01-01

    Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo. PMID:26991273

  7. Engineering higher affinity T cell receptors using a T cell display system

    PubMed Central

    Chervin, Adam S.; Aggen, David H.; Raseman, John M.; Kranz, David M.

    2008-01-01

    The T cell receptor (TCR) determines the cellular response to antigens, which are presented on the surface of target cells in the form of a peptide bound to a product of the major histocompatibility complex (pepMHC). The response of the T cell depends on the affinity of the TCR for the pepMHC, yet many TCRs have been shown to be of low affinity, and some naturally occurring T cell responses are poor due to low affinities. Accordingly, engineering the TCR for increased affinity for pepMHC, particularly tumor-associated antigens, has become an increasingly desirable goal, especially with the advent of adoptive T cell therapies. For largely technical reasons, to date there have been only a handful of TCRs engineered in vitro for higher affinity using well established methods of protein engineering. Here we report the use of a T cell display system, using a retroviral vector, for generating a high affinity TCR from the mouse T cell clone 2C. The method relies on the display of the TCR, in its normal, signaling competent state, as a CD3 complex on the T cell surface. A library in the CDR3α of the 2C TCR was generated in the MSCV retroviral vector and transduced into a TCR-negative hybridoma. Selection of a high affinity, CD8-independent TCR was accomplished after only two rounds of flow cytometric sorting using the pepMHC SIYRYYGL/Kb (SIY/Kb). The selected TCR contained a sequence motif in the CDR3α with characteristics of several other TCRs previously selected by yeast display. In addition, it was possible to directly use the selected T cell hybridoma in functional assays without the need for sub-cloning, revealing that the selected TCR was capable of mediating CD8-independent activity. The method may be useful in the direct isolation and characterization of TCRs that could be used in therapies with adoptive transferred T cells. PMID:18854190

  8. Interaction of nicotinic receptor affinity reagents with central nervous system. cap alpha. -bungarotoxin-binding entities

    SciTech Connect

    Lukas, R.J.; Bennett, E.L.

    1980-01-01

    Membrane-bound ..cap alpha..-bungarotoxin-binding entities derived from rat brain are found to interact specifically with the affinity reagents maleimidobenzyltrimethylammonium (MBTA) and bromoacetylcholine (BAC), originally designed to label nicotinic acetylcholine receptors from electroplax and skeletal muscle. Following treatment of membranes with dithiothreitol, all specific toxin binding sites are irreversibly blocked by reaction with MBTA or BAC. Affinity reagent labeling of dithiothreitol-reduced membranes is prevented (toxin binding sites are not blocked) by prior alkylaction with N-ethylmaleimide, by prior oxidation with dithiobis(2-nitrobenzoic acid), or by incubation with neurotoxin. Reversibly associating cholinergic agonists and antagonists retard the rate of affinity reagent interaction with toxin receptors. The apparent rates of affinity reagent alkylation of toxin receptors, and the influences of other sulfhydryl/disulfide reagents on affinity labeling are comparable to those observed for reaction with nicotinic acetylcholine receptors in the periphery. The results provide further evidence that central nervous system ..cap alpha..-bungarotoxin receptors share a remarkable number of biochemical properties with nicotinic receptors from the periphery.

  9. Fibronectin purification from human plasma in a packed-bed column system with gelatin immobilized PHEMA microspheres.

    PubMed

    Kayirhan-Denizli, F; Arica, M Y; Denizli, A

    2001-01-01

    Bioaffinity chromatography has a unique and powerful role that is used as a purification tool in the production of therapeutic plasma protein derivatives. In this study, a bioaffinity-ligand, i.e. gelatin, was covalently immobilized with PHEMA microspheres (150-200 microm in diameter). The affinity sorbent carrying 7.5 mg gelatin g(-1) polymer was then used to separate fibronectin from human plasma in a packed-bed column system. Fibronectin separation from human plasma on unmodified PHEMA microspheres was 0.45 mg g(-1), while much higher adsorption values, up to 21.8 mg g(-1), were obtained with gelatin-immobilized microspheres. The fibronectin adsorption capacity of the microspheres decreased with an increase in the recirculation rate of plasma. Fibronectin adsorption increased with decreasing temperature, and the maximum adsorption achieved at 4 degrees C (26.3 mg fibronectin g(-1)). Up to 94.7% of the adsorbed fibronectin was desorbed by using 2 M urea in the presence of 1 M sodium chloride as elution agent. The adsorption-desorption cycle was repeated ten times using the same affinity column. There was no remarkable reduction in the adsorption capacity of the gelatin-immobilized PHEMA microspheres. PMID:11469779

  10. Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems.

    PubMed

    Li, Mian; Peeples, Tonya L

    2004-07-25

    Purification of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide-propylene oxide random copolymer (PEO-PPO)/(NH(4))(2)SO(4), and poly(ethylene glycol) (PEG)/(NH(4))(2)SO(4) aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes. PMID:15177162

  11. Magnetically ultraresponsive nanoscavengers for next-generation water purification systems

    PubMed Central

    Zhang, Mingliang; Xie, Xing; Tang, Mary; Criddle, Craig S.; Cui, Yi; Wang, Shan X.

    2014-01-01

    The development of sustainable, robust and energy efficient water purification technology is still challenging. Although use of nanoparticles is promising, methods are needed for their efficient recovery post treatment. Here we address this issue by fabrication of magnetically ultraresponsive ‘nanoscavengers’, nanoparticles containing synthetic antiferromagnetic core layers and functional capping layers. When dispersed in water, the nanoscavengers efficiently interact with contaminants to remove them from the water. They are then quickly collected (<5 min) with a permanent magnet, owing to their magnetically ultraresponsive core layers. Specifically, we demonstrate fabrication and deployment of Ag-capped nanoscavengers for disinfection followed by application of an external magnetic field for separation. We also develop and validate a collision-based model for pathogen inactivation, and propose a cyclical water purification scheme in which nanoscavengers are recovered and recycled for contaminant removal. PMID:23673651

  12. Sodium coolant purification systems for a nuclear power station equipped with a BN-1200 reactor

    NASA Astrophysics Data System (ADS)

    Alekseev, V. V.; Kovalev, Yu. P.; Kalyakin, S. G.; Kozlov, F. A.; Kumaev, V. Ya.; Kondrat'ev, A. S.; Matyukhin, V. V.; Pirogov, E. P.; Sergeev, G. P.; Sorokin, A. P.; Torbenkova, I. Yu.

    2013-05-01

    Both traditional coolant purification methods (by means of traps and sorbents for removing cesium), the use of which supported successful operation of nuclear power installations equipped with fast-neutron reactors with a sodium coolant, and the possibility of removing oxygen from sodium through the use of hot traps are analyzed in substantiating the purification system for a nuclear power station equipped with a BN-1200 reactor. It is shown that a cold trap built into the reactor vessel must be a mandatory component of the reactor plant primary coolant circuit's purification system. The use of hot traps allows oxygen to be removed from the sodium coolant down to permissible concentrations when the nuclear power station operates in its rated mode. The main lines of works aimed at improving the performance characteristics of cold traps are suggested based on the results of performed investigations.

  13. Bromelain purification through unconventional aqueous two-phase system (PEG/ammonium sulphate).

    PubMed

    Coelho, D F; Silveira, E; Pessoa Junior, A; Tambourgi, E B

    2013-02-01

    This paper focuses on the feasibility of unconventional aqueous two-phase systems for bromelain purification from pineapple processing waste. The main difference in comparison with conventional systems is the integration of the liquid-liquid extraction technique with fractional precipitation, which can decrease the protein content with no loss of biological activity by removing of unwanted molecules. The analysis of the results was based on the response surface methodology and revealed that the use of the desirability optimisation methodology (DOM) was necessary to achieve higher purification factor values and greater bromelain recovery. The use of DOM yielded an 11.80-fold purification factor and 66.38 % biological activity recovery using poly(ethylene glycol) (PEG) with a molar mass of 4,000, 10.86 % PEG concentration (m/m) and 36.21 % saturation of ammonium sulphate.

  14. A Lyapunov method for stability analysis of piecewise-affine systems over non-invariant domains

    NASA Astrophysics Data System (ADS)

    Rubagotti, Matteo; Zaccarian, Luca; Bemporad, Alberto

    2016-05-01

    This paper analyses stability of discrete-time piecewise-affine systems, defined on possibly non-invariant domains, taking into account the possible presence of multiple dynamics in each of the polytopic regions of the system. An algorithm based on linear programming is proposed, in order to prove exponential stability of the origin and to find a positively invariant estimate of its region of attraction. The results are based on the definition of a piecewise-affine Lyapunov function, which is in general discontinuous on the boundaries of the regions. The proposed method is proven to lead to feasible solutions in a broader range of cases as compared to a previously proposed approach. Two numerical examples are shown, among which a case where the proposed method is applied to a closed-loop system, to which model predictive control was applied without a-priori guarantee of stability.

  15. 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).

  16. 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). PMID:26096503

  17. Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography ▿ † ‡

    PubMed Central

    Robichon, Carine; Luo, Jianying; Causey, Thomas B.; Benner, Jack S.; Samuelson, James C.

    2011-01-01

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein. PMID:21602383

  18. New results on robust stability for differential-difference systems with affine linear parametric uncertainty

    NASA Astrophysics Data System (ADS)

    Romero, Gerardo; Díaz, Iván; Pérez, Irma; Guerrero, Alfredo; Lara, David; Rivera, José

    2013-01-01

    This article presents sufficient conditions to verify the robust stability property of convex combinations for quasipolynomials that represent the characteristic equation of differential-difference dynamics systems. It considers affine linear parametric uncertainty structure in the coefficients of quasipolynomials and also, interval uncertainty in the time delay. First of all, a transformation of the delay's operator is performed in order to get a two variable polynomial; after this, to obtain the robust stability property, a result based on the Hurwitz matrix is applied.

  19. Diacylglycerol-induced translocation of diacylglycerol kinase: use of affinity-purified enzyme in a reconstitution system.

    PubMed

    Besterman, J M; Pollenz, R S; Booker, E L; Cuatrecasas, P

    1986-12-01

    Diacylglycerol-induced translocation of diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from the soluble to the membrane-bound compartments was demonstrated both in crude tissue homogenates and in a reconstituted enzyme-membrane model system. In homogenates of either rat brain or liver, incubation with diacylglycerol or phospholipase C, but not phospholipase A2 or phospholipase D, resulted in the translocation of diacylglycerol kinase activity from the soluble to the particulate fraction. This observation formed the basis for the first step in a two-step purification of diacylglycerol kinase. Enzyme extracted in 1 M salt from membranes of rat brain homogenates made in the presence of phospholipase C was purified further by affinity chromatography on a column containing phosphatidylserine, diacylglycerol, and cholesterol immobilized in polyacrylamide. This step yielded an enzyme preparation (step 2 enzyme) that was 500- to 750-fold purified (relative to the tissue homogenate) and required phosphatidylserine for stability. All other lipids tested failed to stabilize the enzyme. The properties of the enzyme preparation were similar to those of mammalian diacylglycerol kinases described by others. Reconstitution experiments showed that the soluble step 2 enzyme bound to inside-out vesicles of human erythrocytes only in the presence of diacylglycerol or phospholipase C but not phospholipase A2 or D. Redistribution of the kinase from soluble to vesicle-bound forms occurred rapidly and was dependent on the concentration of phospholipase C used to treat the vesicles. Physiological concentrations of calcium (50-1000 nM) did not enhance the phospholipase C-mediated translocation of the kinase. Thus, diacylglycerol kinase can translocate from cytosol to membranes in a manner dependent on the content of membrane-bound diacylglycerol but independent of the ambient concentration of calcium.

  20. Field Testing of a Small Water Purification System for Non-PRASA Rural Communities

    EPA Science Inventory

    Small, rural communities typically do not have adequate water purification systems to sustain their life quality and residents are exposed to pathogens present in drinking water. In Puerto Rico (PR), approximately 4% of the population does not have access to drinking water provi...

  1. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes.

  2. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes. PMID:25764651

  3. Regenerable Incinerator Exhaust Purification and Trace Contaminant Control System

    NASA Technical Reports Server (NTRS)

    Finn, John E.; Cho, Shelia Y.; LeVan, M. Douglas

    2003-01-01

    In this novel approach to air purification, contaminants removed from a process air stream by a high-capacity adsorbent are displaced periodically by a warm, high-humidity, reverse-flow air stream. Displaced contaminants flow into a closed regeneration loop, in which organic compounds are oxidized catalytically and acid gases are removed by a gas- water contactor (which also serves as the source of the water vapor). These features are expected to result in a design that has few expendables and lower energy consumption than alternative regenerable techniques. The joint project between NASA Ames Research Center and Vanderbilt University has completed its third year. Breadboard development continues at NASA Ames, while Vanderbilt has completed most of its adsorption equilibria development. Vanderbilt has completed its fixed-bed apparatus for investigation of dynamic adsorption and desorption processes for trace organic compounds and water vapor, and is continuing its development of the mathematical model describing the column dynamics.

  4. 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

  5. Radiosynthesis binding affinity and biodistribution of 3-[F-18]fluoro-N-({alpha},{alpha},{alpha}-trifluoro-m-tolyl)piperazine (FTFMPP), a radioligand for the Serotonin system

    SciTech Connect

    Mishani, E.; Cristel, M.E.; McCarthy, T.J.; Welch, M.J.

    1996-05-01

    The serotonin agonist N({alpha},{alpha},{alpha}-trifluoro-m-tolyl)piperazine (TFMPP) is a potent ligand for the serotonin system. Angelini and co-workers previously synthesized the c.a [F-18]TFMPP but the low specific activity (less than 0.2GBq/mmol) limited the application of this ligand. We have recently reported the formation of phenylpiperazines by a novel alumina supported bis-alkylation. We report the application of this method and biological evaluation of 3-[F-18]FTFMPP, a fluoro derivative of TFMPP. Reaction of [F-18]fluoride with 3,5-dinitrobenzotrifluoride gave the 3-[F-18]fluoro-5-nitrobenzotrifluoride in 70% yield. Reduction of the nitro group with Raney nickel and hydrazine hydrate gave the [F-18]aniline derivative in 70% yield. Finally, the phenylpiperazine was constructed by reaction of the [F-18]aniline derivative with bis-2-bromoethyl-N-(ethoxy carbonyl)amine on basic alumina (pH=9) as a solid support. After extraction of the activity with basic MeOH and HPLC purification on normal phase the final product- [F-18]FTFMPP was obtained in 50% yield (98% radiochemical purity). The specific activity of the final product was 100GBq/mmol. The binding affinity of FTFMPP to 5-HT receptor was determined (Ki = 80-100 nM) and found to be similar to the binding affinity of the TFMPP (160-180 nM). The biodistribution of [F-18]FTFMPP was performed in rats.

  6. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  7. Hamiltonian purification

    SciTech Connect

    Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo; Pascazio, Saverio; Nakazato, Hiromichi; Yuasa, Kazuya; Giovannetti, Vittorio

    2015-12-15

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians (h{sub 1}, …, h{sub m}) operating on a d-dimensional quantum system ℋ{sub d}, the problem consists in identifying a set of commuting Hamiltonians (H{sub 1}, …, H{sub m}) operating on a larger d{sub E}-dimensional system ℋ{sub d{sub E}} which embeds ℋ{sub d} as a proper subspace, such that h{sub j} = PH{sub j}P with P being the projection which allows one to recover ℋ{sub d} from ℋ{sub d{sub E}}. The notions of spanning-set purification and generator purification of an algebra are also introduced and optimal solutions for u(d) are provided.

  8. Hamiltonian purification

    NASA Astrophysics Data System (ADS)

    Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo; Nakazato, Hiromichi; Pascazio, Saverio; Yuasa, Kazuya; Giovannetti, Vittorio

    2015-12-01

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians {h1, …, hm} operating on a d-dimensional quantum system ℋd, the problem consists in identifying a set of commuting Hamiltonians {H1, …, Hm} operating on a larger dE-dimensional system ℋdE which embeds ℋd as a proper subspace, such that hj = PHjP with P being the projection which allows one to recover ℋd from ℋdE. The notions of spanning-set purification and generator purification of an algebra are also introduced and optimal solutions for 𝔲(d) are provided.

  9. Characterization of the Rhizobium (Sinorhizobium) meliloti high- and low-affinity phosphate uptake systems.

    PubMed Central

    Voegele, R T; Bardin, S; Finan, T M

    1997-01-01

    Genetic studies have suggested that Rhizobium (Sinorhizobium) meliloti contains two distinct phosphate (Pi) transport systems, encoded by the phoCDET genes and the orfA-pit genes, respectively. Here we present data which show that the ABC-type PhoCDET system has a high affinity for Pi (Km, 0.2 microM) and that Pi uptake by this system is severely inhibited by phosphonates. This high-affinity uptake system was induced under Pi-limiting conditions and was repressed in the presence of excess Pi. Uptake via the OrfA-Pit system was examined in (i) a phoC mutant which showed increased expression of the orfA-pit genes as a result of a promoter-up mutation and (ii) a phoB mutant (PhoB is required for phoCDET expression). Pi uptake in both strains exhibited saturation kinetics (Km, 1 to 2 microM) and was not inhibited by phosphonates. This uptake system was active in wild-type cells grown with excess Pi and appeared to be repressed when the cells were starved for Pi. Thus, our biochemical data show that the OrfA-Pit and PhoCDET uptake systems are differentially expressed depending on the state of the cell with respect to phosphate availability. PMID:9393684

  10. Combined cooling and purification system for nuclear reactor spent fuel pit, refueling cavity, and refueling water storage tank

    DOEpatents

    Corletti, M.M.; Lau, L.K.; Schulz, T.L.

    1993-12-14

    The spent fuel pit of a pressured water reactor (PWR) nuclear power plant has sufficient coolant capacity that a safety rated cooling system is not required. A non-safety rated combined cooling and purification system with redundant branches selectively provides simultaneously cooling and purification for the spent fuel pit, the refueling cavity, and the refueling water storage tank, and transfers coolant from the refueling water storage tank to the refueling cavity without it passing through the reactor core. Skimmers on the suction piping of the combined cooling and purification system eliminate the need for separate skimmer circuits with dedicated pumps. 1 figures.

  11. Combined cooling and purification system for nuclear reactor spent fuel pit, refueling cavity, and refueling water storage tank

    DOEpatents

    Corletti, Michael M.; Lau, Louis K.; Schulz, Terry L.

    1993-01-01

    The spent fuel pit of a pressured water reactor (PWR) nuclear power plant has sufficient coolant capacity that a safety rated cooling system is not required. A non-safety rated combined cooling and purification system with redundant branches selectively provides simultaneously cooling and purification for the spent fuel pit, the refueling cavity, and the refueling water storage tank, and transfers coolant from the refueling water storage tank to the refueling cavity without it passing through the reactor core. Skimmers on the suction piping of the combined cooling and purification system eliminate the need for separate skimmer circuits with dedicated pumps.

  12. Advanced Water Purification System for In Situ Resource Utilization

    NASA Technical Reports Server (NTRS)

    Anthony, Stephen M.; Jolley, Scott T.; Captain, James G.

    2013-01-01

    A main goal in the field of In Situ Resource Utilization is to develop technologies that produce oxygen from regolith to provide consumables to an extraterrestrial outpost. The processes developed reduce metal oxides in the regolith to produce water, which is then electrolyzed to produce oxygen. Hydrochloric and hydrofluoric acids are byproducts of the reduction processes, which must be removed to meet electrolysis purity standards. We previously characterized Nation, a highly water selective polymeric proton-exchange membrane, as a filtration material to recover pure water from the contaminated solution. While the membranes successfully removed both acid contaminants, the removal efficiency of and water flow rate through the membranes were not sufficient to produce large volumes of electrolysis-grade water. In the present study, we investigated electrodialysis as a potential acid removal technique. Our studies have shown a rapid and significant reduction in chloride and fluoride concentrations in the feed solution, while generating a relatively small volume of concentrated waste water. Electrodialysis has shown significant promise as the primary separation technique in ISRU water purification processes.

  13. Advanced Water Purification System for In Situ Resource Utilization Project

    NASA Technical Reports Server (NTRS)

    Anthony, Stephen M.

    2014-01-01

    A main goal in the field of In Situ Resource Utilization is to develop technologies that produce oxygen from regolith to provide consumables to an extratrrestrial outpost. The processes developed reduce metal oxides in the regolith to produce water, which is then electrolyzed to produce oxygen. Hydrochloric and hydrofluoric acids are byproducts of the reduction processes, which must be removed to meet electrolysis purity standards. We previously characterized Nation, a highly water selective polymeric proton-exchange membrane, as a filtrtion material to recover pure water from the contaminated solution. While the membranes successfully removed both acid contaminants, the removal efficiency of and water flow rate through the membranes were not sufficient to produce large volumes of electrolysis-grade water. In the present study, we investigated electrodialysis as a potential acid removable technique. Our studies have show a rapid and significant reduction in chloride and fluoride concentrations in the feed solution, while generating a relatively small volume of concentrated waste water. Electrodialysis has shown significant promise as the primary separation technique in ISRU water purification processes.

  14. On the existence of bifocal heteroclinic cycles in a class of four-dimensional piecewise affine systems

    NASA Astrophysics Data System (ADS)

    Wu, Tiantian; Yang, Xiao-Song

    2016-05-01

    Based on mathematical analysis, this paper provides a methodology to ensure the existence of heteroclinic cycles in a class of four-dimensional piecewise affine systems. In addition, examples are provided to illustrate the effectiveness of the method.

  15. Efficiency in removing pollutants by constructed wetland purification systems in Poland.

    PubMed

    Samecka-Cymerman, A; Stepien, D; Kempers, A J

    2004-02-27

    The aim of this study was to compare the efficiency between Phragmites communis, Salix viminalis, and Populus canadensis in removing the heavy metals Al, Ba, Mn, Ni, Sr, V, Zn, Cd, Cu, and Pb and the eutrophying macroelements phosphate, nitrate, nitrite, ammonia, chloride, sulfate, Ca, Mg, K, and Fe from sewage in subsurface flow constructed wetlands in Poland. The effectiveness of the sewage treatment system was higher in summer compared to winter for the removal of (1) all heavy metals, phosphate (P) and mineral nitrogen (N) for all of species, (2) sulfates (S) for Phragmites and Salix, (3) iron (Fe) for Salix, and (4) chloride (Cl) for Salix and Populus. Analysis of variance indicated that there was no significant difference between the purifications systems in phytoremediation of Mn; so all species were equally effective (99%, prob. level 0.001). The Salix wetland system was most effective in purification of water and removal of macroelements (24-82% in summer, 10-80% in winter with Fe 97%), Cd (58-71%), V (100%), and Zn (84-92%). The Phragmites system was most effective in purification and removal of Al (81-97%), Ba (70-95%), Pb (64-81%), and Sr (24-51%), while in the case of Cu (49-60%) and Ni (55-67%) the Populus wetland system proved most effective. The outflowing water of the wetlands contained elements in amounts exceeding the admissible levels as established for unpolluted water both in winter and summer. Therefore the effectiveness of the observed phytoremediation systems in this study was not sufficient alone to remove these elements and can be considered as a supplemental tool in purification of sewage.

  16. Purification of uranothorite solid solutions from polyphase systems

    NASA Astrophysics Data System (ADS)

    Clavier, Nicolas; Szenknect, Stéphanie; Costin, Dan Tiberiu; Mesbah, Adel; Ravaux, Johann; Poinssot, Christophe; Dacheux, Nicolas

    2013-10-01

    The mineral coffinite, nominally USiO4, and associated Th1-xUxSiO4 uranothorite solid solutions are of great interest from a geochemical point of view and in the case of the direct storage of spent nuclear fuels. Nevertheless, they clearly exhibit a lack in the evaluation of their thermodynamic data, mainly because of the difficulties linked with their preparation as pure phases. This paper thus presents physical and chemical methods aiming to separate uranothorite solid solutions from oxide additional phases such as amorphous SiO2 and nanometric crystallized Th1-yUyO2. The repetition of centrifugation steps envisaged in first place was rapidly dropped due to poor recovery yields, to the benefit of successive washings in acid then basic media. Under both static and dynamic flow rates (i.e. low or high rate of leachate renewal), ICP-AES (Inductively Coupled Plasma - Atomic Emission Spectroscopy) analyses revealed the systematic elimination of Th1-yUyO2 in acid media and of SiO2 in basic media. Nevertheless, two successive steps were always needed to reach pure samples. On this basis, a first cycle performed in static conditions was chosen to eliminate the major part of the accessory phases while a second one, in dynamic conditions, allowed the elimination of the residual impurities. The complete purification of the samples was finally evidenced through the characterization of the samples by the means of PXRD (Powder X-Ray Diffraction), SEM (Scanning Electron Microscopy) observations and X-EDS (X-Ray Energy Dispersive Spectroscopy) analyses.

  17. Purification and enrichment of virus samples utilizing magnetic beads on a microfluidic system.

    PubMed

    Lien, Kang-Yi; Lin, Jr-Lung; Liu, Cheng-Yu; Lei, Huan-Yao; Lee, Gwo-Bin

    2007-07-01

    This study reports a new microfluidic system with three integrated functional devices for pumping, mixing and separation of bio-samples by utilizing micro-electro-mechanical-systems technology. By using antibody-conjugated magnetic beads, the developed system can be used to purify and enrich virus samples such that the subsequent detection of viruses can be performed with a higher sensitivity. The target viruses were first captured by the antibody coated onto the magnetic beads by using a rotary micromixer which performed the incubation process. The viruses were then purified and enriched by a magnetic field generated by planar microcoils. The integrated microfluidic system can perform the whole purification and enrichment process automatically using a rotary micropump and appropriate microvalves. In addition, a numerical simulation was also employed to optimize the design of the microcoils and to investigate the magnetic field strength and distribution. The simulation results were consistent with experimental observations. Finally, the developed system was used to successfully perform the purification and enrichment of Dengue viruses. The detectable limit of Dengue viruses was found to be as low as 10(2) pfu ml(-1) by using this approach. Therefore, the integrated microsystem can perform incubation, transportation, mixing and purification of virus samples, possibly making it a promising platform for future biological and medical applications.

  18. Expression of bioactive soluble human stem cell factor (SCF) from recombinant Escherichia coli by coproduction of thioredoxin and efficient purification using arginine in affinity chromatography.

    PubMed

    Akuta, Teruo; Kikuchi-Ueda, Takane; Imaizumi, Keitaro; Oshikane, Hiroyuki; Nakaki, Toshio; Okada, Yoko; Sultana, Sara; Kobayashi, Kenichiro; Kiyokawa, Nobutaka; Ono, Yasuo

    2015-01-01

    Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5M l-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l-arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method.

  19. Integrated extraction and purification of soy isoflavones by using aqueous micellar systems.

    PubMed

    Cordisco, Estefanía; Haidar, Carla N; Coscueta, Ezequiel R; Nerli, Bibiana B; Malpiedi, Luciana P

    2016-12-15

    In this work, an integration of solid-liquid and liquid-liquid extractions by using aqueous micellar two-phase systems was evaluated as potential tool to purify soy isoflavones. Additionally, the proposed methodology aimed to preserve the protein content of the processed soy flour. The extractive assays were performed in AMTPS formed by Triton X-114 and sodium tartrate. In order to optimize the purification process, temperature and time were evaluated as independent variables. Under optimal working conditions, i.e. 100min and 33°C of incubation, IF were purified with a recovery percentage of 93 and a purification factor of almost 10. More importantly, the obtained sample presented an aglycone proportion superior to the reported by other methodologies. These results open perspectives to the use of aqueous micellar two-phase systems as an integrative methodology to extract, concentrate and purify isoflavones. PMID:27451211

  20. Genetic evidence of a high-affinity cyanuric acid transport system in Pseudomonas sp. ADP.

    PubMed

    Platero, Ana I; Santero, Eduardo; Govantes, Fernando

    2014-03-01

    The Pseudomonas sp. ADP plasmid pADP-1 encodes the activities involved in the hydrolytic degradation of the s-triazine herbicide atrazine. Here, we explore the presence of a specific transport system for the central intermediate of the atrazine utilization pathway, cyanuric acid, in Pseudomonas sp. ADP. Growth in fed-batch cultures containing limiting cyanuric acid concentrations is consistent with high-affinity transport of this substrate. Acquisition of the ability to grow at low cyanuric acid concentrations upon conjugal transfer of pADP1 to the nondegrading host Pseudomonas putida KT2442 suggests that all activities required for this phenotype are encoded in this plasmid. Co-expression of the pADP1-borne atzDEF and atzTUVW genes, encoding the cyanuric acid utilization pathway and the subunits of an ABC-type solute transport system, in P. putida KT2442 was sufficient to promote growth at cyanuric acid concentrations as low as 50 μM in batch culture. Taken together, our results strongly suggest that the atzTUVW gene products are involved in high-affinity transport of cyanuric acid.

  1. Pool Purification

    NASA Technical Reports Server (NTRS)

    1988-01-01

    Caribbean Clear, Inc. used NASA's silver ion technology as a basis for its automatic pool purifier. System offers alternative approach to conventional purification chemicals. Caribbean Clear's principal markets are swimming pool owners who want to eliminate chlorine and bromine. Purifiers in Caribbean Clear System are same silver ions used in Apollo System to kill bacteria, plus copper ions to kill algae. They produce spa or pool water that exceeds EPA Standards for drinking water.

  2. Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti(4+)-SPE enrichment for mass spectrometric analysis.

    PubMed

    Zhang, Ying; Peng, Ye; Bin, Zhichao; Wang, Huijie; Lu, Haojie

    2016-08-31

    N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti(4+)-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti(4+)-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified. PMID:27506354

  3. Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti(4+)-SPE enrichment for mass spectrometric analysis.

    PubMed

    Zhang, Ying; Peng, Ye; Bin, Zhichao; Wang, Huijie; Lu, Haojie

    2016-08-31

    N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti(4+)-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti(4+)-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified.

  4. 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. PMID:26739785

  5. Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation.

    PubMed

    Burdman, S; Dulguerova, G; Okon, Y; Jurkevitch, E

    2001-04-01

    The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

  6. Artificial immunoglobulin G-binding protein mimetic to staphylococcal protein A. Its production and application to affinity purification of immunoglobulin G.

    PubMed

    Kihira, Y; Aiba, S

    1992-04-24

    Staphylococcal protein A consists of a single polypeptide with five immunoglobulin G (IgG)-binding domains, which are linked as E-D-A-B-C in this order from the amino terminal. The DNA coding domains A-B were polymerized one to six times linearly, taking advantage of the non-palindromic nucleotide sequence of the AccI recognition site and the resultant DNAs were inserted in pTRP vector carrying trp promoter. The artificial IgG-binding proteins [pA(AB)1-6], which had been expressed in Escherichia coli JM109, were purified by methods involving IgG-Sepharose affinity chromatography. Among pA(AB)1-6 immobilized on cyanogen bromide-Sepharose, pA(AB)4-Sepharose was the highest in IgG-binding capacity at the same level of mg protein per ml gel, about 30% higher than protein A-Sepharose. At 8 mg protein per ml gel, it bound and eluted about 24 mg of IgG from rabbit serum. Its IgG-binding capacities were the highest with porcine, rabbit, human and guinea pig sera, intermediate with bovine, horse and sheep sera and the lowest with mouse, goat, rat and chicken sera.

  7. Final LDRD report :ultraviolet water purification systems for rural environments and mobile applications.

    SciTech Connect

    Banas, Michael Anthony; Crawford, Mary Hagerott; Ruby, Douglas Scott; Ross, Michael P.; Nelson, Jeffrey Scott; Allerman, Andrew Alan; Boucher, Ray

    2005-11-01

    We present the results of a one year LDRD program that has focused on evaluating the use of newly developed deep ultraviolet LEDs in water purification. We describe our development efforts that have produced an LED-based water exposure set-up and enumerate the advances that have been made in deep UV LED performance throughout the project. The results of E. coli inactivation with 270-295 nm LEDs are presented along with an assessment of the potential for applying deep ultraviolet LED-based water purification to mobile point-of-use applications as well as to rural and international environments where the benefits of photovoltaic-powered systems can be realized.

  8. Identification of high- and low-affinity NGF receptors during development of the chicken central nervous system

    SciTech Connect

    Escandon, E.; Chao, M.V. )

    1990-12-01

    In order to study regulation of the nerve growth factor (NGF) receptor during embryogenesis in chick brain, we have used affinity crosslinking of tissues with {sup 125}I-NGF. NGF interacts with high- and low-affinity receptors; high-affinity receptors are required for the majority of NGF's actions. Most measurements of receptor levels do not distinguish between high- and low-affinity forms of the receptor. We have used the lipophilic crosslinking agent HSAB to identify the high-affinity, functional receptor during development of the chicken central nervous system. A peak of expression during Embryonic Days 5-10 was detected in all regions of the chicken central nervous system, but, shortly after birth, only the cerebellar region displays significant levels of NGF receptor protein. The time course of expression confirms the dramatic regulation of the NGF receptor gene during defined embryonic periods. The detection of high-affinity NGF receptors in brain and neural retina provides strong evidence that NGF is involved in essential ontogenetic events in the development of the chicken central nervous system.

  9. A portable and high energy efficient desalination/purification system by ion concentration polarization

    NASA Astrophysics Data System (ADS)

    Kim, Sung Jae; Kim, Bumjoo; Kwak, Rhokyun; Kim, Geunbae; Han, Jongyoon

    2012-10-01

    The shortage of fresh water is one of the acute challenges that the world is facing now and, thus, energy efficient desalination strategies can provide substantial answers for the water-crisis. Current desalination methods utilizing reverse-osmosis and electrodialysis mechanisms required high power consumptions/large-scale infrastructures which do not make them appropriate for disaster-stricken area or underdeveloped countries. In addition, groundwater contamination by heavy metal compounds, such as arsenic, cadmium and lead, poses significant public health challenges, especially in developing countries. Existing water purification strategies for heavy metal removal are not readily applicable due to technological, environmental, and economical barriers. This presentation elucidates a novel desalination/purification process, where a continuous contaminated stream is divided into filtered and concentrated stream by the ion concentration polarization. The key distinct feature is that both salts and larger particles (cells, viruses, and microorganisms) are pushed away from the membrane, in continuous flow operations, eliminating the membrane fouling that plagues the membrane filtration methods. The power consumption is less than 5Wh/L, comparable to any existing systems. The energy and removal efficiency, and low cost manufacturability hold strong promises for portable, self-powered water purification/desalination system that can have significant impacts on water shortage in developing/rural part of the world.

  10. The Effectiveness of Home Water Purification Systems on the Amount of Fluoride in Drinking Water

    PubMed Central

    Eftekhar, Behrooz; Skini, Masoume; Shamohammadi, Milad; Ghaffaripour, Jaber; Nilchian, Firoozeh

    2015-01-01

    Statement of the Problem Water purification systems for domestic use have drawn significant attention over the past few years. This can be related to the improvement of public health and concern for water contamination.  Purpose The aim of this study was to evaluate whether home water purification systems eliminate the essential materials such as fluoride besides filtrating the heavy ions and other unwanted particles out of water. Materials and Method In this experimental study, six most frequently used commercial brands of water purifiers were evaluated and compared. Specimens were collected right before and after setting up the device, and 6 months later. Then, spectrophotometry (the Harrison device) was performed to compare fluoride clearance by each home water cleaner device. Results Based on the data collected from all water purification devices in different locations, the amount of fluoride was significantly different before and right after using home water purifier and six months later (p= 0.001 and p= 0.00, respectively). Conclusion The filtration of water significantly decreased its fluoride concentration. The fluoride content of purified water was approximately as much as zero in some cases. PMID:26535409

  11. Adaptive neural network control of unknown nonlinear affine systems with input deadzone and output constraint.

    PubMed

    He, Wei; Dong, Yiting; Sun, Changyin

    2015-09-01

    In this paper, we aim to solve the control problem of nonlinear affine systems, under the condition of the input deadzone and output constraint with the external unknown disturbance. To eliminate the effects of the input deadzone, a Radial Basis Function Neural Network (RBFNN) is introduced to compensate for the negative impact of input deadzone. Meanwhile, we design a barrier Lyapunov function to ensure that the output parameters are restricted. In support of the barrier Lyapunov method, we build an adaptive neural network controller based on state feedback and output feedback methods. The stability of the closed-loop system is proven via the Lyapunov method and the performance of the expected effects is verified in simulation.

  12. Identification and affinity of very high affinity binding sites for the phenylalkylamine series of Ca/sup +/ channel blockers in the Drosophila nervous system

    SciTech Connect

    Pauron, D.; Qar, J.; Barhanin, J.; Fournier, D.; Cuany, A.; Pralavorio, M.; Berge, J.B.; Lazdunski, M.

    1987-10-06

    The interaction of putative Ca/sup 2 +/ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-(/sup 3/H)D888 and (+/-)-(/sup 3/H)verapamil. These ligands recognize a single class of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a K/sub d/ value as exceptional low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca/sup 2 +/ channel blockers as well as bepridil inhibited (-)-(/sup 3/H)D888 binding in a competitive way with K/sub d/ values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor was used in photoaffinity experiments. A protein of M/sub r/ 135,000 +/- 5000 was specifically labeled after ultraviolet irradiation.

  13. Biofilm bacterial communities in urban drinking water distribution systems transporting waters with different purification strategies.

    PubMed

    Wu, Huiting; Zhang, Jingxu; Mi, Zilong; Xie, Shuguang; Chen, Chao; Zhang, Xiaojian

    2015-02-01

    Biofilm formation in drinking water distribution systems (DWDS) has many adverse consequences. Knowledge of microbial community structure of DWDS biofilm can aid in the design of an effective control strategy. However, biofilm bacterial community in real DWDS and the impact of drinking water purification strategy remain unclear. The present study investigated the composition and diversity of biofilm bacterial community in real DWDSs transporting waters with different purification strategies (conventional treatment and integrated treatment). High-throughput Illumina MiSeq sequencing analysis illustrated a large shift in the diversity and structure of biofilm bacterial community in real DWDS. Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Nitrospirae, and Cyanobacteria were the major components of biofilm bacterial community. Proteobacteria (mainly Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) predominated in each DWDS biofilm, but the compositions of the dominant proteobacterial classes and genera and their proportions varied among biofilm samples. Drinking water purification strategy could shape DWDS biofilm bacterial community. Moreover, Pearson's correlation analysis indicated that Actinobacteria was positively correlated with the levels of total alkalinity and dissolved organic carbon in tap water, while Firmicutes had a significant positive correlation with nitrite nitrogen.

  14. Expression and purification of recombinant antibody formats and antibody fusion proteins.

    PubMed

    Siegemund, Martin; Richter, Fabian; Seifert, Oliver; Unverdorben, Felix; Kontermann, Roland E

    2014-01-01

    In the laboratory-scale production of antibody fragments or antibody fusion proteins, it is often difficult to keep track on the most suitable affinity tags for protein purification from either prokaryotic or eukaryotic host systems. Here, we describe how such recombinant proteins derived from Escherichia coli lysates as well as HEK293 cell culture supernatants are purified by IMAC and by different affinity chromatography methods based on fusions to FLAG-tag, Strep-tag, and Fc domains. PMID:24515473

  15. Carbon Dioxide Collection and Purification System for Mars

    NASA Technical Reports Server (NTRS)

    Clark, D. Larry; Trevathan, Joseph R.

    2001-01-01

    One of the most abundant resources available on Mars is the atmosphere. The primary constituent, carbon dioxide, can be used to produce a wide variety of consumables including propellants and breathing air. The residual gases can be used for additional pressurization tasks including supplementing the oxygen partial pressure in human habitats. A system is presented that supplies pure, high-pressure carbon dioxide and a separate stream of residual gases ready for further processing. This power-efficient method freezes the carbon dioxide directly from the atmosphere using a pulse-tube cryocooler. The resulting CO2 mass is later thawed in a closed pressure vessel, resulting in a compact source of liquefied gas at the vapor pressure of the bulk fluid. Results from a demonstration system are presented along with analysis and system scaling factors for implementation at larger scales. Trace gases in the Martian atmosphere challenge the system designer for all carbon dioxide acquisitions concepts. The approximately five percent of other gases build up as local concentrations of CO2 are removed, resulting in diminished performance of the collection process. The presented system takes advantage of this fact and draws the concentrated residual gases away as a useful byproduct. The presented system represents an excelient volume and mass solution for collecting and compressing this valuable Martian resource. Recent advances in pulse-tube cryocooler technology have enabled this concept to be realized in a reliable, low power implementation.

  16. Distribution of bacteria within operating laboratory water purification systems.

    PubMed Central

    McFeters, G A; Broadaway, S C; Pyle, B H; Egozy, Y

    1993-01-01

    Experiments were conducted to measure communities of bacteria within operating ultrapure water treatment systems intended for laboratory use. Samples from various locations within Milli-Q Plus and Milli-Q UV Plus systems were analyzed for populations of planktonic bacteria at weekly intervals over 3 months of operation. Relatively high initial densities of planktonic bacteria (10(2) to 10(3) bacteria per ml) were seen within both units when they were challenged with source water of poor quality, although the product water continued to be acceptable with regard to bacterial numbers, resistivity, and endotoxin concentration. Under more normal operating conditions, significant differences were seen in planktonic populations throughout the systems with excellent product water quality. A great deal of variability was observed in biofilm populations analyzed from various system surfaces after 3 months of operation. The concentrations of planktonic bacteria and biofilm densities were much lower in the unit containing a UV lamp. These findings suggest that a range of microenvironmental conditions exist within purified water systems, leading to variable populations of bacteria. However, product water of excellent quality was obtained despite the bacterial communities. PMID:8517737

  17. Reinforcement learning controller design for affine nonlinear discrete-time systems using online approximators.

    PubMed

    Yang, Qinmin; Jagannathan, Sarangapani

    2012-04-01

    In this paper, reinforcement learning state- and output-feedback-based adaptive critic controller designs are proposed by using the online approximators (OLAs) for a general multi-input and multioutput affine unknown nonlinear discretetime systems in the presence of bounded disturbances. The proposed controller design has two entities, an action network that is designed to produce optimal signal and a critic network that evaluates the performance of the action network. The critic estimates the cost-to-go function which is tuned online using recursive equations derived from heuristic dynamic programming. Here, neural networks (NNs) are used both for the action and critic whereas any OLAs, such as radial basis functions, splines, fuzzy logic, etc., can be utilized. For the output-feedback counterpart, an additional NN is designated as the observer to estimate the unavailable system states, and thus, separation principle is not required. The NN weight tuning laws for the controller schemes are also derived while ensuring uniform ultimate boundedness of the closed-loop system using Lyapunov theory. Finally, the effectiveness of the two controllers is tested in simulation on a pendulum balancing system and a two-link robotic arm system.

  18. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  19. Polonium purification

    SciTech Connect

    Baker, J.D.

    1996-09-01

    Three processes for the purification of {sup 210}Po from irradiated bismuth targets are described. Safety equipment includes shielded hotcells for the initial separation from other activation products, gloveboxes for handling the volatile and highly toxic materials, and provisions for ventilation. All chemical separations must be performed under vacuum or in inerted systems. Two of the processes require large amounts of electricity; the third requires vessels made from exotic materials.

  20. Preparation of λN-GST fusion protein for affinity immobilization of RNA.

    PubMed

    Di Tomasso, Geneviève; Lampron, Philipe; Omichinski, James G; Legault, Pascale

    2012-01-01

    Affinity purification of in vitro transcribed RNA is becoming an attractive alternative to purification using standard denaturing gel electrophoresis. Affinity purification is particularly advantageous because it can be performed in a few hours under non-denaturing conditions. However, the performance of affinity purification methods can vary tremendously depending on the RNA immobilization matrix. It was previously shown that RNA immobilization via an optimized λN-GST fusion protein bound to glutathione-Sepharose resin allows affinity purification of RNA with very high purity and yield. This Chapter outlines the experimental procedure employed to prepare the λN-GST fusion protein used for RNA immobilization in successful affinity purifications of RNA. It describes the details of protein expression and purification as well as routine quality control analyses. PMID:23065558

  1. Membrane-based systems for carbon capture and hydrogen purification

    SciTech Connect

    Berchtold, Kathryn A

    2010-11-24

    This presentation describes the activities being conducted at Los Alamos National Laboratory to develop carbon capture technologies for power systems. This work is aimed at continued development and demonstration of a membrane based pre- and post-combustion carbon capture technology and separation schemes. Our primary work entails the development and demonstration of an innovative membrane technology for pre-combustion capture of carbon dioxide that operates over a broad range of conditions relevant to the power industry while meeting the US DOE's Carbon Sequestration Program goals of 90% CO{sub 2} capture at less than a 10% increase in the cost of energy services. Separating and capturing carbon dioxide from mixed gas streams is a first and critical step in carbon sequestration. To be technically and economically viable, a successful separation method must be applicable to industrially relevant gas streams at realistic temperatures and pressures as well as be compatible with large gas volumes. Our project team is developing polymer membranes based on polybenzimidazole (PBI) chemistries that can purify hydrogen and capture CO{sub 2} at industrially relevant temperatures. Our primary objectives are to develop and demonstrate polymer-based membrane chemistries, structures, deployment platforms, and sealing technologies that achieve the critical combination of high selectivity, high permeability, chemical stability, and mechanical stability all at elevated temperatures (> 150 C) and packaged in a scalable, economically viable, high area density system amenable to incorporation into an advanced Integrated Gasification Combined-Cycle (IGCC) plant for pre-combustion CO{sub 2} capture. Stability requirements are focused on tolerance to the primary synthesis gas components and impurities at various locations in the IGCC process. Since the process stream compositions and conditions (temperature and pressure) vary throughout the IGCC process, the project is focused on the

  2. Integral reinforcement learning for continuous-time input-affine nonlinear systems with simultaneous invariant explorations.

    PubMed

    Lee, Jae Young; Park, Jin Bae; Choi, Yoon Ho

    2015-05-01

    This paper focuses on a class of reinforcement learning (RL) algorithms, named integral RL (I-RL), that solve continuous-time (CT) nonlinear optimal control problems with input-affine system dynamics. First, we extend the concepts of exploration, integral temporal difference, and invariant admissibility to the target CT nonlinear system that is governed by a control policy plus a probing signal called an exploration. Then, we show input-to-state stability (ISS) and invariant admissibility of the closed-loop systems with the policies generated by integral policy iteration (I-PI) or invariantly admissible PI (IA-PI) method. Based on these, three online I-RL algorithms named explorized I-PI and integral Q -learning I, II are proposed, all of which generate the same convergent sequences as I-PI and IA-PI under the required excitation condition on the exploration. All the proposed methods are partially or completely model free, and can simultaneously explore the state space in a stable manner during the online learning processes. ISS, invariant admissibility, and convergence properties of the proposed methods are also investigated, and related with these, we show the design principles of the exploration for safe learning. Neural-network-based implementation methods for the proposed schemes are also presented in this paper. Finally, several numerical simulations are carried out to verify the effectiveness of the proposed methods.

  3. Enrichment of circulating tumor cells using a centrifugal affinity plate system.

    PubMed

    Lee, Sung-Woo; Hyun, Kyung-A; Kim, Seung-Il; Kang, Ji-Yoon; Jung, Hyo-Il

    2014-12-19

    Circulating tumor cells (CTCs) are defined as cells that have detached from a primary tumor and are circulating in the bloodstream. Their isolation and quantification is of great value for cancer prognoses and drug testing. Here, the development of a centrifugal affinity plate (CAP) system is described, in which centrifugal force and antibody-based capture are exploited to enrich CTCs on one plate and hematological cells on the other. The CAP is rotated to exert centrifugal force on the cells in a blood sample, quickly transporting them to the anti-epithelial adhesion molecule (EpCAM)-coated and anti-CD45-coated surface of the CAP to shorten the reaction time and increase the adhesion force between the tumor and blood cells and each antibody. The effect of a rotating process on cell capture was investigated, and the capture efficiency was demonstrated using blood samples from healthy donors spiked with human non-small cell lung cancer (NCI-H1650) and breast cancer (MCF-7) cells. The CAP system was capable of rapid isolation and identification of CTCs without the requirement for pretreatment of blood samples. Finally, the CAP system was tested to evaluate the detection efficiency of CTCs in the blood samples of breast cancer patients. The number of captured CTCs in only 1ml of blood varied from 6 to 10.

  4. Purification and crystallization of components of the protein-synthesizing system from Thermus thermophilus

    NASA Astrophysics Data System (ADS)

    Garber, M. B.; Agalarov, S. Ch.; Eliseikina, I. A.; Sedelnikova, S. E.; Tishchenko, S. V.; Shirokov, V. A.; Yusupov, M. M.; Reshetnikova, L. S.; Trakhanov, S. D.; Tukalo, M. A.; Yaremchuk, A. D.

    1991-03-01

    An extreme thermophilic bacterium Thermus thermophilus has been chosen as a source for the isolation of components of the protein-synthesizing system to investigate their structures by X-ray crystallographic methods. The scheme of simultaneous isolation of ribosomes, tRNA, three elongation factors, several aminoacyl-tRNA synthetases and several enzymes has been developed. Methods of purification of ribosomes and individual ribosomal proteins without denaturation were elaborated. Crystals of the elongation factor G, the 70S ribosome, the 30S ribosomal subunit, six ribosomal proteins and three aminoacyl-tRNA synthetases have been obtained. Structural investigations of EF-G and the 70S ribosome are underway.

  5. Systematic development and validation of sanitization protocols for a chromatographic system designed for biotherapeutics purification.

    PubMed

    Burgoyne, R F; Priest, M C; Roche, K L; Vella, G

    1993-01-01

    Production of protein therapeutics through the application of genetic engineering and biotechnology techniques requires comprehensive attention to good manufacturing practice and good laboratory practice (GMP/GLP) guidelines for product recovery and purification. Validated clean-in-place procedures are part of the master method and require analysis of microbial bioburden to assess the efficacy of cleaning protocols. This article describes the extensive microbial challenge of a chromatography system, the use of membrane filtration methods for high sensitivity microbial contamination measurement, and the effectiveness of sodium hydroxide and ethanol solutions in achieving multilog reduction of microbial contamination. PMID:8123748

  6. Novel lipase purification methods - a review of the latest developments.

    PubMed

    Tan, Chung Hong; Show, Pau Loke; Ooi, Chien Wei; Ng, Eng-Poh; Lan, John Chi-Wei; Ling, Tau Chuan

    2015-01-01

    Microbial lipases are popular biocatalysts due to their ability to catalyse diverse reactions such as hydrolysis, esterification, and acidolysis. Lipases function efficiently on various substrates in aqueous and non-aqueous media. Lipases are chemo-, regio-, and enantio-specific, and are useful in various industries, including those manufacturing food, detergents, and pharmaceuticals. A large number of lipases from fungal and bacterial sources have been isolated and purified to homogeneity. This success is attributed to the development of both conventional and novel purification techniques. This review highlights the use of these techniques in lipase purification, including conventional techniques such as: (i) ammonium sulphate fractionation; (ii) ion-exchange; (iii) gel filtration and affinity chromatography; as well as novel techniques such as (iv) reverse micellar system; (v) membrane processes; (vi) immunopurification; (vi) aqueous two-phase system; and (vii) aqueous two-phase floatation. A summary of the purification schemes for various bacterial and fungal lipases are also provided. PMID:25273633

  7. Hydrogen Purification and Recycling for an Integrated Oxygen Recovery System Architecture

    NASA Technical Reports Server (NTRS)

    Abney, Morgan B.; Greenwood, Zachary; Wall, Terry; Nur, Mononita; Wheeler, Richard R., Jr.; Preston, Joshua; Molter, Trent

    2016-01-01

    The United States Atmosphere Revitalization life support system on the International Space Station (ISS) performs several services for the crew including oxygen generation, trace contaminant control, carbon dioxide (CO2) removal, and oxygen recovery. Oxygen recovery is performed using a Sabatier reactor developed by Hamilton Sundstrand, wherein CO2 is reduced with hydrogen in a catalytic reactor to produce methane and water. The water product is purified in the Water Purification Assembly and recycled to the Oxygen Generation Assembly (OGA) to provide O2 to the crew. This architecture results in a theoretical maximum oxygen recovery from CO2 of approx.54% due to the loss of reactant hydrogen in Sabatier-produced methane that is currently vented outside of ISS. Plasma Pyrolysis Assembly (PPA) technology, developed by Umpqua Research Company, provides the capability to further close the Atmosphere Revitalization oxygen loop by recovering hydrogen from Sabatier-produced methane. A key aspect of this technology approach is the need to purify the hydrogen from the PPA product stream which includes acetylene, unreacted methane and byproduct water and carbon monoxide. In 2015, four sub-scale hydrogen separation systems were delivered to NASA for evaluation. These included two electrolysis single-cell hydrogen purification cell stacks developed by Sustainable Innovations, LLC, a sorbent-based hydrogen purification unit using microwave power for sorbent regeneration developed by Umpqua Research Company, and a LaNi4.6Sn0.4 metal hydride produced by Hydrogen Consultants, Inc. Here we report the results of these evaluations to-date, discuss potential architecture options, and propose future work.

  8. Hydrogen Purification and Recycling for an Integrated Oxygen Recovery System Architecture

    NASA Technical Reports Server (NTRS)

    Abney, Morgan B.; Greenwood, Zachary; Wall, Terry; Miller, Lee; Wheeler, Ray

    2016-01-01

    The United States Atmosphere Revitalization life support system on the International Space Station (ISS) performs several services for the crew including oxygen generation, trace contaminant control, carbon dioxide (CO2) removal, and oxygen recovery. Oxygen recovery is performed using a Sabatier reactor developed by Hamilton Sundstrand, wherein CO2 is reduced with hydrogen in a catalytic reactor to produce methane and water. The water product is purified in the Water Purification Assembly and recycled to the Oxygen Generation Assembly (OGA) to provide O2 to the crew. This architecture results in a theoretical maximum oxygen recovery from CO2 of approximately 54% due to the loss of reactant hydrogen in Sabatier-produced methane that is currently vented outside of ISS. Plasma Methane Pyrolysis technology (PPA), developed by Umpqua Research Company, provides the capability to further close the Atmosphere Revitalization oxygen loop by recovering hydrogen from Sabatier-produced methane. A key aspect of this technology approach is to purify the hydrogen from the PPA product stream which includes acetylene, unreacted methane and byproduct water and carbon monoxide. In 2015, four sub-scale hydrogen separation systems were delivered to NASA for evaluation. These included two electrolysis single-cell hydrogen purification cell stacks developed by Sustainable Innovations, LLC, a sorbent-based hydrogen purification unit using microwave power for sorbent regeneration developed by Umpqua Research Company, and a LaNi4.6Sn0.4 metal hydride produced by Hydrogen Consultants, Inc. Here we report the results of these evaluations, discuss potential architecture options, and propose future work.

  9. Water Purification

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Silver ionization water purification technology was originally developed for Apollo spacecraft. It was later used to cleanse swimming pools and has now been applied to industrial cooling towers and process coolers. Sensible Technologies, Inc. has added two other technologies to the system, which occupies only six square feet. It is manufactured in three capacities, and larger models are custom built on request. The system eliminates scale, corrosion, algae, bacteria and debris, and because of the NASA technology, viruses and waterborne bacteria are also destroyed. Applications include a General Motors cooling tower, amusement parks, ice manufacture and a closed-loop process cooling system.

  10. Regulation of a high-affinity diamine transport system in Trypanosoma cruzi epimastigotes.

    PubMed Central

    Le Quesne, S A; Fairlamb, A H

    1996-01-01

    Trypanosoma cruzi epimastigotes take up exogenous [3H]putrescine and [3H]cadaverine by a rapid, high-affinity, transport system that exhibits saturable kinetics (putrescine K(m) 2.0 microM, V(max) 3.3 nmol/min per 10(8) cells; cadaverine K(m) 13.4 microM, V(max) 3.9 nmol/min per 10(8) cells). Putrescine transport is temperature dependent and requires the presence of a membrane potential and thiol groups for activity. Its activity is altered in response to extracellular putrescine levels and as the cells proceed through the growth cycle. This transporter shows high specificity for the diamines putrescine and cadaverine, but low specificity for the polyamines spermidine and spermine. The existence of rapid diamine/polyamine transport systems whose activity can be adjusted in response to the growth conditions is of particular importance, as they seem unable to synthesize their own putrescine [Hunter, Le Quesne and Fairlamb (1994) Eur. J. Biochem. 226, 1019-1027]. PMID:8687391

  11. Gγ recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets.

    PubMed

    Kaishima, Misato; Ishii, Jun; Fukuda, Nobuo; Kondo, Akihiko

    2015-11-19

    Protein-protein interactions (PPIs) are crucial for the vast majority of biological processes. We previously constructed a Gγ recruitment system to screen PPI candidate proteins and desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. The methods utilized a target protein fused to a mutated G-protein γ subunit (Gγcyto) lacking the ability to localize to the inner leaflet of the plasma membrane. However, the previous systems were adapted to use only soluble cytosolic proteins as targets. Recently, membrane proteins have been found to form the principal nodes of signaling involved in diseases and have attracted a great deal of interest as primary drug targets. Here, we describe new protocols for the Gγ recruitment systems that are specifically designed to use membrane proteins as targets to overcome previous limitations. These systems represent an attractive approach to exploring novel interacting candidates and affinity-altered protein variants and their interactions with proteins on the inner side of the plasma membrane, with high specificity and selectivity.

  12. Gγ recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets

    PubMed Central

    Kaishima, Misato; Ishii, Jun; Fukuda, Nobuo; Kondo, Akihiko

    2015-01-01

    Protein-protein interactions (PPIs) are crucial for the vast majority of biological processes. We previously constructed a Gγ recruitment system to screen PPI candidate proteins and desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. The methods utilized a target protein fused to a mutated G-protein γ subunit (Gγcyto) lacking the ability to localize to the inner leaflet of the plasma membrane. However, the previous systems were adapted to use only soluble cytosolic proteins as targets. Recently, membrane proteins have been found to form the principal nodes of signaling involved in diseases and have attracted a great deal of interest as primary drug targets. Here, we describe new protocols for the Gγ recruitment systems that are specifically designed to use membrane proteins as targets to overcome previous limitations. These systems represent an attractive approach to exploring novel interacting candidates and affinity-altered protein variants and their interactions with proteins on the inner side of the plasma membrane, with high specificity and selectivity. PMID:26581329

  13. Purification and characterisation of immunoglobulins from the Australian black flying fox (Pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of IgA.

    PubMed

    Wynne, James W; Di Rubbo, Antonio; Shiell, Brian J; Beddome, Gary; Cowled, Christopher; Peck, Grantley R; Huang, Jing; Grimley, Samantha L; Baker, Michelle L; Michalski, Wojtek P

    2013-01-01

    There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.

  14. Purification and Characterisation of Immunoglobulins from the Australian Black Flying Fox (Pteropus alecto) Using Anti-Fab Affinity Chromatography Reveals the Low Abundance of IgA

    PubMed Central

    Shiell, Brian J.; Beddome, Gary; Cowled, Christopher; Peck, Grantley R.; Huang, Jing; Grimley, Samantha L.; Baker, Michelle L.; Michalski, Wojtek P.

    2013-01-01

    There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host. PMID:23308125

  15. Purification and characterisation of immunoglobulins from the Australian black flying fox (Pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of IgA.

    PubMed

    Wynne, James W; Di Rubbo, Antonio; Shiell, Brian J; Beddome, Gary; Cowled, Christopher; Peck, Grantley R; Huang, Jing; Grimley, Samantha L; Baker, Michelle L; Michalski, Wojtek P

    2013-01-01

    There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host. PMID:23308125

  16. Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing

    NASA Astrophysics Data System (ADS)

    Lane, Rebecca E.; Korbie, Darren; Anderson, Will; Vaidyanathan, Ramanathan; Trau, Matt

    2015-01-01

    Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.

  17. Synthesis of new bivalent peptides for applications in the Affinity Enhancement System.

    PubMed

    Morandeau, L; Benoist, E; Loussouarn, A; Ouadi, A; Lesaec, P; Mougin, M; Faivre-Chauvet, A; Le Boterff, J; Chatal, J F; Barbet, J; Gestin, J F

    2005-01-01

    The feasibility of two-step radioimmunotherapy (RIT) of cancer by the Affinity Enhancement System (AES) has been demonstrated in experimental and clinical studies. This technique, associating a bispecific antibody and a bivalent peptide radiolabeled with iodine-131, has been developed to reduce toxicity and to improve therapeutic efficacy compared to one-step targeting methods. The use of AES with different beta-emitters such as rhenium-188, samarium-153, or lutetium-177 or alpha-emitters such as actinium-225 or bismuth-213 is now considered. Thus three new peptides, designed to allow for the coupling of a variety of bifunctional chelating agents BCA, were synthesized by associating two glycyl-succinyl-histamine (GSH) arms, which are recognized by the 679 monoclonal antibody (mAb-679), with different binding agents, such as p-nitrophenylalanine or N,N-bis(carboxymethyl)-4-N'-(9-fluorenylmethyloxycarbonyl)aminobenzylamine. Immunoreactivity and serum stability evaluation were performed for each synthesized peptide. One of the three peptides (LM218) proved to be more stable than the others, and three different BCAs were coupled to LM218 (CITC-DTPA, CITC-TTHA, and CITC-CHXA''DTPA). One of these products, LM218-BzTTHA was radiolabeled with indium-111 without loss of immunoreactivity toward the mAb-679. These new peptides will allow pretargeted RIT with a large variety of radionuclides, to adapt the choice of the radionuclide (LET, half-life, penetrating emission) to the nature and size of targeted tumors. PMID:15656590

  18. "Clickable" agarose for affinity chromatography.

    PubMed

    Punna, Sreenivas; Kaltgrad, Eiton; Finn, M G

    2005-01-01

    Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.

  19. Hyperentanglement purification for two-photon six-qubit quantum systems

    NASA Astrophysics Data System (ADS)

    Wang, Guan-Yu; Liu, Qian; Deng, Fu-Guo

    2016-09-01

    Recently, two-photon six-qubit hyperentangled states were produced in experiment and they can improve the channel capacity of quantum communication largely. Here we present a scheme for the hyperentanglement purification of nonlocal two-photon systems in three degrees of freedom (DOFs), including the polarization, the first-longitudinal-momentum, and the second-longitudinal-momentum DOFs. Our hyperentanglement purification protocol (hyper-EPP) is constructed with two steps resorting to parity-check quantum nondemolition measurement on the three DOFs and swap gates, respectively. With these two steps, the bit-flip errors in the three DOFs can be corrected efficiently. Also, we show that using swap gates is a universal method for hyper-EPP in the polarization DOF and multiple-longitudinal-momentum DOFs. The implementation of our hyper-EPP is assisted by nitrogen-vacancy centers in optical microcavities, which could be achieved with current techniques. It is useful for long-distance high-capacity quantum communication with two-photon six-qubit hyperentanglement.

  20. A biosensor-based approach toward purification and crystallization of G protein-coupled receptors.

    PubMed

    Navratilova, Iva; Pancera, Marie; Wyatt, Richard T; Myszka, David G

    2006-06-15

    Biacore technology was used to develop an affinity purification method and screen cocrystallization conditions for the chemokine receptor CCR5. We characterized the binding of nine HIV gp120 variants and identified a truncated construct (YU2DV1V2) that bound CCR5 independent of CD4. This construct was used in an affinity purification step to improve the activity of detergent-solubilized receptor by approximately 300%. The biosensor was also used to screen receptor binding activity automatically under 50 different crystallization conditions. We found that high-molecular-weight polyethylene glycols (PEGs 4,000 and 8,000 Da) most often stabilized the receptor and improved complex formation with potential cocrystallization partners such as conformationally sensitive monoclonal antibodies and gp120. Our results show how biosensors can provide unique insights into receptor purification methods and reveal the effects of crystallization conditions on complex formation. Importantly, these methods can be readily applied to other systems.

  1. Zero-sum two-player game theoretic formulation of affine nonlinear discrete-time systems using neural networks.

    PubMed

    Mehraeen, Shahab; Dierks, Travis; Jagannathan, S; Crow, Mariesa L

    2013-12-01

    In this paper, the nearly optimal solution for discrete-time (DT) affine nonlinear control systems in the presence of partially unknown internal system dynamics and disturbances is considered. The approach is based on successive approximate solution of the Hamilton-Jacobi-Isaacs (HJI) equation, which appears in optimal control. Successive approximation approach for updating control and disturbance inputs for DT nonlinear affine systems are proposed. Moreover, sufficient conditions for the convergence of the approximate HJI solution to the saddle point are derived, and an iterative approach to approximate the HJI equation using a neural network (NN) is presented. Then, the requirement of full knowledge of the internal dynamics of the nonlinear DT system is relaxed by using a second NN online approximator. The result is a closed-loop optimal NN controller via offline learning. A numerical example is provided illustrating the effectiveness of the approach. PMID:24273142

  2. Thermal-destruction products of coal in the blast-furnace gas-purification system

    SciTech Connect

    A.M. Amdur; M.V. Shibanova; E.V. Ental'tsev

    2008-10-15

    The lean, poorly clinkering coal and anthracite used to replace coke in blast furnaces has a considerable content of volatile components (low-molecular thermaldestruction products), which enter the water and sludge of the blast-furnace gas-purification system as petroleum products. Therefore, it is important to study the influence of coal on the petroleum-product content in the water and sludge within this system. The liberation of primary thermal-destruction products is investigated for anthracite with around 4 wt % volatiles, using a STA 449C Jupiter thermoanalyzer equipped with a QMC 230 mass spectrometer. The thermoanalyzer determines small changes in mass and thermal effects with high accuracy (weighing accuracy 10{sup -8} g; error in measuring thermal effects 1 mV). This permits experiments with single layers of coal particles, eliminating secondary reactions of its thermal-destruction products.

  3. Ribonucleic acid purification.

    PubMed

    Martins, R; Queiroz, J A; Sousa, F

    2014-08-15

    Research on RNA has led to many important biological discoveries and improvement of therapeutic technologies. From basic to applied research, many procedures employ pure and intact RNA molecules; however their isolation and purification are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality. The current techniques to isolate and purify RNA molecules still have several limitations and the requirement for new methods able to improve RNA quality to meet regulatory demands is growing. In fact, as basic research improves the understanding of biological roles of RNAs, the biopharmaceutical industry starts to focus on them as a biotherapeutic tools. Chromatographic bioseparation is a high selective unit operation and is the major option in the purification of biological compounds, requiring high purity degree. In addition, its application in biopharmaceutical manufacturing is well established. This paper discusses the importance and the progress of RNA isolation and purification, considering RNA applicability both in research and clinical fields. In particular and in view of the high specificity, affinity chromatography has been recently applied to RNA purification processes. Accordingly, recent chromatographic investigations based on biorecognition phenomena occurring between RNA and amino acids are focused. Histidine and arginine have been used as amino acid ligands, and their ability to isolate different RNA species demonstrated a multipurpose applicability in molecular biology analysis and RNA therapeutics preparation, highlighting the potential contribution of these methods to overcome the challenges of RNA purification. PMID:24951289

  4. Interval-type and affine arithmetic-type techniques for handling uncertainty in expert systems

    NASA Astrophysics Data System (ADS)

    Ceberio, Martine; Kreinovich, Vladik; Chopra, Sanjeev; Longpre, Luc; Nguyen, Hung T.; Ludascher, Bertram; Baral, Chitta

    2007-02-01

    Expert knowledge consists of statements Sj (facts and rules). The facts and rules are often only true with some probability. For example, if we are interested in oil, we should look at seismic data. If in 90% of the cases, the seismic data were indeed helpful in locating oil, then we can say that if we are interested in oil, then with probability 90% it is helpful to look at the seismic data. In more formal terms, we can say that the implication "if oil then seismic" holds with probability 90%. Another example: a bank A trusts a client B, so if we trust the bank A, we should trust B too; if statistically this trust was justified in 99% of the cases, we can conclude that the corresponding implication holds with probability 99%. If a query Q is deducible from facts and rules, what is the resulting probability p(Q) in Q? We can describe the truth of Q as a propositional formula F in terms of Sj, i.e., as a combination of statements Sj linked by operators like &, [logical or], and [not sign]; computing p(Q) exactly is NP-hard, so heuristics are needed. Traditionally, expert systems use technique similar to straightforward interval computations: we parse F and replace each computation step with corresponding probability operation. Problem: at each step, we ignore the dependence between the intermediate results Fj; hence intervals are too wide. Example: the estimate for P(A[logical or][not sign]A) is not 1. Solution: similar to affine arithmetic, besides P(Fj), we also compute P(Fj&Fi) (or P(Fj1&...&Fjd)), and on each step, use all combinations of l such probabilities to get new estimates. Results: e.g., P(A[logical or][not sign]A) is estimated as 1.

  5. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  6. Development of concept for concurrent biocide generation and water system purification. [with application to Skylab water tanks

    NASA Technical Reports Server (NTRS)

    1974-01-01

    An attempt was made to construct an electrochemical system, using iodine, for water purification in Skylab. Data cover measurements of iodine production rates, effect of electrode size and geometry on iodine production rates, and feasibility of using stainless steels as reference electrodes.

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

    PubMed Central

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

    2014-01-01

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

  8. Generation of dimethylnitrosamine in water purification systems. Detection in human blood samples during hemodialysis.

    PubMed

    Simenhoff, M L; Dunn, S R; Fiddler, W; Pensabene, J W; Smiley, J

    1983-10-21

    Dimethylnitrosamine (DMNA), a carcinogen, was detected at levels up to 32 micrograms/L in dialysate from five of 16 dialysis units surveyed. Blood drawn from patients at one of these units in which DMNA was raised in the dialysate showed a significant increase in the amount of DMNA in the patient's blood when predialysis levels were compared with 15-minute intradialysis levels. The presence of a mixed-bed deionizer without an antecedent carbon filter appeared to be necessary for DMNA production. These data suggest that DMNA is generated in certain water purification systems and may then diffuse into the patient's blood. Guidelines for deionizer-treated water should be revised to include an activated carbon filter.

  9. NO.sub.x catalyst and method of suppressing sulfate formation in an exhaust purification system

    DOEpatents

    Balmer-Millar, Mari Lou; Park, Paul W.; Panov, Alexander G.

    2007-06-26

    The activity and durability of a zeolite lean-burn NOx catalyst can be increased by loading metal cations on the outer surface of the zeolite. However, the metal loadings can also oxidize sulfur dioxide to cause sulfate formation in the exhaust. The present invention is a method of suppressing sulfate formation in an exhaust purification system including a NO.sub.x catalyst. The NO.sub.x catalyst includes a zeolite loaded with at least one metal. The metal is selected from among an alkali metal, an alkaline earth metal, a lanthanide metal, a noble metal, and a transition metal. In order to suppress sulfate formation, at least a portion of the loaded metal is complexed with at least one of sulfate, phosphate, and carbonate.

  10. NO.sub.x catalyst and method of suppressing sulfate formation in an exhaust purification system

    DOEpatents

    Balmer-Millar, Mari Lou; Park, Paul W.; Panov, Alexander G.

    2006-08-22

    The activity and durability of a zeolite lean-bum NOx catalyst can be increased by loading metal cations on the outer surface of the zeolite. However, the metal loadings can also oxidize sulfur dioxide to cause sulfate formation in the exhaust. The present invention is a method of suppressing sulfate formation in an exhaust purification system including a NO.sub.x catalyst. The NO.sub.x catalyst includes a zeolite loaded with at least one metal. The metal is selected from among an alkali metal, an alkaline earth metal, a lanthanide metal, a noble metal, and a transition metal. In order to suppress sulfate formation, at least a portion of the loaded metal is complexed with at least one of sulfate, phosphate, and carbonate.

  11. [Selection of winter plant species for wetlands constructed as sewage treatment systems and evaluation of their wastewater purification potentials].

    PubMed

    Chen, Yong-hua; Wu, Xiao-fu; Chen, Ming-li; Jiang, Li-juan; Li, Ke-lin; Lei, Dian; Wang, Hai-bin

    2010-08-01

    In order to establish an evaluation system for selection of winter wetland plants possessing high wastewater purification potentials in subtropics areas, designed sewage treatment experiments were carried out by introducing into the constructed wetlands 25 species of winter wetland plants. Cluster analysis was performed by including harmful environment-resistant enzyme and substrate enzyme activities into the commonly applied plant screening and assessment indexes system. The obtained results indicated that there were significant differences among the tested winter plants in their root length and vigor, leaf malonaldehyde (MDA), biomass, average nitrogen and phosphorus concentration and uptake, and urease and phosphoric acid enzyme activities in the root areas. Based on the established evaluation system, the tested plants were clustered into 3 groups. The plants in the 1st group possessing high purification potentials are Oenanthe javanica, Brassicacapestris, Juncus effusu, Saxifragaceae, Iris pseudoacorus, Osmanthus fragrans and Iris ensata; those in the 2nd group possessing moderate purification potentials are Brassica oleracea var acephala, Calendula officinalis, Aucuba japonica, Ligustrum lucidu, Beta vulgaris, Rhododendron simsii and Ilex latifolia; and those in the 3rd group with low purification potentials are Brassica oleracea var acephala, Calistephus chinensis, Rosa chinensis, Antirrhinums, Liriope palatyphylla, Zephyranthes candida, Fatshedera lizei, Petunia hybrida, Ilex quihoui, Dianthus caryophyllus and Loropetalum chinensis.

  12. Improving the extraction and purification of immunoglobulin G by the use of ionic liquids as adjuvants in aqueous biphasic systems.

    PubMed

    Ferreira, Ana M; Faustino, Vânia F M; Mondal, Dibyendu; Coutinho, João A P; Freire, Mara G

    2016-10-20

    Immunoglobulins G (IgG) could become widespread biopharmaceuticals if cost-efficient processes for their extraction and purification are available. In this work, aqueous biphasic systems (ABS) composed of polyethylene glycols and a buffered salt, and with ionic liquids (ILs) as adjuvants, have been studied as alternative extraction and purification platforms of IgG from a rabbit serum source. Eleven ILs were investigated to provide insights on the chemical features which maximize the IgG partitioning. It is shown that in polymer-salt systems pure IgG preferentially partitions to the polymer-rich phase; yet, the complete extraction was never attained. Remarkably, after the addition of 5wt% of adequate ILs to polymer-salt ABS, the complete extraction of pure IgG in a single-step was accomplished. The best systems and conditions were then applied to the extraction and purification of IgG directly from rabbit serum samples. The complete extraction of IgG in a single-step was maintained while its purity in the polymer-rich phase was enhanced by ca. 37% as compared to the IL-free ABS. The antibody stability was also evaluated revealing that appropriate ILs are able to maintain the IgG stability and can be used as phase-forming components of ABS when envisaging the purification of high-cost biopharmaceuticals.

  13. [Selection of winter plant species for wetlands constructed as sewage treatment systems and evaluation of their wastewater purification potentials].

    PubMed

    Chen, Yong-hua; Wu, Xiao-fu; Chen, Ming-li; Jiang, Li-juan; Li, Ke-lin; Lei, Dian; Wang, Hai-bin

    2010-08-01

    In order to establish an evaluation system for selection of winter wetland plants possessing high wastewater purification potentials in subtropics areas, designed sewage treatment experiments were carried out by introducing into the constructed wetlands 25 species of winter wetland plants. Cluster analysis was performed by including harmful environment-resistant enzyme and substrate enzyme activities into the commonly applied plant screening and assessment indexes system. The obtained results indicated that there were significant differences among the tested winter plants in their root length and vigor, leaf malonaldehyde (MDA), biomass, average nitrogen and phosphorus concentration and uptake, and urease and phosphoric acid enzyme activities in the root areas. Based on the established evaluation system, the tested plants were clustered into 3 groups. The plants in the 1st group possessing high purification potentials are Oenanthe javanica, Brassicacapestris, Juncus effusu, Saxifragaceae, Iris pseudoacorus, Osmanthus fragrans and Iris ensata; those in the 2nd group possessing moderate purification potentials are Brassica oleracea var acephala, Calendula officinalis, Aucuba japonica, Ligustrum lucidu, Beta vulgaris, Rhododendron simsii and Ilex latifolia; and those in the 3rd group with low purification potentials are Brassica oleracea var acephala, Calistephus chinensis, Rosa chinensis, Antirrhinums, Liriope palatyphylla, Zephyranthes candida, Fatshedera lizei, Petunia hybrida, Ilex quihoui, Dianthus caryophyllus and Loropetalum chinensis. PMID:21090294

  14. Single-Step Purification of Ovalbumin from Egg White Using Aqueous Biphasic Systems

    PubMed Central

    Pereira, Matheus M.; Cruz, Rafaela A. P.; Almeida, Mafalda R.; Lima, Álvaro S.; Coutinho, João A. P.; Freire, Mara G.

    2016-01-01

    The ability of aqueous biphasic systems (ABS) composed of polyethylene glycols of different molecular weights (PEG 400, 600 and 1000) and buffered aqueous solutions of potassium citrate/citric acid (pH = 5.0 - 8.0) to selectively extract ovalbumin from egg white was here investigated. Phase diagrams, tie-lines and tie-line lengths were determined at 25ºC and the partitioning of ovalbumin in these systems was then evaluated. Aiming at optimizing the selective extraction of ovalbumin in the studied ABS, factors such as pH, PEG molecular weight and amount of the phase-forming components were initially investigated with pure commercial ovalbumin. In almost all ABS, it was observed a preferential partitioning of ovalbumin to the polymer-rich phase, with extraction efficiencies higher than 90%. The best ABS were then applied in the purification of ovalbumin from the real egg white matrix. In order to ascertain on the ovalbumin purity and yield, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high performance liquid chromatography (SE-HPLC) analyses were conducted, confirming that the isolation/purification of ovalbumin from egg white was completely achieved in a single-step with a recovery yield of 65%. The results obtained show that polymer-salt-based ABS allow the selective extraction of ovalbumin from egg white with a simpler approach and better performance than previously reported. Finally, it is shown that ovalbumin can be completely recovered from the PEG-rich phase by an induced precipitation using an inexpensive and sustainable separation platform which can be easily applied on an industrial scale. PMID:27642253

  15. Minimization of the root of a quadratic functional under a system of affine equality constraints with application to portfolio management

    NASA Astrophysics Data System (ADS)

    Landsman, Zinoviy

    2008-10-01

    We present an explicit closed form solution of the problem of minimizing the root of a quadratic functional subject to a system of affine constraints. The result generalizes Z. Landsman, Minimization of the root of a quadratic functional under an affine equality constraint, J. Comput. Appl. Math. 2007, to appear, see , articles in press, where the optimization problem was solved under only one linear constraint. This is of interest for solving significant problems pertaining to financial economics as well as some classes of feasibility and optimization problems which frequently occur in tomography and other fields. The results are illustrated in the problem of optimal portfolio selection and the particular case when the expected return of finance portfolio is certain is discussed.

  16. An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

    SciTech Connect

    AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

    2015-11-19

    Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database in which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the

  17. An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

    DOE PAGES

    AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

    2015-11-19

    Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database inmore » which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the

  18. Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

    PubMed Central

    Mazzola, Priscila G; Martins, Alzira MS; Penna, Thereza CV

    2006-01-01

    Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite

  19. High temperature metal purification using a compact portable rf heating and levitation system on the wake shield

    NASA Technical Reports Server (NTRS)

    Hahs, C. A.

    1990-01-01

    The Wake Shield Facility (WSF) can provide an ideal vacuum environment for the purification of high temperature metals in space. The Modular Electromagnetic Levitator (MEL), will provide the opportunity to study undercooling of metals in space and allow to determine material properties in space. The battery powered rf levitation and heating system developed for the MEL demonstrated efficiency of 36 percent. This system is being considered to purify metals at temperatures below 3000 C.

  20. Thermodynamic optimization of a solar system for cogeneration of water heating/purification and absorption cooling

    NASA Astrophysics Data System (ADS)

    Hovsapian, Zohrob O.

    This dissertation presents a contribution to understanding the behavior of solar powered air conditioning and refrigeration systems with a view to determining the manner in which refrigeration rate; mass flows, heat transfer areas, and internal architecture are related. A cogeneration system consisting of a solar concentrator, a cavity-type receiver, a gas burner, and a thermal storage reservoir is devised to simultaneously produce water heating/purification and cooling (absorption refrigerator system). A simplified mathematical model, which combines fundamental and empirical correlations, and principles of classical thermodynamics, mass and heat transfer, is developed. An experimental setup was built to adjust and validate the numerical results obtained with the mathematical model. The proposed model is then utilized to simulate numerically the system transient and steady state response under different operating and design conditions. A system global optimization for maximum performance (or minimum exergy destruction) in the search for minimum pull-down and pull-up times, and maximum system second law efficiency is performed with low computational time. Appropriate dimensionless groups are identified and the results presented in normalized charts for general application. The numerical results show that the three way maximized system second law efficiency, etaII,max,max,max, occurs when three system characteristic mass flow rates are optimally selected in general terms as dimensionless heat capacity rates, i.e., (Psisps , Psiwxwx, PsiHs)opt ≅ (1.43, 0.17, 0.19). The minimum pull-down and pull-up times, and maximum second law efficiencies found with respect to the optimized operating parameters are sharp and, therefore important to be considered in actual design. As a result, the model is expected to be a useful tool for simulation, design, and optimization of solar energy systems in the context of distributed power generation.

  1. 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.

  2. Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions.

    PubMed

    Kang, Yu; Gohlke, Ulrich; Engström, Olof; Hamark, Christoffer; Scheidt, Tom; Kunstmann, Sonja; Heinemann, Udo; Widmalm, Göran; Santer, Mark; Barbirz, Stefanie

    2016-07-27

    Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system. PMID:27045683

  3. A circulating hydrogen ultra-high purification system for the MuCap experiment

    NASA Astrophysics Data System (ADS)

    Ganzha, V. A.; Kravtsov, P. A.; Maev, O. E.; Schapkin, G. N.; Semenchuk, G. G.; Trofimov, V. A.; Vasilyev, A. A.; Vznuzdaev, M. E.; Clayton, S. M.; Kammel, P.; Kiburg, B.; Hildebrandt, M.; Petitjean, C.; Banks, T. I.; Lauss, B.

    2007-08-01

    The MuCap experiment is a high-precision measurement of the rate for the basic electroweak process of muon capture, μ-+p→n+ ν μ. The experimental approach is based on an active target consisting of a time projection chamber (TPC) operating with pure hydrogen gas. The hydrogen has to be kept extremely pure and at a stable pressure. A Circulating Hydrogen Ultra-high Purification System was designed at the Petersburg Nuclear Physics Institute (PNPI) to continuously clean the hydrogen from impurities. The system is based on an adsorption cryo pump to stimulate the hydrogen flow and on a cold adsorbent for the hydrogen cleaning. It was installed at the Paul Scherrer Institute (PSI) in 2004 and performed reliably during three experiment runs. During several months long operating periods the system maintained the hydrogen purity in the detector on the level of 20 ppb for moisture, which is the main contaminant, and of better than 7 and 5 ppb for nitrogen and oxygen, respectively. The pressure inside the TPC was stabilized to within 0.024% of 10 bar at a hydrogen flow rate of three standard liters per minute.

  4. Determination of the kinetic rate constant of cyclodextrin supramolecular systems by high-performance affinity chromatography.

    PubMed

    Zhang, Jiwen; Li, Haiyan; Sun, Lixin; Wang, Caifen

    2015-01-01

    The kinetics of the association and dissociation are fundamental kinetic processes for the host-guest interactions (such as the drug-target and drug-excipient interactions) and the in vivo performance of supramolecules. With advantages of rapid speed, high precision and ease of automation, the high-performance affinity chromatography (HPAC) is one of the best techniques to measure the interaction kinetics of weak to moderate affinities, such as the typical host-guest interactions of drug and cyclodextrins by using a cyclodextrin-immobilized column. The measurement involves the equilibration of the cyclodextrin column, the upload and elution of the samples (non-retained substances and retained solutes) at different flow rates on the cyclodextrin and control column, and data analysis. It has been indicated that cyclodextrin-immobilized chromatography is a cost-efficient high-throughput tool for the measurement of (small molecule) drug-cyclodextrin interactions as well as the dissociation of other supramolecules with relatively weak, fast, and extensive interactions. PMID:25749964

  5. Simulation of a hydrogen production and purification system for a PEM fuel-cell using bioethanol as raw material

    NASA Astrophysics Data System (ADS)

    Giunta, Pablo; Mosquera, Carlos; Amadeo, Norma; Laborde, Miguel

    A process to produce "fuel-cell grade" hydrogen from ethanol steam reforming is analyzed from a thermodynamic point of view. The hydrogen purification process consists of WGS and COPROX reactors. Equations to evaluate the efficiency of the system, including the fuel cell, are presented. A heat exchange network is proposed in order to improve the exploitation of the available power. The effect of key variables such as the reformer temperature and the ethanol/water molar feed ratio on the fuel-cell efficiency is discussed. Results show that it is feasible to carry out the energy integration of the hydrogen catalytic production and purification-PEM fuel-cell system, using ethanol as raw material. The technology of "fuel-cell grade" hydrogen production using ethanol as raw material is a very attractive alternative to those technologies based in fossil fuels.

  6. Evaluation of a Surface Sampling Probe Electrospray Mass Spectrometry System for the Analysis of Surface Deposited and Affinity Captured Proteins

    SciTech Connect

    Van Berkel, Gary J; Ford, Michael J; Doktycz, Mitchel John; Kennel, Steve J

    2006-01-01

    A combined self-aspirating electrospray emitter/surfacing-sampling probe coupled with an ion trap mass spectrometer was used to sample and mass analyze proteins from surfaces. The sampling probe mass spectrometer system was used to sample and detect lysozyme that had been deposited onto a glass slide using a piezoelectric spotter or murine gamma-interferon affinity captured on a glass slide using surface-immobilized anti-gamma-interferon antibody. The detection level for surface-deposited lysozyme (spot size {le}200 {micro}m) was approximately 1.0 fmol ({approx}100 fmol/mm{sup 2}) as determined from the ability to measure accurately the protein molecular mass from the mass spectrum acquired by sampling the deposit. These detection limits may be sufficient for certain applications in which protein fractions from a separation method are collected onto a surface. Radiolabeled proteins were used to quantify the surface density of immobilized antibody and the efficiency of capture of the gamma-interferon on glass and higher surface area ceramic supports. The capture density of gamma-interferon at surface saturation ranged from about 23 to 50 fmol/mm{sup 2} depending on the capture surface. Nonetheless, mass spectrometric detection of affinity capture protein was successful in some cases, but the results were not reproducible. Thus, improvement of the sampling system, ionization efficiency and/or capture density will be necessary for practical sampling of affinity-captured proteins. The means to accomplish improved sampling system detection limits and to increase the absolute amounts of protein captured per unit area are discussed.

  7. An improved LMI-based approach for stability of piecewise affine time-delay systems with uncertainty

    NASA Astrophysics Data System (ADS)

    Duan, Shiming; Ni, Jun; Galip Ulsoy, A.

    2012-09-01

    The stability problem for uncertain piecewise affine (PWA) time-delay systems is investigated in this article. It is assumed that there exists a known constant time delay in the system and the uncertainly is norm-bounded. Sufficient conditions for the stability of nominal systems and the stability of systems subject to uncertainty are derived using the Lyapunov-Krasovskii functional with a triple integration term. This approach handles switching based on the delayed states (in addition to the states) for a PWA time-delay system, considers structured as well as unstructured uncertainty and reduces the conservativeness of previous approaches. The effectiveness of the proposed approach is demonstrated by comparing with the existing methods through numerical examples.

  8. Disposable and removable nucleic acid extraction and purification cartridges for automated flow-through systems

    SciTech Connect

    Regan, John Frederick

    2014-09-09

    Removable cartridges are used on automated flow-through systems for the purpose of extracting and purifying genetic material from complex matrices. Different types of cartridges are paired with specific automated protocols to concentrate, extract, and purifying pathogenic or human genetic material. Their flow-through nature allows large quantities sample to be processed. Matrices may be filtered using size exclusion and/or affinity filters to concentrate the pathogen of interest. Lysed material is ultimately passed through a filter to remove the insoluble material before the soluble genetic material is delivered past a silica-like membrane that binds the genetic material, where it is washed, dried, and eluted. Cartridges are inserted into the housing areas of flow-through automated instruments, which are equipped with sensors to ensure proper placement and usage of the cartridges. Properly inserted cartridges create fluid- and air-tight seals with the flow lines of an automated instrument.

  9. Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system.

    PubMed

    Caldwell, James T; Melkani, Girish C; Huxford, Tom; Bernstein, Sanford I

    2012-01-01

    Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.

  10. Automated sample preparation facilitated by PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins.

    PubMed

    Prater, Bradley D; Anumula, Kalyan R; Hutchins, Jeff T

    2007-10-15

    A reproducible high-throughput sample cleanup method for fluorescent oligosaccharide mapping of glycoproteins is described. Oligosaccharides are released from glycoproteins using PNGase F and labeled with 2-aminobenzoic acid (anthranilic acid, AA). A PhyNexus MEA system was adapted for automated isolation of the fluorescently labeled oligosaccharides from the reaction mixture prior to mapping by HPLC. The oligosaccharide purification uses a normal-phase polyamide resin (DPA-6S) in custom-made pipette tips. The resin volume, wash, and elution steps involved were optimized to obtain high recovery of oligosaccharides with the least amount of contaminating free fluorescent dye in the shortest amount of time. The automated protocol for sample cleanup eliminated all manual manipulations with a recycle time of 23 min. We have reduced the amount of excess AA by 150-fold, allowing quantitative oligosaccharide mapping from as little as 500 ng digested recombinant immunoglobulin G (rIgG). This low sample requirement allows early selection of a cell line with desired characteristics (e.g., oligosaccharide profile and high specific productivity) for the production of glycoprotein drugs. In addition, the use of Tecan or another robotic platform in conjunction with this method should allow the cleanup of 96 samples in 23 min, a significant decrease in the amount of time currently required to process such a large number of samples.

  11. C-X...H contacts in biomolecular systems: how they contribute to protein-ligand binding affinity.

    PubMed

    Lu, Yunxiang; Wang, Yong; Xu, Zhijian; Yan, Xiuhua; Luo, Xiaoming; Jiang, Hualiang; Zhu, Weiliang

    2009-09-17

    The hydrogen bond acceptor capability of halogens has long been underappreciated in the field of biology. In this work, we have surveyed structures of protein complexes with halogenated ligands to characterize geometrical preferences of C-X...H contacts and contributions of such interactions to protein-ligand binding affinity. Notably, F...H interactions in biomolecules exhibit a remarkably different behavior as compared to three other kinds of X...H (X = Cl, Br, I) interactions, which has been rationalized by means of ab initio calculations using simple model systems. The C-X...H contacts in biological systems are characterized as weak hydrogen bonding interactions. Furthermore, the electrophile "head on" and nucleophile "side on" interactions of halogens have been extensively investigated through the examination of interactions in protein structures and a two-layer ONIOM-based QM/MM method. In biomolecular systems, C-X...H contacts are recognized as secondary interaction contributions to C-X...O halogen bonds that play important roles in conferring specificity and affinity for halogenated ligands. The results presented here are within the context of their potential applications in drug design, including relevance to the development of accurate force fields for halogens.

  12. Metal-affinity separations: A new dimension in protein processing

    SciTech Connect

    Arnold, F.H. )

    1991-02-01

    Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

  13. Extraction and purification of wheat-esterase using aqueous two-phase systems of ionic liquid and salt.

    PubMed

    Jiang, Bin; Feng, Zhibiao; Liu, Chunhong; Xu, Yingcao; Li, Dongmei; Ji, Guo

    2015-05-01

    To explore a new and simple rapid extraction and purification technique for wheat-esterase, an ionic liquids (ILs)-based aqueous two-phase system (ATPS) was developed for the purification of wheat-esterase from wheat extracts. Effects of various process parameters such as the concentrations of [Bmim]BF4, the types and concentrations of phase-forming salt, the system pH and the temperature on partitioning of wheat-esterase were evaluated. The obtained data indicated that wheat-esterase was preferentially partitioned into the ILs-rich phase and the ATPS composed of 20 % [Bmim]BF4 (w/w) and 25 % (w/w) NaH2PO4(pH = 4.8) showed good selectivity on wheat-esterase. Under the optimum conditions, wheat-esterase was purified with an acceptable yield (88.93 %), but produced wheat-esterase was 4.23 times as pure. It was obvious that temperature shows little influence on the purification between 10 and 50 °C. Sephadex G-150FF revealed that the band intensity of contaminating proteins in ATPS fraction almost disappeared. Therefore, ILs-based ATPS was an effective method for partitioning and recovery of wheat-esterase from wheat crude extracts. PMID:25892786

  14. AM-DMC-AMPS Multi-Functionalized Magnetic Nanoparticles for Efficient Purification of Complex Multiphase Water System.

    PubMed

    Ge, Yuru; Li, Yushu; Zu, Baiyi; Zhou, Chaoyu; Dou, Xincun

    2016-12-01

    Complex multiphase waste system purification, as one of the major challenges in many industrial fields, urgently needs an efficient one-step purification method to remove several pollutants simultaneously and efficiently. Multi-functionalized magnetic nanoparticles, Fe3O4@SiO2-MPS-AM-DMC-AMPS, were facilely prepared via a one-pot in situ polymerization of three different functional monomers, AM, DMC, and AMPS, on a Fe3O4@SiO2-MPS core-shell structure. The multi-functionalized magnetic nanoparticles (MNPs) are proven to be a highly effective purification agent for oilfield wastewater, an ideal example of industrial complex multiphase waste system containing cations, anions, and organic pollutants. Excellent overall removal efficiencies for both cations, including K(+), Ca(2+), Na(+), and Mg(2+) of 80.68 %, and anions, namely Cl(-) and SO4 (2-), of 85.18 % along with oil of 97.4 % were shown. The high removal efficiencies are attributed to the effective binding of the functional groups from the selected monomers with cations, anions, and oil emulsions. PMID:27102906

  15. AM-DMC-AMPS Multi-Functionalized Magnetic Nanoparticles for Efficient Purification of Complex Multiphase Water System

    NASA Astrophysics Data System (ADS)

    Ge, Yuru; Li, Yushu; Zu, Baiyi; Zhou, Chaoyu; Dou, Xincun

    2016-04-01

    Complex multiphase waste system purification, as one of the major challenges in many industrial fields, urgently needs an efficient one-step purification method to remove several pollutants simultaneously and efficiently. Multi-functionalized magnetic nanoparticles, Fe3O4@SiO2-MPS-AM-DMC-AMPS, were facilely prepared via a one-pot in situ polymerization of three different functional monomers, AM, DMC, and AMPS, on a Fe3O4@SiO2-MPS core-shell structure. The multi-functionalized magnetic nanoparticles (MNPs) are proven to be a highly effective purification agent for oilfield wastewater, an ideal example of industrial complex multiphase waste system containing cations, anions, and organic pollutants. Excellent overall removal efficiencies for both cations, including K+, Ca2+, Na+, and Mg2+ of 80.68 %, and anions, namely Cl- and SO4 2-, of 85.18 % along with oil of 97.4 % were shown. The high removal efficiencies are attributed to the effective binding of the functional groups from the selected monomers with cations, anions, and oil emulsions.

  16. DOE Hydrogen, Fuel Cells and Infrastructure Technologies Program Integrated Hydrogen Production, Purification and Compression System

    SciTech Connect

    Tamhankar, Satish; Gulamhusein, Ali; Boyd, Tony; DaCosta, David; Golben, Mark

    2011-06-30

    The project was started in April 2005 with the objective to meet the DOE target of delivered hydrogen of <$1.50/gge, which was later revised by DOE to $2-$3/gge range for hydrogen to be competitive with gasoline as a fuel for vehicles. For small, on-site hydrogen plants being evaluated at the time for refueling stations (the 'forecourt'), it was determined that capital cost is the main contributor to the high cost of delivered hydrogen. The concept of this project was to reduce the cost by combining unit operations for the entire generation, purification, and compression system (refer to Figure 1). To accomplish this, the Fluid Bed Membrane Reactor (FBMR) developed by MRT was used. The FBMR has hydrogen selective, palladium-alloy membrane modules immersed in the reformer vessel, thereby directly producing high purity hydrogen in a single step. The continuous removal of pure hydrogen from the reformer pushes the equilibrium 'forward', thereby maximizing the productivity with an associated reduction in the cost of product hydrogen. Additional gains were envisaged by the integration of the novel Metal Hydride Hydrogen Compressor (MHC) developed by Ergenics, which compresses hydrogen from 0.5 bar (7 psia) to 350 bar (5,076 psia) or higher in a single unit using thermal energy. Excess energy from the reformer provides up to 25% of the power used for driving the hydride compressor so that system integration improved efficiency. Hydrogen from the membrane reformer is of very high, fuel cell vehicle (FCV) quality (purity over 99.99%), eliminating the need for a separate purification step. The hydride compressor maintains hydrogen purity because it does not have dynamic seals or lubricating oil. The project team set out to integrate the membrane reformer developed by MRT and the hydride compression system developed by Ergenics in a single package. This was expected to result in lower cost and higher efficiency compared to conventional hydrogen production technologies. The

  17. Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system.

    PubMed

    Barbosa, José Murillo P; Souza, Ranyere L; Fricks, Alini T; Zanin, Gisella Maria; Soares, Cleide Mara F; Lima, Alvaro S

    2011-12-15

    This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution. PMID:22100550

  18. Independent characterization of thymidine transport and subsequent metabolism in Hymenolepis diminuta--II. Purification and preliminary analysis of thymidine kinase.

    PubMed

    Insler, G D; Halikias, F J

    1991-01-01

    1. An affinity column for the purification of thymidine kinase (TK) from the cestode Hymenolepis diminuta is described. Using an epoxy-activated Sepharose 6B affinity column containing thymidine as a ligand, a 698-fold purification of thymidine kinase was obtained. 2. Thymidine kinase eluted from this affinity column was partially characterized as having an apparent Km value of 3.94 microM thymidine. This value is very similar to those observed in mammalian systems. 3. Thymidine kinase appears to be an extremely active and ubiquitous enzyme, whose primary function is to rapidly phosphorylate incoming thymidine and thus "trap" it for the cell's use, reducing efflux to a minimum. 4. The apparent Km for TK is two orders of magnitude lower than the Kt for thymidine transport. Thus, theories postulating that long-term (2 min) uptake kinetics for thymidine actually represent subsequent metabolism must look further along the thymidine phosphorylating pathway, beyond TK and its very active role.

  19. DNA-affinity-purified chip (DAP-chip) method to determine gene targets for bacterial two component regulatory systems.

    PubMed

    Rajeev, Lara; Luning, Eric G; Mukhopadhyay, Aindrila

    2014-07-21

    In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

  20. The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli.

    PubMed

    Patzer, S I; Hantke, K

    1998-06-01

    In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5mM EGTA, only the parental strain was able to take up 65Zn2+. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI) and localized at 42 min on the genetic map of E. coli. At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 microM Zn2+. A znuA-lacZ operon fusion was repressed by 5 microM Zn2+ and showed a more than 20-fold increase in beta-galactosidase activity when Zn2+ was bound to 1.5 microM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB. PMID:9680209

  1. Online adaptive policy learning algorithm for H∞ state feedback control of unknown affine nonlinear discrete-time systems.

    PubMed

    Zhang, Huaguang; Qin, Chunbin; Jiang, Bin; Luo, Yanhong

    2014-12-01

    The problem of H∞ state feedback control of affine nonlinear discrete-time systems with unknown dynamics is investigated in this paper. An online adaptive policy learning algorithm (APLA) based on adaptive dynamic programming (ADP) is proposed for learning in real-time the solution to the Hamilton-Jacobi-Isaacs (HJI) equation, which appears in the H∞ control problem. In the proposed algorithm, three neural networks (NNs) are utilized to find suitable approximations of the optimal value function and the saddle point feedback control and disturbance policies. Novel weight updating laws are given to tune the critic, actor, and disturbance NNs simultaneously by using data generated in real-time along the system trajectories. Considering NN approximation errors, we provide the stability analysis of the proposed algorithm with Lyapunov approach. Moreover, the need of the system input dynamics for the proposed algorithm is relaxed by using a NN identification scheme. Finally, simulation examples show the effectiveness of the proposed algorithm. PMID:25095274

  2. Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

    PubMed Central

    Cheng, Hao; Fan, Chen; Zhang, Si-wei; Wu, Zhong-shan; Cui, Zhi-cheng; Melcher, Karsten; Zhang, Cheng-hai; Jiang, Yi; Cong, Yao; Xu, H Eric

    2015-01-01

    Aim: To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target. Methods: α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis. Results: Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel. Conclusion: We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis. PMID:26073323

  3. Strategies for automated sample preparation, nucleic acid purification, and concentration of low-target-number nucleic acids in environmental and food processing samples

    NASA Astrophysics Data System (ADS)

    Bruckner-Lea, Cynthia J.; Holman, David A.; Schuck, Beatrice L.; Brockman, Fred J.; Chandler, Darrell P.

    1999-01-01

    The purpose of this work is to develop a rapid, automated system for nucleic acid purification and concentration from environmental and food processing samples. Our current approach involves off-line filtration and cell lysis (ballistic disintegration) functions in appropriate buffers followed by automated nucleic acid capture and purification on renewable affinity matrix microcolumns. Physical cell lysis and renewable affinity microcolumns eliminate the need for toxic organic solvents, enzyme digestions or other time- consuming sample manipulations. Within the renewable affinity microcolumn, we have examined nucleic acid capture and purification efficiency with various microbead matrices (glass, polymer, paramagnetic), surface derivitization (sequence-specific capture oligonucleotides or peptide nucleic acids), and DNA target size and concentration under variable solution conditions and temperatures. Results will be presented comparing automated system performance relative to benchtop procedures for both clean (pure DNA from a laboratory culture) and environmental (soil extract) samples, including results which demonstrate 8 minute purification and elution of low-copy nucleic acid targets from a crude soil extract in a form suitable for PCR or microarray-based detectors. Future research will involve the development of improved affinity reagents and complete system integration, including upstream cell concentration and cell lysis functions and downstream, gene-based detectors. Results of this research will ultimately lead to improved processes and instrumentation for on-line, automated monitors for pathogenic micro-organisms in food, water, air, and soil samples.

  4. Optimal control of unknown affine nonlinear discrete-time systems using offline-trained neural networks with proof of convergence.

    PubMed

    Dierks, Travis; Thumati, Balaje T; Jagannathan, S

    2009-01-01

    The optimal control of linear systems accompanied by quadratic cost functions can be achieved by solving the well-known Riccati equation. However, the optimal control of nonlinear discrete-time systems is a much more challenging task that often requires solving the nonlinear Hamilton-Jacobi-Bellman (HJB) equation. In the recent literature, discrete-time approximate dynamic programming (ADP) techniques have been widely used to determine the optimal or near optimal control policies for affine nonlinear discrete-time systems. However, an inherent assumption of ADP requires the value of the controlled system one step ahead and at least partial knowledge of the system dynamics to be known. In this work, the need of the partial knowledge of the nonlinear system dynamics is relaxed in the development of a novel approach to ADP using a two part process: online system identification and offline optimal control training. First, in the system identification process, a neural network (NN) is tuned online using novel tuning laws to learn the complete plant dynamics so that a local asymptotic stability of the identification error can be shown. Then, using only the learned NN system model, offline ADP is attempted resulting in a novel optimal control law. The proposed scheme does not require explicit knowledge of the system dynamics as only the learned NN model is needed. The proof of convergence is demonstrated. Simulation results verify theoretical conjecture. PMID:19596551

  5. Second-Order Perturbation Theory for Fractional Occupation Systems: Applications to Ionization Potential and Electron Affinity Calculations.

    PubMed

    Su, Neil Qiang; Xu, Xin

    2016-05-10

    Recently, we have developed an integration approach for the calculations of ionization potentials (IPs) and electron affinities (EAs) of molecular systems at the level of second-order Møller-Plesset (MP2) (Su, N. Q.; Xu, X. J. Chem. Theory Comput. 11, 4677, 2015), where the full MP2 energy gradient with respect to the orbital occupation numbers was derived but only at integer occupations. The theory is completed here to cover the fractional occupation systems, such that Slater's transition state concept can be used to have accurate predictions of IPs and EAs. Antisymmetrized Goldstone diagrams have been employed for interpretations and better understanding of the derived equations, where two additional rules were introduced in the present work specifically for hole or particle lines with fractional occupation numbers.

  6. Band gaps, ionization potentials, and electron affinities of periodic electron systems via the adiabatic-connection fluctuation-dissipation theorem

    NASA Astrophysics Data System (ADS)

    Trushin, Egor; Betzinger, Markus; Blügel, Stefan; Görling, Andreas

    2016-08-01

    An approach to calculate fundamental band gaps, ionization energies, and electron affinities of periodic electron systems is explored. Starting from total energies obtained with the help of the adiabatic-connection fluctuation-dissipation (ACFD) theorem, these physical observables are calculated according to their basic definition by differences of the total energies of the N -, (N -1 ) -, and (N +1 ) -electron system. The response functions entering the ACFD theorem are approximated here by the direct random phase approximation (dRPA). For a set of prototypical semiconductors and insulators it is shown that even with this quite drastic approximation the resulting band gaps are very close to experiment and of a similar quality to those from the computationally more involved G W approximation. By going beyond the dRPA in the future the accuracy of the calculated band gaps may be significantly improved further.

  7. Second-Order Perturbation Theory for Fractional Occupation Systems: Applications to Ionization Potential and Electron Affinity Calculations.

    PubMed

    Su, Neil Qiang; Xu, Xin

    2016-05-10

    Recently, we have developed an integration approach for the calculations of ionization potentials (IPs) and electron affinities (EAs) of molecular systems at the level of second-order Møller-Plesset (MP2) (Su, N. Q.; Xu, X. J. Chem. Theory Comput. 11, 4677, 2015), where the full MP2 energy gradient with respect to the orbital occupation numbers was derived but only at integer occupations. The theory is completed here to cover the fractional occupation systems, such that Slater's transition state concept can be used to have accurate predictions of IPs and EAs. Antisymmetrized Goldstone diagrams have been employed for interpretations and better understanding of the derived equations, where two additional rules were introduced in the present work specifically for hole or particle lines with fractional occupation numbers. PMID:27010405

  8. Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system

    PubMed Central

    Penna, Vessoni Thereza Christina; Martins, Silva Alzira Maria; Mazzola, Priscila Gava

    2002-01-01

    Background A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. Methodology Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. Results The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. Conclusions We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments. PMID:12182763

  9. Purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilin-inspired polypeptides.

    PubMed

    Lyons, Russell E; Elvin, Christopher M; Taylor, Karin; Lekieffre, Nicolas; Ramshaw, John A M

    2012-12-01

    Polypeptides containing between 4 and 32 repeats of a resilin-inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence-related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold-coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein-poor phase. We show that purification of recombinant proteins by cold-coacervation can be performed when engineered as a fusion partner to a resilin-inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time-consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold-coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature.

  10. Automated small‐scale protein purification and analysis for accelerated development of protein therapeutics

    PubMed Central

    LeSaout, Xavier; Costioli, Matteo; Jordan, Lynn; Lambert, Jeremy; Beighley, Ross; Provencher, Laurel; McGuire, Kevin; Verlinden, Nico; Barry, Andrew

    2015-01-01

    Small‐scale protein purification presents opportunities for accelerated process development of biotherapeutic molecules. Miniaturization of purification conditions reduces time and allows for parallel processing of samples, thus offering increased statistical significance and greater breadth of variables. The ability of the miniaturized platform to be predictive of larger scale purification schemes is of critical importance. The PerkinElmer JANUS BioTx Pro and Pro‐Plus workstations were developed as intuitive, flexible, and automated devices capable of performing parallel small‐scale analytical protein purification. Preprogrammed methods automate a variety of commercially available ion exchange and affinity chromatography solutions, including miniaturized chromatography columns, resin‐packed pipette tips, and resin‐filled microtiter vacuum filtration plates. Here, we present a comparison of microscale chromatography versus standard fast protein LC (FPLC) methods for process optimization. In this study, we evaluated the capabilities of the JANUS BioTx Pro‐Plus robotic platform for miniaturized chromatographic purification of proteins with the GE ӒKTA Express system. We were able to demonstrate predictive analysis similar to that of larger scale purification platforms, while offering advantages in speed and number of samples processed. This approach is predictive of scale‐up conditions, resulting in shorter biotherapeutic development cycles and less consumed material than traditional FPLC methods, thus reducing time‐to‐market from discovery to manufacturing.

  11. Optimization of conditions for the single step IMAC purification of miraculin from Synsepalum dulcificum.

    PubMed

    He, Zuxing; Tan, Joo Shun; Lai, Oi Ming; Ariff, Arbakariya B

    2015-08-15

    In this study, the methods for extraction and purification of miraculin from Synsepalum dulcificum were investigated. For extraction, the effect of different extraction buffers (phosphate buffer saline, Tris-HCl and NaCl) on the extraction efficiency of total protein was evaluated. Immobilized metal ion affinity chromatography (IMAC) with nickel-NTA was used for the purification of the extracted protein, where the influence of binding buffer pH, crude extract pH and imidazole concentration in elution buffer upon the purification performance was explored. The total amount of protein extracted from miracle fruit was found to be 4 times higher using 0.5M NaCl as compared to Tris-HCl and phosphate buffer saline. On the other hand, the use of Tris-HCl as binding buffer gave higher purification performance than sodium phosphate and citrate-phosphate buffers in IMAC system. The optimum purification condition of miraculin using IMAC was achieved with crude extract at pH 7, Tris-HCl binding buffer at pH 7 and the use of 300 mM imidazole as elution buffer, which gave the overall yield of 80.3% and purity of 97.5%. IMAC with nickel-NTA was successfully used as a single step process for the purification of miraculin from crude extract of S. dulcificum. PMID:25794715

  12. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking.

    PubMed

    Dolgosheina, Elena V; Jeng, Sunny C Y; Panchapakesan, Shanker Shyam S; Cojocaru, Razvan; Chen, Patrick S K; Wilson, Peter D; Hawkins, Nancy; Wiggins, Paul A; Unrau, Peter J

    2014-10-17

    Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes. PMID:25101481

  13. Purification effects of two eco-ditch systems on Chinese soft-shelled turtle greenhouse culture wastewater pollution.

    PubMed

    Wu, Xiang; Wu, Hao; Ye, Jinyun

    2014-04-01

    The present study used an eco-ditch system that employed Eichhornia crassipes, Bacillus subtilis, and Bellamya aeruginosa (E-B-B) during the summer and fall (high temperature) seasons and a second eco-ditch system that employed Elodea nuttallii, a compound microbial preparation called "EM bacteria", and Hypophthalmichthys molitrix (E-E-H) during the winter and spring (low temperature) seasons successively to purify the discharged wastewater produced by Chinese soft-shelled turtle greenhouse cultivation. The wastewater was sampled, and the dynamic changes in the major nutrient pollutant indicators over several months were analysed. After the E-B-B and E-E-H eco-ditch purification systems were operated for nearly 140 days each, the following results were observed: the total nitrogen (TN) removal rates in the wastewater were 75 % and 69 %, respectively; the total phosphorus (TP) removal rates were 82 % and 86 %, respectively; the NH4 (+)-N removal rates were 91 % and 75 %, respectively; the chemical oxygen demand (CODcr) decreased 54 % and 44 %, respectively; the dissolved oxygen (DO) contents increased nearly 3 to 4 times; and the wastewater was maintained at neutral or alkaline pH values. The wastewater physical traits gradually changed from being yellow, brown, and muddy to being pale yellow, slightly turbid, and odourless. Both eco-ditch systems were observed to have a relatively favourable effect on the purification of Chinese soft-shelled turtle aquaculture wastewater. The continuous use of both eco-ditch systems could result in a year-round purification effect on Chinese soft-shelled turtle greenhouse aquaculture wastewater; therefore, this method has good prospects for promotion and application.

  14. Recombinant Dragline Silk-Like Proteins-Expression and Purification.

    PubMed

    Gaines, William A; Marcotte, William R

    2011-03-01

    Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.

  15. Recombinant Dragline Silk-Like Proteins—Expression and Purification

    PubMed Central

    Gaines, William A.; Marcotte, William R.

    2011-01-01

    Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification. PMID:23914141

  16. Improving impurities clearance by amino acids addition to buffer solutions for chromatographic purifications of monoclonal antibodies.

    PubMed

    Ishihara, Takashi; Hosono, Mareto

    2015-07-15

    The performance of amino acids in Protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography for monoclonal antibody purification was investigated. Glycine, threonine, arginine, glutamate, and histidine were used as buffer components in the equilibration, washing, and elution steps of these chromatographies. Improved clearance of impurity, high molecular weight species (HMW) and host cell proteins (HCP) was observed in the purification processes when using the amino acids as base-buffer constituents, additives or eluents compared with that of buffers without these amino acids. In addition, we designed a buffer system in which the mobile phases were composed of only a single amino acid, histidine, and applied it to the above three chromatographies. Effective HMW and HCP clearance was also obtained in this manner. These results suggest that amino acids may enhance impurity clearance during the purification of monoclonal antibodies. PMID:26057847

  17. AMP/GMP Analogs as Affinity ESIPT Probes for Highly Selective Sensing of Alkaline Phosphatase Activity in Living Systems.

    PubMed

    Jia, Yan; Li, Peng; Han, Keli

    2015-11-01

    Current probes for alkaline phosphatase (ALP) detection had been developed mainly by adding a phosphate group to a dye, which would lead to indistinct performance when implemented in a living system as several phosphatases exist together. In this study, the nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) were introduced into 2'-(2'-hydroxyphenyl)-benzothiazole-based probes, and highly fluorescent turn-on probes with good selectivity towards ALP over several phosphatases, as well as high affinity and low toxicity were obtained. In the presence of L-phenylalanine, an ALP inhibitor, a strong decrease in fluorescence recovery was observed. These probes allowed for real-time imaging of endogenous ALP activity in living cells as well as in a zebrafish model.

  18. Robust fuzzy output feedback controller for affine nonlinear systems via T-S fuzzy bilinear model: CSTR benchmark.

    PubMed

    Hamdy, M; Hamdan, I

    2015-07-01

    In this paper, a robust H∞ fuzzy output feedback controller is designed for a class of affine nonlinear systems with disturbance via Takagi-Sugeno (T-S) fuzzy bilinear model. The parallel distributed compensation (PDC) technique is utilized to design a fuzzy controller. The stability conditions of the overall closed loop T-S fuzzy bilinear model are formulated in terms of Lyapunov function via linear matrix inequality (LMI). The control law is robustified by H∞ sense to attenuate external disturbance. Moreover, the desired controller gains can be obtained by solving a set of LMI. A continuous stirred tank reactor (CSTR), which is a benchmark problem in nonlinear process control, is discussed in detail to verify the effectiveness of the proposed approach with a comparative study.

  19. Generalized hamilton-jacobi-bellman formulation -based neural network control of affine nonlinear discrete-time systems.

    PubMed

    Chen, Zheng; Jagannathan, Sarangapani

    2008-01-01

    In this paper, we consider the use of nonlinear networks towards obtaining nearly optimal solutions to the control of nonlinear discrete-time (DT) systems. The method is based on least squares successive approximation solution of the generalized Hamilton-Jacobi-Bellman (GHJB) equation which appears in optimization problems. Successive approximation using the GHJB has not been applied for nonlinear DT systems. The proposed recursive method solves the GHJB equation in DT on a well-defined region of attraction. The definition of GHJB, pre-Hamiltonian function, HJB equation, and method of updating the control function for the affine nonlinear DT systems under small perturbation assumption are proposed. A neural network (NN) is used to approximate the GHJB solution. It is shown that the result is a closed-loop control based on an NN that has been tuned a priori in offline mode. Numerical examples show that, for the linear DT system, the updated control laws will converge to the optimal control, and for nonlinear DT systems, the updated control laws will converge to the suboptimal control. PMID:18269941

  20. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    SciTech Connect

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

  1. Data-based fault-tolerant control for affine nonlinear systems with actuator faults.

    PubMed

    Xie, Chun-Hua; Yang, Guang-Hong

    2016-09-01

    This paper investigates the fault-tolerant control (FTC) problem for unknown nonlinear systems with actuator faults including stuck, outage, bias and loss of effectiveness. The upper bounds of stuck faults, bias faults and loss of effectiveness faults are unknown. A new data-based FTC scheme is proposed. It consists of the online estimations of the bounds and a state-dependent function. The estimations are adjusted online to compensate automatically the actuator faults. The state-dependent function solved by using real system data helps to stabilize the system. Furthermore, all signals in the resulting closed-loop system are uniformly bounded and the states converge asymptotically to zero. Compared with the existing results, the proposed approach is data-based. Finally, two simulation examples are provided to show the effectiveness of the proposed approach. PMID:27180025

  2. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  3. Two plant bacteria, S. meliloti and Ca. Liberibacter asiaticus, share functional znuABC homologues that encode for a high affinity Zinc uptake system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not bee...

  4. A pH-induced, intein-mediated expression and purification of recombinant human epidermal growth factor in Escherichia coli.

    PubMed

    Zhang, Yuejuan; Zhang, Kun; Wan, Yi; Zi, Jing; Wang, Yan; Wang, Jun; Wang, Lili; Xue, Xiaochang

    2015-01-01

    Human epidermal growth factor (hEGF) is a cellular factor that promotes cell proliferation and has been widely used for the treatment of wounds, corneal injuries, and gastric ulcers. Recombinant hEGF (rhEGF) has previously been expressed using the pTWIN1 system with pH-induced intein and a chitin-binding domain. The rhEGF protein can be purified by chitin affinity chromatography because of the high affinity between the chitin-binding domain fusion-tag and the column. However, uncontrolled cleavage presents a major problem with this method. To overcome this problem, a novel purification method has been developed for a pH-induced intein tag rhEGF that is expressed in Escherichia coli. Following purification by denaturation of inclusion bodies, the fusion protein is renatured and simultaneously induced to self-cleave by dialysis. Further purification of rhEGF is achieved by heat treatment and ion-exchange chromatography. Our results show that the purity of rhEGF obtained through this method is over 98% and the quantity of purified rhEGF is 248 mg from a 1 L culture or 2,967 mg from a 12 L culture. Therefore, we conclude that we have developed an efficient purification method of rhEGF, which may be used for the purification of other heat-resistant and acid-resistant recombinant proteins.

  5. Neural network-based finite-horizon optimal control of uncertain affine nonlinear discrete-time systems.

    PubMed

    Zhao, Qiming; Xu, Hao; Jagannathan, Sarangapani

    2015-03-01

    In this paper, the finite-horizon optimal control design for nonlinear discrete-time systems in affine form is presented. In contrast with the traditional approximate dynamic programming methodology, which requires at least partial knowledge of the system dynamics, in this paper, the complete system dynamics are relaxed utilizing a neural network (NN)-based identifier to learn the control coefficient matrix. The identifier is then used together with the actor-critic-based scheme to learn the time-varying solution, referred to as the value function, of the Hamilton-Jacobi-Bellman (HJB) equation in an online and forward-in-time manner. Since the solution of HJB is time-varying, NNs with constant weights and time-varying activation functions are considered. To properly satisfy the terminal constraint, an additional error term is incorporated in the novel update law such that the terminal constraint error is also minimized over time. Policy and/or value iterations are not needed and the NN weights are updated once a sampling instant. The uniform ultimate boundedness of the closed-loop system is verified by standard Lyapunov stability theory under nonautonomous analysis. Numerical examples are provided to illustrate the effectiveness of the proposed method. PMID:25720005

  6. Disposable on-chip microfluidic system for buccal cell lysis, DNA purification, and polymerase chain reaction.

    PubMed

    Cho, Woong; Maeng, Joon-Ho; Ahn, Yoomin; Hwang, Seung Yong

    2013-09-01

    This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off-chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.

  7. Water purification in Borexino

    SciTech Connect

    Giammarchi, M.; Balata, M.; Ioannucci, L.; Nisi, S.; Goretti, A.; Ianni, A.; Miramonti, L.

    2013-08-08

    Astroparticle Physics and Underground experiments searching for rare nuclear events, need high purity materials to act as detectors or detector shielding. Water has the advantage of being cheap, dense and easily available. Most of all, water can be purified to the goal of obatining a high level of radiopurity. Water Purification can be achieved by means of a combination of processes, including filtration, reverse osmosis, deionization and gas stripping. The Water Purification System for the Borexino experiment, will be described together with its main performances.

  8. Comparison of material flows in sewage-free and sewage-generating flue-gas purification systems of municipal waste incineration plants

    SciTech Connect

    Achternbosch, M.; Richers, U.

    1998-07-01

    During incineration of waste in waste incineration plants, polluted flue gases are generated which have to be subjected to flue gas purification. Although the legal requirements are nearly unambiguous, the question of whether wet flue gas purification is to be performed in a sewage-free or sewage-generating manner is discussed controversially by experts in the Federal Republic of Germany. As a contribution to this discussion, material flow studies of sewage-free and sewage-generating flue gas purification processes in waste incineration plants were performed by ITAS in cooperation with ITC-TAB. The study covered three waste incineration plants, two of which were operated in a sewage-generating and one in a sewage-free manner. The data and information submitted by most of the plant operators are not sufficient for a comprehensive balancing of flue gas purification systems in waste incineration plants. For this reason, plant operation often is not optimally tailored to the substances prevailing. During operation, at least temporary strong superstoichiometric dosage of auxiliary chemicals cannot be excluded. By means of plausibility assumptions and model calculations, closed balancing of most plants could be achieved. Moreover, it was demonstrated by the balancing of technical-scale waste incineration plants that the material flows in wet flue gas purification re less dependent on the design of the flue gas purification section (sewage-free/sewage-generating), but considerably affected by the operation of the flue gas purification system (e.g., volume of absorption agents used). Hence, material flows can be controlled in a certain range.

  9. Global multi-method analysis of affinities and cooperativity in complex systems of macromolecular interactions.

    PubMed

    Zhao, Huaying; Schuck, Peter

    2012-11-01

    Cooperativity, multisite, and multicomponent interactions are hallmarks of biological systems of interacting macromolecules. Their thermodynamic characterization is often very challenging due to the notoriously low information content of binding isotherms. We introduce a strategy for the global multimethod analysis of data from multiple techniques (GMMA) that exploits enhanced information content emerging from the mutual constraints of the simultaneous modeling of orthogonal observables from calorimetric, spectroscopic, hydrodynamic, biosensing, or other thermodynamic binding experiments. We describe new approaches to address statistical problems that arise in the analysis of dissimilar data sets. The GMMA approach can significantly increase the complexity of interacting systems that can be accurately thermodynamically characterized.

  10. ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera.

    PubMed

    Crimmins, Dan L; Brada, Nancy A; Lockwood, Christina M; Griest, Terry A; Waldemer, Rachel J; Cervinski, Mark A; Ohlendorf, Matthew F; McQuillan, Jay J; Ladenson, Jack H

    2010-12-01

    Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33 kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time. PMID:21054278

  11. Biogas recirculation for simultaneous calcium removal and biogas purification within an expanded granular sludge bed system treating leachate.

    PubMed

    Luo, Jinghuan; Lu, Xueqin; Liu, Jianyong; Qian, Guangren; Lu, Yongsheng

    2014-12-01

    Biogas, generated from an expanded granular sludge bed (EGSB) reactor treating municipal solid waste (MSW) leachate, was recirculated for calcium removal from the leachate via a carbonation process with simultaneous biogas purification. Batch trials were performed to optimize the solution pH and imported biogas (CO2) for CaCO3 precipitation. With applicable pH of 10-11 obtained, continuous trials achieved final calcium concentrations of 181-375 mg/L (removal efficiencies≈92.8-96.5%) in the leachate and methane contents of 87.1-91.4% (purification efficiencies≈65.4-82.2%) in the biogas. Calcium-balance study indicates that 23-986 mg Ca/d was released from the bio-system under the carbonized condition where CaCO3 precipitating was moved outside the bioreactor, whereas 7918-9517 mg Ca/d was trapped into the system for the controlled one. These findings demonstrate that carbonation removal of calcium by biogas recirculation could be a promising alternative to pretreat calcium-rich MSW leachate and synergistically to improve methane content.

  12. Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli.

    PubMed

    Chiang, Chung-Jen; Chen, Hong-Chen; Chao, Yun-Peng; Tzen, Jason T C

    2005-06-15

    Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.

  13. Characterization of the high affinity binding of epsilon toxin from Clostridium perfringens to the renal system.

    PubMed

    Dorca-Arévalo, Jonatan; Martín-Satué, Mireia; Blasi, Juan

    2012-05-25

    Epsilon toxin (ε-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxaemia in livestock. In the renal system, the toxin binds to target cells before oligomerization, pore formation and cell death. Still, there is little information about the cellular and molecular mechanism involved in the initial steps of the cytotoxic action of ε-toxin, including the specific binding to the target sensitive cells. In the present report, the binding step of ε-toxin to the MDCK cell line is characterized by means of an ELISA-based binding assay with recombinant ε-toxin-green fluorescence protein (ε-toxin-GFP) and ε-prototoxin-GFP. In addition, different treatments with Pronase E, detergents, N-glycosidase F and beta-elimination on MDCK cells and renal cryosections have been performed to further characterize the ε-toxin binding. The ELISA assays revealed a single binding site with a similar dissociation constant (K(d)) for ε-toxin-GFP and ε-prototoxin-GFP, but a three-fold increase in B(max) levels in the case of ε-toxin-GFP. Double staining on kidney cryoslices with lectins and ε-prototoxin-GFP revealed specific binding to distal and collecting tubule cells. In addition, experiments on kidney and bladder cryoslices demonstrated the specific binding to distal tubule of a range of mammalian renal systems. Pronase E and beta-elimination treatments on kidney cryoslices and MDCK cells revealed that the binding of ε-toxin in renal system is mediated by a O-glycoprotein. Detergent treatments revealed that the integrity of the plasma membrane is required for the binding of ε-toxin to its receptor.

  14. Purification of specific loci for proteomic analysis

    PubMed Central

    Byrum, Stephanie D.; Taverna, Sean D.; Tackett, Alan J.

    2015-01-01

    Purification of small, native chromatin sections for proteomic identification of specifically bound proteins and histone posttranslational modifications is a powerful approach for studying mechanisms of chromosome metabolism. We detail a Chromatin Affinity Purification with Mass Spectrometry (ChAP-MS) approach for affinity purification of ~1 kb sections of chromatin for targeted proteomic analysis. This approach utilizes quantitative, high resolution mass spectrometry to categorize proteins and histone posttranslational modifications co-enriching with the given chromatin section as either “specific” to the targeted chromatin or “non-specific” contamination. In this way, the ChAP-MS approach can help define and re-define mechanisms of chromatin-templated activities. PMID:25311124

  15. Recovery and purification of ethylene

    DOEpatents

    Reyneke, Rian; Foral, Michael J.; Lee, Guang-Chung; Eng, Wayne W. Y.; Sinclair, Iain; Lodgson, Jeffery S.

    2008-10-21

    A process for the recovery and purification of ethylene and optionally propylene from a stream containing lighter and heavier components that employs an ethylene distributor column and a partially thermally coupled distributed distillation system.

  16. Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems.

    PubMed

    Birkenmeier, G; Vijayalakshmi, M A; Stigbrand, T; Kopperschläger, G

    1991-02-22

    Immobilized metal ions were used for the affinity extraction of proteins in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran or PEG and salt. Soluble chelating polymers were prepared by covalent attachment of metal-chelating groups to PEG. The effect on the partitioning of proteins of such chelating PEG derivatives coordinated with different metal ions is demonstrated. The proteins studied were alpha 2-macroglobulin, tissue plasminogen activator, superoxide dismutase and monoclonal antibodies. The results indicate that immobilized metal ion affinity partitioning provides excellent potential for the extraction of proteins. PMID:1710621

  17. A multi-purpose system for water purification and sea-water softening.

    PubMed

    Barsky, L; Rubinstein, J; Barsky, S; Kirzhner, F; Bodul, O

    1998-01-01

    A novel technique that can be used for reacting toxic carbon dioxide (CO2) emissions from power plants and other combustion wastes with sea water is described. A chemical interaction between CO2 and the cations in sea water, with the pH electrolytically regulated, can precipitate almost all the calcium and magnesium ions, as well as some sodium and potassium ions, as carbonates and bicarbonates. The carbonates and bicarbonates thus prepared can then be mixed with ash to yield a building material. Sulfur ions will be neutralized with calcium and magnesium, and the remaining ions can be removed using reverse osmosis or some other method. The technology and equipment for purification are based on modules that can be used for industrial waste-water, sea water, solutions, and otherwise. The module for separation of sand and suspended coarse substances consists of a tank for flocculation, coagulation, and precipitation of solid particles; and a low-pressure hydrocyclone. The module for purification from oil and fine suspensions is based on column flotation, flotation with a special ejector, and adhesion flotation. The module for ions and colloids consists of an absorbing filter with zeolite, fly ash, and other absorbing materials. Using a laboratory model consisting of a special mini-plant, we processed 10 L of factory-waste water containing more than 20 g/L organic content (compare with the upper limit of 0.02 g/L allowed by the Ministry of Environmental Protection in Israel). After the experimental solution was treated and evaporated to a small bulk, the water obtained was almost clear. On the basis of the results in the model, we present a scaled-up process for the design, development, and production of equipment for and the assembly of a large installation for drainage and water purification.

  18. Telomerase repeat amplification protocol (TRAP) activity upon recombinant expression and purification of human telomerase in a bacterial system.

    PubMed

    Hansen, Debra T; Thiyagarajan, Thirumagal; Larson, Amy C; Hansen, Jeffrey L

    2016-07-01

    Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a ∼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting. PMID:26965413

  19. Identification of a PutP proline permease gene homolog from Staphylococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli.

    PubMed Central

    Wengender, P A; Miller, K J

    1995-01-01

    The important food-borne pathogen Staphylococcus aureus is distinguished by its ability to grow at low water activity values. Previous work in our laboratory and by others has revealed that proline accumulation via transport is an important osmoregulatory strategy employed by this bacterium. Furthermore, proline uptake by this bacterium has been shown to be mediated by two distinct transport systems: a high-affinity system and a low-affinity system (J.-H. Bae, and K. J. Miller, Appl. Environ. Microbiol. 58:471-475, 1992; D. E. Townsend and B. J. Wilkinson, J. Bacteriol. 174:2702-2710, 1992). In the present study, we report the cloning of the high-affinity proline transport system of S. aureus by functional expression in an Escherichia coli host. The sequence of the staphylococcal proline permease gene was predicted to encode a protein of 497 amino acids which shares 49% identity with the PutP high-affinity proline permease of E. coli. Analysis of hydropathy also indicated a common overall structure for these proteins. PMID:7887605

  20. Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Anderson, Alexander J.

    1988-01-01

    Discusses a laboratory technique of enzyme purification by affinity chromatography as part of an undergraduate biochemical methodology course. Provides preparation details of the rat muscle homogenate and reagents. Proposes column requirements and assaying information. (MVL)

  1. New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast*

    PubMed Central

    Martin, D. Christian; Kim, Hyemin; Mackin, Nancy A.; Maldonado-Báez, Lymarie; Evangelista, Carlos C.; Beaudry, Veronica G.; Dudgeon, Drew D.; Naiman, Daniel Q.; Erdman, Scott E.; Cunningham, Kyle W.

    2011-01-01

    The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca2+ influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca2+ after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis. PMID:21252230

  2. Direct purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel using a PEG/salt-based Aqueous Two Phase System.

    PubMed

    Mehrnoush, Amid; Sarker, Md Zaidul Islam; Mustafa, Shuhaimi; Yazid, Abdul Manap Mohd

    2011-10-10

    An Aqueous Two-Phase System (ATPS) was employed for the first time for the separation and purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel. The effects of different parameters such as molecular weight of the polymer (polyethylene glycol, 2,000-10,000), potassium phosphate composition (12-20%, w/w), system pH (6-9), and addition of different concentrations of neutral salts (0-8%, w/w) on partition behavior of pectinase were investigated. The partition coefficient of the enzyme was decreased by increasing the PEG molecular weight. Additionally, the phase composition showed a significant effect on purification factor and yield of the enzyme. Optimum conditions for purification of pectinase from mango peel were achieved in a 14% PEG 4000-14% potassium phosphate system using 3% (w/w) NaCl addition at pH 7.0. Based on this system, the purification factor of pectinase was increased to 13.2 with a high yield of (97.6%). Thus, this study proves that ATPS can be an inexpensive and effective method for partitioning of pectinase from mango peel.

  3. Antibody-based affinity cryo-EM grid.

    PubMed

    Yu, Guimei; Li, Kunpeng; Jiang, Wen

    2016-05-01

    The Affinity Grid technique combines sample purification and cryo-Electron Microscopy (cryo-EM) grid preparation into a single step. Several types of affinity surfaces, including functionalized lipids monolayers, streptavidin 2D crystals, and covalently functionalized carbon surfaces have been reported. More recently, we presented a new affinity cryo-EM approach, cryo-SPIEM, which applies the traditional Solid Phase Immune Electron Microscopy (SPIEM) technique to cryo-EM. This approach significantly simplifies the preparation of affinity grids and directly works with native macromolecular complexes without need of target modifications. With wide availability of high affinity and high specificity antibodies, the antibody-based affinity grid would enable cryo-EM studies of the native samples directly from cell cultures, targets of low abundance, and unstable or short-lived intermediate states.

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

    SciTech Connect

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

    2011-02-25

    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 small 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.

  5. Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate).

    PubMed

    Nascimento, Thiago Pajeú; Sales, Amanda Emmanuelle; Porto, Camila Souza; Brandão, Romero Marcos Pedrosa; de Campos-Takaki, Galba Maria; Teixeira, José Antônio Couto; Porto, Tatiana Souza; Porto, Ana Lúcia Figueiredo; Converti, Attilio

    2016-07-01

    A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 2(3) full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37°C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification. PMID:27183214

  6. Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification.

    PubMed

    Johansson, H O; Persson, J; Tjerneld, F

    1999-01-01

    In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14 degrees C. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By

  7. Development of a sensitive method for selection of affinity ligand for trypsin using quartz crystal microbalance sensor.

    PubMed

    Bayramoglu, Gulay; Yakup Arica, M

    2012-03-01

    In this work, a new methodology is developed for selection of affinity ligands towards the enzyme "trypsin" using quartz crystals microbalance (QCM) technique. To achieve this goal, the surface amination of gold plated QCM crystals was achieved in 13.56 MHz plasma polymerization system by using ethylenediamine. Three different ligands (i.e., 4-aminobenzamidine, 4-aminobenzoic acid, and phenylalanine) were immobilized on the aminated QCM crystals surface via glutaraldehyde coupling. All three ligand immobilized QCM crystals were characterized and compared under different experimental conditions. It was observed that the benzamidine ligand showed higher affinity to trypsin with a dissociation constant on the order of 1.76 × 10(-9) M, which is within the range of 10(-4)-10(-8) M for affinity ligands. Thus, its selectivity was suitable for purification of trypsin from biological fluids. PMID:21853329

  8. A gas circulation and purification system for gas-cell-based low-energy RI-beam production.

    PubMed

    Sonoda, T; Tsubota, T; Wada, M; Katayama, I; Kojima, T M; Reponen, M

    2016-06-01

    A gas circulation and purification system was developed at the RIKEN Radioactive Isotope Beam Factory that can be used for gas-cell-based low-energy RI-beam production. A high-flow-rate gas cell filled with one atmosphere of buffer gas (argon or helium) is used for the deceleration and thermalization of high-energy RI-beams. The exhausted buffer gas is efficiently collected using a compact dry pump and returned to the gas cell with a recovery efficiency of >97%. The buffer gas is efficiently purified using two gas purifiers as well as collision cleaning, which eliminates impurities in the gas. An impurity level of one part per billion is achieved with this method. PMID:27370494

  9. A gas circulation and purification system for gas-cell-based low-energy RI-beam production

    NASA Astrophysics Data System (ADS)

    Sonoda, T.; Tsubota, T.; Wada, M.; Katayama, I.; Kojima, T. M.; Reponen, M.

    2016-06-01

    A gas circulation and purification system was developed at the RIKEN Radioactive Isotope Beam Factory that can be used for gas-cell-based low-energy RI-beam production. A high-flow-rate gas cell filled with one atmosphere of buffer gas (argon or helium) is used for the deceleration and thermalization of high-energy RI-beams. The exhausted buffer gas is efficiently collected using a compact dry pump and returned to the gas cell with a recovery efficiency of >97%. The buffer gas is efficiently purified using two gas purifiers as well as collision cleaning, which eliminates impurities in the gas. An impurity level of one part per billion is achieved with this method.

  10. Enhancement of a novel extracellular uricase production by media optimization and partial purification by aqueous three-phase system.

    PubMed

    Ram, Senthoor K; Raval, Keyur; JagadeeshBabu, P E

    2015-01-01

    Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3), an enzyme belonging to the class of oxidoreductases, catalyzes the enzymatic oxidation of uric acid to allantoin and finds a wide variety of application as therapeutic and clinical reagent. In this study, uricase production ability of the bacterial strains isolated from deep litter poultry soil is investigated. The strain with maximum extracellular uricase production capability was identified as Xanthomonas fuscans subsp. aurantifolii based on 16S rRNA sequencing. Effect of various carbon and nitrogen sources on uricase productivity was investigated. The uricase production for this strain was optimized using statistically based experimental designs and resulted in uricase activity of 306 U/L, which is 2 times higher than initial uricase activity. Two-step purification, such as ammonium sulfate precipitation and aqueous two-phase system, was carried out and a twofold increase in yield and specific activity was observed.

  11. Optimization of serine protease purification from mango (Mangifera indica cv. Chokanan) peel in polyethylene glycol/dextran aqueous two phase system.

    PubMed

    Mehrnoush, Amid; Mustafa, Shuhaimi; Sarker, Md Zaidul Islam; Yazid, Abdul Manap Mohd

    2012-01-01

    Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000-12,000 g·mol(-1)), tie line length (-3.42-35.27%), NaCl (-2.5-11.5%) and pH (4.5-10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol(-1) of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing.

  12. 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.

  13. 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. PMID:25277090

  14. A complementary method to CD4 counting: measurement of CD4+/CD8+ T lymphocyte ratio in a tandem affinity microfluidic system.

    PubMed

    Li, Wenjie; Gao, Yan; Pappas, Dimitri

    2015-12-01

    We describe a tandem affinity microfluidic separation that measures the ratio of CD4+/CD8+ T lymphocytes from blood samples. It is performed by injecting 2 μL of lysed blood samples at 1800-2700 cells μL(-1) into a microfluidic device containing two serially linked affinity regions, followed with a stop flow incubation that captures CD4+/CD8+ T lymphocytes on the corresponding affinity regions. Fluorophore conjugated antibodies are then injected at a controlled shear stress of 1.7 dyn cm(-2) to label target cells while eluting non-specific cells; and at last the CD4/CD8 ratio is calculated after the cell enumeration. The ratio of CD4+/CD8+ T lymphocytes achieved by our tandem affinity microfluidic system was in close agreement with that performed using conventional flow cytometry (R (2)  = 0.97) over a wide range (0.4-2.5) that covered the reference values from immune deficient patients to healthy people. This approach may represent an inexpensive and powerful tool in diagnosis of immunodeficiency disorders including HIV or mycobacterium tuberculosis.

  15. Argon Purification Reference and Recommendation

    SciTech Connect

    Wu, J.; /Fermilab

    1991-05-23

    This engineering note is a reference for future consideration on the purification of argon. The original concern was for the possibility of argon contamination from components in the cryostats over long-term storage. An argon purification system could also be useful for purifying the contents of the argon dewar. The general conclusion is that most of the systems researched are too expensive at this time, but the recommended choice would be Centorr Furnaces. There were three basic types of purification systems which were to be considered. The first was the molecular sieve. This method would have been the preferred one, because it was claimed that it could purify liquid argon, removing liquid oxygen from the argon. However, none of the commercial companies researched provided this type of purification for use with liquid argon. Most companies said that this type of purification was impossible, and tests at IB-4 confirmed this. The second system contained a copper oxide to remove gaseous oxygen from argon gas. The disadvantage of this system wass that the argon had to be heated to a gas, and then cooled back down to liquid. The third system was similar to the second, except that it used tungsten or another material like titanium. This system also needed to heat the argon to gas, however the advantage of this system was that it supposedly removed all contaminants, that is, everything except for inert gases. Of the three systems, the third is the type manufactured by Centorr Furnaces, which uses a titanium charge.

  16. SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications.

    PubMed

    Haney, Paul J; Draveling, Connie; Durski, Wendy; Romanowich, Kathryn; Qoronfleh, M Walid

    2003-04-01

    Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry. PMID:12699691

  17. Purification of pectinase from mango (Mangifera indica L. cv. Chokanan) waste using an aqueous organic phase system: a potential low cost source of the enzyme.

    PubMed

    Amid, Mehrnoush; Abdul Manap, Mohd Yazid; Mustafa, Shuhaimi

    2013-07-15

    As a novel method of purification, an aqueous organic phase system (AOPS) was employed to purify pectinase from mango waste. The effect of different parameters, such as the alcohol concentration (ethanol, 1-propanol, and 2-propanol), the salt type and concentration (ammonium sulfate, potassium phosphate and sodium citrate), the feed stock crude load, the aqueous phase pH and NaCl concentration, were investigated in the recovery of pectinase from mango peel. The partition coefficient (K), selectivity (S), purification factor (PF) and yield (Y, %) were investigated in this study as important parameters for the evaluation of enzyme recovery. The desirable partition efficiency for pectinase purification was achieved in an AOPS of 19% (w/w) ethanol and 22% (w/w) potassium phosphate in the presence of 5% (w/w) NaCl at pH 7.0. Based on the system, the purification factor of pectinase was enhanced 11.7, with a high yield of 97.1%.

  18. PURIFICATION PROCESS

    DOEpatents

    Wibbles, H.L.; Miller, E.I.

    1958-01-14

    This patent deals with the separation of uranium from molybdenum compounds, and in particular with their separation from ether solutions containing the molybdenum in the form of acids, such as silicomolybdic and phosphomolybdic acids. After the nitric acid leach of pitchblende, the molybdenum values present in the ore are found in the leach solution in the form of complex acids. The uranium bearing solution may be purified of this molybdenum content by comtacting it with activated charcoal. The purification is improved when the acidity of the solution is low ad agitation is also beneficial. The molybdenum may subsequently be recovered from the charcosl ad the charcoal reused.

  19. Synthesis of isomers of 18F-labelled amino acid radiopharmaceutical: position 2- and 3-L-18F-alpha-methyltyrosine using a separation and purification system.

    PubMed

    Tomiyoshi, K; Amed, K; Muhammad, S; Higuchi, T; Inoue, T; Endo, K; Yang, D

    1997-02-01

    To diagnose cancers with radiolabelled amino acid using positron emission tomography, we have constructed a separation and purification system for the production of L-18F-alpha-methyltyrosine (L-18FAmT). This system could provide radioprotection and consistent production of L-18FAmT. L-18FAmT was synthesized and purified and the efficiency of the system was examined. The radiochemical yield of L-18FAmT was 20.3 +/- 5.1% (n = 5) based on the radioactivity trapped in the reaction vessel. The radiochemical purity was greater than 99.4 +/- 0.3% (n = 5). The radiochemical stability in phosphate-buffered saline and human plasma was examined and little decomposition was observed by HPLC analysis. Our results indicate that the separation and purification system gave simple and quick synthesis of L-18FAmT with a large reduction in radiation exposure and consistent production of L-18FAmT.

  20. Ionic Liquid-Based Ultrasonic-Assisted Extraction of Secoisolariciresinol Diglucoside from Flaxseed (Linum usitatissimum L.) with Further Purification by an Aqueous Two-Phase System.

    PubMed

    Tan, Zhi-Jian; Wang, Chao-Yun; Yang, Zi-Zhen; Yi, Yong-Jian; Wang, Hong-Ying; Zhou, Wan-Lai; Li, Fen-Fang

    2015-01-01

    In this work, a two-step extraction methodology of ionic liquid-based ultrasonic-assisted extraction (IL-UAE) and ionic liquid-based aqueous two-phase system (IL-ATPS) was developed for the extraction and purification of secoisolariciresinol diglucoside (SDG) from flaxseed. In the IL-UAE step, several kinds of ILs were investigated as the extractants, to identify the IL that affords the optimum extraction yield. The extraction conditions such as IL concentration, ultrasonic irradiation time, and liquid-solid ratio were optimized using response surface methodology (RSM). In the IL-ATPS step, ATPS formed by adding kosmotropic salts to the IL extract was used for further separation and purification of SDG. The most influential parameters (type and concentration of salt, temperature, and pH) were investigated to obtain the optimum extraction efficiency. The maximum extraction efficiency was 93.35% under the optimal conditions of 45.86% (w/w) IL and 8.27% (w/w) Na₂SO₄ at 22 °C and pH 11.0. Thus, the combination of IL-UAE and IL-ATPS makes up a simple and effective methodology for the extraction and purification of SDG. This process is also expected to be highly useful for the extraction and purification of bioactive compounds from other important medicinal plants. PMID:26437389

  1. Ionic Liquid-Based Ultrasonic-Assisted Extraction of Secoisolariciresinol Diglucoside from Flaxseed (Linum usitatissimum L.) with Further Purification by an Aqueous Two-Phase System.

    PubMed

    Tan, Zhi-Jian; Wang, Chao-Yun; Yang, Zi-Zhen; Yi, Yong-Jian; Wang, Hong-Ying; Zhou, Wan-Lai; Li, Fen-Fang

    2015-01-01

    In this work, a two-step extraction methodology of ionic liquid-based ultrasonic-assisted extraction (IL-UAE) and ionic liquid-based aqueous two-phase system (IL-ATPS) was developed for the extraction and purification of secoisolariciresinol diglucoside (SDG) from flaxseed. In the IL-UAE step, several kinds of ILs were investigated as the extractants, to identify the IL that affords the optimum extraction yield. The extraction conditions such as IL concentration, ultrasonic irradiation time, and liquid-solid ratio were optimized using response surface methodology (RSM). In the IL-ATPS step, ATPS formed by adding kosmotropic salts to the IL extract was used for further separation and purification of SDG. The most influential parameters (type and concentration of salt, temperature, and pH) were investigated to obtain the optimum extraction efficiency. The maximum extraction efficiency was 93.35% under the optimal conditions of 45.86% (w/w) IL and 8.27% (w/w) Na₂SO₄ at 22 °C and pH 11.0. Thus, the combination of IL-UAE and IL-ATPS makes up a simple and effective methodology for the extraction and purification of SDG. This process is also expected to be highly useful for the extraction and purification of bioactive compounds from other important medicinal plants.

  2. Functional expression, purification, and characterization of human Flt3 ligand in the Pichia pastoris system.

    PubMed

    Zhang, Yan-Li; Chen, Song-Sen; Yang, Ke-Gong; Su, Lin; Deng, Yan-Chun; Liu, Chang-Zheng

    2005-08-01

    Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30 mg/L. rhFL was purified to about 95% purity with overnight dialysis, filtration and an anion-exchange step. Further purification steps employing Sephacryl S-200 and reverse-phase HPLC raised the purity to over 99%. The purified rhFL possessed correct N-terminal amino acid sequence and positive Western blotting bands. SDS-PAGE and mass spectrometry analysis showed molecular weight of rhFL was about 21 and 34 kDa, suggesting that rhFL was glycosylated. The result of capillary electrophoresis showed that its pI is 3.12-4.72. Endo H deglycosylation analysis indicated that there was O-glycosylation besides N-glycosylation in rhFL secreted from P. pastoris. Bioactivity assay showed that the purified rhFL had dose-dependent expansion activity on bone marrow nucleated cells. PMID:15914030

  3. Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system.

    PubMed

    Finn, Michael W; Clayberger, Carol; Krensky, Alan M

    2011-01-01

    Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.

  4. Purification of bioethanol effluent in an UASB reactor system with simultaneous biogas formation.

    PubMed

    Torry-Smith, M; Sommer, P; Ahring, B K

    2003-10-01

    In this study, the prospect of using an Upflow Anaerobic Sludge Blanket (UASB) reactor for detoxification of process water derived from bioethanol production has been investigated. The bioethanol effluent (BEE) originated from wet oxidized wheat straw fermented by Saccharomyces cerevisiae and Thermoanaerobacter mathranii A3M4 to produce ethanol from glucose and xylose, respectively. In batch experiments the methane potential of BEE was determined to 529 mL-CH(4)/g-VS. In batch degradation experiments it was shown that the presence of BEE had a positive influence on the removal of the inhibitors 2-furoic acid, 4-hydroxyacetophenone, and acetovanillone as compared to conversion of the inhibitors as sole substrate in synthetic media. Furthermore, experiments were carried out treating BEE in a laboratory-scale UASB reactor. The results showed a Chemical Oxygen Demand (COD) removal of 80% (w/w) at an organic loading rate of 29 g-COD/(L. d). GC analysis of the lignocellulosic related potentially inhibitory compounds 2-furoic acid, vanillic acid, homovanillic acid, acetovanillone, syringic acid, acetosyringone, syringol, 4-hydroxybenzoic acid, and 4-hydroxybenzaldehyde showed that all of these compounds were removed from the BEE in the reactor. Implementation of a UASB purification step was found to be a promising approach to detoxify process water from bioethanol production allowing for recirculation of the process water and reduced production costs. PMID:12910537

  5. Application of the aqueous two-phase systems of ethylene and propylene oxide copolymer-maltodextrin for protein purification.

    PubMed

    Bolognese, Belén; Nerli, Bibiana; Picó, Guillermo

    2005-01-25

    In this study, the effect of several factors that govern the partitioning behaviour of three model proteins, such as bovine serum albumin, lysozyme and trypsin was analysed in a two-phase system formed by maltodextrin and a copolymer of ethylene and propylene oxides. The protein partition coefficient (K(r)) showed to be very sensitive to temperature changes, protein molecular weight, pH medium and the lyotropic ion presence. The phase diagram obtained for these novel polymer-polymer two-phase systems shows two phases with high polymer concentrations. The maltodextrin is enriched in the bottom phase while the copolymer of ethylene and propylene oxides is found in the upper phase. Since this copolymer is thermoreactive, the upper phase can be removed and heated above the copolymer's cloud point resulting in the formation of a new two-phase system with a lower water phase, containing the target protein and an upper copolymer-rich phase. Our results show that systems formed by maltodextrin and a copolymer of ethylene and propylene oxides may be considered as an interesting alternative to be used in protein purification due to their low cost, and also because they offer a viable solution to problems of polymer removal and recycling.

  6. Peri-implant and systemic effects of high-/low-affinity bisphosphonate-hydroxyapatite composite coatings in a rabbit model with peri-implant high bone turnover

    PubMed Central

    2012-01-01

    Background Hydroxyapatite (HA) coatings composed with bisphosphonates (BPs) which have high mineral-binding affinities have been confirmed to successfully enhance implant stability. However, few previous studies focused on HA coatings composed with low-affinity BPs or on systemic effects of locally released BPs. Methods In this long-term study, we developed two kinds of BP-HA composite coatings using either high-affinity BP (alendronate, ALN) or low-affinity BP (risedronate, RIS). Thirty-six rabbits were divided into three groups according to different coating applications (group I: HA, group II: ALN-HA, and group III: RIS-HA). Implants were inserted into the proximal region of the medullary cavity of the left tibiay. At insertion, 2 × 108 wear particles were injected around implants to induce a peri-implant high bone turnover environment. Both local (left tibias) and systemic (right tibias and lumbar vertebrae) inhibitory effect on bone resorption were compared, including bone-implant integration, bone architecture, bone mineral density (BMD), implant stability, and serum levels of bone turnover markers. Results The results indicated that ALN-HA composite coating, which could induce higher bone-implant contact (BIC) ratio, bone mass augmentation, BMD, and implant stability in the peri-implant region, was more potent on peri-implant bone, while RIS-HA composite coating, which had significant systemic effect, was more potent on non-peri-implant bone, especially lumbar vertebrae. Conclusions It is instructive and meaningful to further clinical studies that we could choose different BP-HA composite coatings according to the patient’s condition. PMID:22686414

  7. [Evaluation of the binding affinity and RNA interference of low-molecular-weight chitosan/siRNA complexes using an imaging system].

    PubMed

    Kawaguchi, Yasuhisa; Okuda, Tomoyuki; Ban, Tatsunori; Danjo, Kazumi; Okamoto, Hirokazu

    2009-04-01

    Chitosan is one of the attractive non-viral carriers for gene delivery including siRNA. However, common chitosan, which has a relatively high molecular weight, is insoluble in water, which might make it difficult to apply clinically. In this study, we investigated the efficacy of low-molecular-weight chitosan (LMWC), which is soluble in water, as a carrier for siRNA delivery. To evaluate the binding affinity and RNA interference (RNAi) of LMWC/siRNA complexes, a multi-well imaging system (IVIS) was adapted. CT26 cells stably expressing firefly luciferase (CT26/Luc cells) were established to evaluate RNAi. Evaluation of RNAi using lipofectamine(TM) 2000 was carried out by employing a luminometer with cell lysis and IVIS without cell lysis. The results were closely correlated, suggesting the advantages of the multi-well imaging system regarding screening, the visualization of results, and nondestructive evaluation. Fluorescence generated by ethidium bromide intercalated in the double strand of siRNA was markedly quenched at a higher ratio of LMWC to siRNA (N/P) and lower pH. Evaluation of the particle size and zeta potential of LMWC/siRNA complexes also indicated the higher binding affinity of LMWC with siRNA. At N/P=300 and pH 6.5, which satisfied the high-level binding affinity of LMWC with siRNA, significantly lower luminescence was detected in CT26/Luc cells treated with LMWC/siRNA compared with those treated with LMWC alone, suggesting the presence of RNAi. These results suggested that LMWC may be an effective carrier for siRNA delivery, and that the multi-well imaging system may be a powerful tool to evaluate the binding affinity and RNAi.

  8. Preparation and characterisation of surfaces properties of poly(hydroxyethylmethacrylate-co-methacrylolyamido-histidine) membranes: application for purification of human immunoglobulin G.

    PubMed

    Arica, M Yakup; Yalçin, Emine; Bayramoğlu, Gülay

    2004-08-01

    In this study, an affinity membrane containing L-histidine as an amino acid ligand was used in separation and purification of human immunoglobulin G (HIgG) from solution and human serum. The polarities and the surface free energies of the affinity membranes were determined by contact angle measurements. HIgG adsorption and purification onto the affinity membranes from aqueous solution and human serum were investigated in a batch and a continuous system. Effect of different system parameters such as ligand density, adsorbent dosage, pH, temperature, ionic strength and HIgG initial concentration on HIgG adsorption were investigated. The maximum adsorption capacity of p(HEMA-MAAH-4) membranes for HIgG was 13.06 mgml(-1). The reversible HIgG adsorption on the affinity membrane obeyed both the Langmuir and Freundlich isotherm models. The adsorption data was analysed using the first- and second-order kinetic model and the experimental data was well described by the first-order equations. In the continuous system, the purity of the eluted HIgG, as determined by HPLC, was 93% with recovery 58% for p(HEMA-MAAH-4) membrane. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.

  9. Californium purification and electrodeposition

    SciTech Connect

    Burns, Jonathan D.; Van Cleve, Shelley M.; Smith, Edward Hamilton; Boll, Rose Ann

    2014-11-30

    The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of the feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO3 and HCl, and Eichrom TEVA resin in NH4SCN. The TEVA NH4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.

  10. Probabilistic theories with purification

    SciTech Connect

    Chiribella, Giulio; D'Ariano, Giacomo Mauro; Perinotti, Paolo

    2010-06-15

    We investigate general probabilistic theories in which every mixed state has a purification, unique up to reversible channels on the purifying system. We show that the purification principle is equivalent to the existence of a reversible realization of every physical process, that is, to the fact that every physical process can be regarded as arising from a reversible interaction of the system with an environment, which is eventually discarded. From the purification principle we also construct an isomorphism between transformations and bipartite states that possesses all structural properties of the Choi-Jamiolkowski isomorphism in quantum theory. Such an isomorphism allows one to prove most of the basic features of quantum theory, like, e.g., existence of pure bipartite states giving perfect correlations in independent experiments, no information without disturbance, no joint discrimination of all pure states, no cloning, teleportation, no programming, no bit commitment, complementarity between correctable channels and deletion channels, characterization of entanglement-breaking channels as measure-and-prepare channels, and others, without resorting to the mathematical framework of Hilbert spaces.

  11. Californium purification and electrodeposition

    DOE PAGES

    Burns, Jonathan D.; Van Cleve, Shelley M.; Smith, Edward Hamilton; Boll, Rose Ann

    2014-11-30

    The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of themore » feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO3 and HCl, and Eichrom TEVA resin in NH4SCN. The TEVA NH4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.« less

  12. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  13. A yeast glycolipid biosurfactant, mannosylerythritol lipid, shows high binding affinity towards lectins on a self-assembled monolayer system.

    PubMed

    Konishi, Masaaki; Imura, Tomohiro; Fukuoka, Tokuma; Morita, Tomotake; Kitamoto, Dai

    2007-03-01

    Mannosylerythritol lipids (MEL), which are glycolipid biosurfactants secreted by the Pseudozyma yeasts, show not only excellent surface-active properties but also versatile biochemical actions including antitumor and cell-differentiation activities. In order to address the biochemical actions, interactions between MEL-A, the major component of MEL, and different lectins were investigated using the surface plasmon resonance spectroscopy. The monolayer of MEL-A showed high binding affinity to concanavalin A (ConA) and Maackia amurensis lectin-I (MAL-I). The observed affinity constants for ConA and MAL-I were estimated to be 9.48 +/- 1.31 x 10(6) and 3.13 +/- 0.274 x 10(6) M(-1), respectively; the value was comparable to that of Manalpha1-6(Manalpha1-3)Man, which is one of the most specific probe to ConA. Significantly, alpha-methyl-D-mannopyranoside (1 mM) exhibited no binding inhibition between MEL-A and ConA. MEL-A is thus likely to self-assemble to give a high affinity surface, where ConA binds to the hydrophilic headgroup in a different manner from that generally observed in lectin-saccharide interactions. The binding manner should be related with the biochemical actions of MEL toward mammalian cells via protein-carbohydrate interactions. PMID:17205206

  14. VLP production in Leishmania tarentolae: A novel expression system for purification and assembly of HPV16 L1.

    PubMed

    Bolhassani, Azam; Shirbaghaee, Zeinab; Agi, Elnaz; Davoudi, Noushin

    2015-12-01

    Viral like particles (VLPs) have been used as immunogen for improvement of preventive vaccines against several viral infections in preclinical and clinical trials. These constructs can stimulate both cellular and humoral immunity. Two prophylactic HPV L1 VLP vaccines known as Gardasil and Cervarix were commercialized worldwide. However, there are main problems for expression and purification of VLPs in eukaryotic expression systems such as baculovirus and yeast leading to high cost of these vaccines. A novel Leishmania protozoan system has been applied to produce different recombinant proteins due to unique properties including generation of similar proteins with mammalian, easy handling, and large-scale culture. In the current study, we developed a novel strategy to produce HPV L1 VLP using stably transfected Leishmania cells. The positive transfectants were analyzed by SDS-PAGE and Western blot analysis. The assembly of purified L1 protein was detected by TEM microscopy. Finally, C57BL/6 mice were immunized by crude VLPs and antibody responses were assessed. The results of electronic microscopy revealed average 55-60 nm for L1 VLP. Furthermore, high IgG1 and IgG2a antibody responses were generated by L1 VLPs in mice similar to L1 VLPs produced in baculovirus-infected insect cells. Regarding the results, the amount of recombinant protein generated by Leishmania was 2-3mg/500 ml media, suggesting further optimization of this system for using in large animals and human.

  15. A novel method for the purification of low soluble recombinant C-type lectin proteins.

    PubMed

    Yin, Chunhui; Jia, Ying; Garcia, Carlos A

    2012-08-31

    Snake venoms contain a complex mixture of many biological molecules including proteins. The purification of recombinant proteins is a key step in studying their function and structure with affinity chromatography as the common method used in their purification. In bacterial expression systems, hydrophobic recombinant proteins are usually precipitated into inclusion bodies, and contaminants are typically associated with tagged proteins after purification. The purpose of this study was to develop a procedure to purify hydrophobic recombinant proteins without an affinity tag. Snake venom mature C-type lectin-like proteins (CLPs) with a tag were cloned, expressed, and purified by repeated sonication and wash steps. The effects of the signal peptide on the expression and solubility of the recombinant protein were investigated. The CLPs in washed inclusion bodies were solubilized and refolded by dialysis. The CLPs without a tag were successfully purified with a yield 38 times higher than the traditional method, and inhibited blood platelet aggregation with an IC(50) of 100.57 μM in whole blood. This novel procedure is a rapid, and inexpensive method to purify functional recombinant hydrophobic CLPs from snake venoms useful in the development of drug therapies. PMID:22867876

  16. A versatile-deployable bacterial detection system for food and environmental safety based on LabTube-automated DNA purification, LabReader-integrated amplification, readout and analysis.

    PubMed

    Hoehl, Melanie M; Bocholt, Eva Schulte; Kloke, Arne; Paust, Nils; von Stetten, Felix; Zengerle, Roland; Steigert, Juergen; Slocum, Alexander H

    2014-06-01

    Contamination of foods is a public health hazard that episodically causes thousands of deaths and sickens millions worldwide. To ensure food safety and quality, rapid, low-cost and easy-to-use detection methods are desirable. Here, the LabSystem is introduced for integrated, automated DNA purification, amplification and detection. It consists of a disposable, centrifugally driven DNA purification platform (LabTube) and a low-cost UV/vis-reader (LabReader). For demonstration of the LabSystem in the context of food safety, purification of Escherichia coli (non-pathogenic E. coli and pathogenic verotoxin-producing E. coli (VTEC)) in water and milk and the product-spoiler Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice was integrated and optimized in the LabTube. Inside the LabReader, the purified DNA was amplified, readout and analyzed using both qualitative isothermal loop-mediated DNA amplification (LAMP) and quantitative real-time PCR. For the LAMP-LabSystem, the combined detection limits for purification and amplification of externally lysed VTEC and A. acidoterrestris are 10(2)-10(3) cell-equivalents. In the PCR-LabSystem for E. coli cells, the quantification limit is 10(2) cell-equivalents including LabTube-integrated lysis. The demonstrated LabSystem only requires a laboratory centrifuge (to operate the disposable, fully closed LabTube) and a low-cost LabReader for DNA amplification, readout and analysis. Compared with commercial DNA amplification devices, the LabReader improves sensitivity and specificity by the simultaneous readout of four wavelengths and the continuous readout during temperature cycling. The use of a detachable eluate tube as an interface affords semi-automation of the LabSystem, which does not require specialized training. It reduces the hands-on time from about 50 to 3 min with only two handling steps: sample input and transfer of the detachable detection tube.

  17. Purification of lipoteichoic acid by chromatography in water-organic solvent systems.

    PubMed Central

    Josephson, S L; Stinson, M W; Millar, S J; Cohen, R E

    1986-01-01

    Lipoteichoic acid (LTA), extracted from Streptococcus mutans 10449 by hot aqueous phenol, was partially purified by Sepharose 6B column chromatography in 0.01 M sodium acetate, pH 6.0, containing 0.25 M sodium chloride and 0.001 M EDTA. Nucleic acid and polysaccharide were precipitated from the LTA-containing column peak by the addition of 2 volumes of chloroform-methanol (1:5). The resulting single-phase chloroform-methanol-water (1:5:3) supernatant contained LTA and small amounts of several contaminating substances as indicated by reverse-phase high-pressure liquid chromatography and chemical analyses. LTA was purified further by DEAE-cellulose chromatography, using a concentration gradient of sodium chloride in chloroform-methanol-water (1:5:3). Two column peaks of LTA were found to contain phosphate, glycerol, glucose, and fatty acids at molar ratios of 1:1:0.11:0.10 and 1:1:0.09:0.04, respectively. The LTA polymers contained 18 and 22 repeating units of unsubstituted glycerophosphate and two glucose residues. The LTA in one column peak had two fatty acids per molecule, whereas that in the second peak contained only one. The yield of LTA was 1.68 mg per g of cell dry weight or 65 mg per g of phenol-water-extracted material. The specific activity of the LTA preparation was increased 128-fold by the purification scheme as determined by a erythrocyte-binding assay. Reverse-phase high-pressure liquid chromatography may be used for rapid separation of LTA molecules containing different numbers of acyl groups. PMID:3943892

  18. Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification.

    PubMed

    von Rechenberg, Moritz; Blake, Brian Kelly; Ho, Yew-Seng J; Zhen, Yuejun; Chepanoske, Cindy Lou; Richardson, Bonnie E; Xu, Nafei; Kery, Vladimir

    2005-05-01

    The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors. PMID:15761956

  19. Method for the Purification of Endogenous Unanchored Polyubiquitin Chains.

    PubMed

    Scott, Daniel; Strachan, Jo; Krishna, Varun Gopala; Shaw, Barry; Tooth, David J; Searle, Mark S; Oldham, Neil J; Layfield, Rob

    2016-01-01

    Unanchored polyubiquitin chains are endogenous non-substrate linked ubiquitin polymers which have emerging roles in the control of cellular physiology. We describe an affinity purification method based on an isolated ubiquitin-binding domain, the ZnF_UBP domain of the deubiquitinating enzyme USP5, which permits the selective purification of mixtures of endogenous unanchored polyubiquitin chains that are amenable to downstream molecular analyses. Further, we present methods for detection of unanchored polyubiquitin chains in purified fractions. PMID:27613037

  20. Abundance and distribution of microorganisms involved in denitrification in sediments of a Myriophyllum elatinoides purification system for treating swine wastewater.

    PubMed

    Li, Xi; Zhang, Miaomiao; Liu, Feng; Li, Yong; He, Yang; Zhang, Shunan; Wu, Jinshui

    2015-11-01

    Environmental pollution from livestock production, particularly swine production, is often managed by the use of constructed wetlands, which incorporate plants such as Myriophyllum elatinoides as a means of treating wastewater. The M. elatinoides purification system has been shown to effectively remove, via nitrification and denitrification, more than 90% of the total nitrogen (TN) and 84% of the NH4 (+)-N produced in swine wastewater. However, the mechanisms of variation in aquatic environmental factors and how the interaction of these factors affects denitrification by microorganisms in sediments remain poorly understood. In this study, the impacts of dissolved oxygen (DO), TN, NH4(+)-N, and NO3(-)-N on the abundance, diversity, and community distribution of denitrifiers in the sediments from different concentrations and types of wastewater including tap water (CK), two strengths of synthetic wastewater: 200 mg NH4(+)-N L(-1) (T1) and 400 mg NH4(+)-N L(-1) (T2), swine wastewater diluted 50% (T3), and swine wastewater (T4) were investigated in a microcosm experiment. A significant improvement was observed in the abundance of denitrification genes (nirK and nirS) in response to increased NO3(-)-N and DO in the swine wastewater sediments. The abundance of these denitrification genes was highest in the T4 sediments compared with other treatments. Terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the DO, TN, and NH4(+)-N positively impacted the richness index (S) of the nirK denitrifiers in T1, whereas the NO3(-)-N negatively affected the Simpson diversity index (D) of nirK and nirS denitrifiers in T3 and T4. However, the NO3(-)-N positively affected the nirK and nirS denitrifier community distribution, whereas the DO negatively affected the nirK and nirS denitrifier distribution in T3 and T4. These findings will be helpful in that they allow us to recognize the effects of environmental factors on the formation of the denitrifiers in