Sample records for rapid affinity purification

  1. Affinity purification of Car9-tagged proteins on silica matrices: Optimization of a rapid and inexpensive protein purification technology. (United States)

    Soto-Rodríguez, Jessica; Coyle, Brandon L; Samuelson, Ariana; Aravagiri, Kannan; Baneyx, François


    Car9, a dodecapeptide identified by cell surface display for its ability to bind to the edge of carbonaceous materials, also binds to silica with high affinity. The interaction can be disrupted with l-lysine or l-arginine, enabling a broad range of technological applications. Previously, we reported that C-terminal Car9 extensions support efficient protein purification on underivatized silica. Here, we show that the Car9 tag is functional and TEV protease-excisable when fused to the N-termini of target proteins, and that it supports affinity purification under denaturing conditions, albeit with reduced yields. We further demonstrate that capture of Car9-tagged proteins is enhanced on small particle size silica gels with large pores, that the concomitant problem of nonspecific protein adsorption can be solved by lysing cells in the presence of 0.3% Tween 20, and that efficient elution is achieved at reduced l-lysine concentrations under alkaline conditions. An optimized small-scale purification kit incorporating the above features allows Car9-tagged proteins to be inexpensively recovered in minutes with better than 90% purity. The Car9 affinity purification technology should prove valuable for laboratory-scale applications requiring rapid access to milligram-quantities of proteins, and for preparative scale purification schemes where cost and productivity are important factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag Technology. (United States)

    Hata, Toshiyuki; Nakayama, Manabu


    Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 microl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple, potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies.

  3. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification. (United States)

    Coyle, Brandon L; Baneyx, François


    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. © 2014 Wiley Periodicals, Inc.

  4. Rapid purification of circular DNA by triplex-mediated affinity capture (United States)

    Ji, H.; Smith, L.M.


    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.

  5. A rapid and simple Sep Pak method for purification of radioiodinated IQNP, a high affinity ligand for the muscarinic receptor

    Energy Technology Data Exchange (ETDEWEB)

    McPherson, D.W. E-mail:; Knapp, F.F


    A simplified procedure for the purification of 1-azabicyclo[2.2.2]oct-3-yl {alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IQNP) stereoisomers utilizing a silica Sep Pak (SSP) is described. Iodine-131-E- and iodine-125-Z-(R,R)-IQNP were isolated after SSP purification in 80% and 75% radiochemical yields, respectively. The biodistribution of iodine-131-E-/iodine-125-Z-(R,R)-IQNP, purified either by SSP or high performance liquid chromatography (HPLC), was evaluated in female rats and demonstrated no significant differences in the uptake in various organs and cerebral regions. The utilization of SSP thus affords a simple and rapid method for the purification of IQNP for use in a variety of animal studies.

  6. Protein purification by affinity precipitation. (United States)

    Hilbrig, Frank; Freitag, Ruth


    Developing the most efficient strategy for the purification of a (recombinant) protein especially at large scale remains a challenge. A typical problem of the downstream process of mammalian cell products is, for instance, the early capture of the highly diluted product from the complex process stream. Affinity precipitation has been suggested in this context. The technique is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation, but also because it seems ideally suited to specific product capture at large scale. The present review gives a comprehensive overview over this technique. Besides an introduction to the basic principle and a brief summary of the historical development, the main focus is on the current state-of-art of the technique, the available materials, important recent applications, as well as process design strategies and operating procedures. Special consideration is given to affinity precipitation for product recovery at large scale.

  7. Rapid purification of recombinant histones.

    Directory of Open Access Journals (Sweden)

    Henrike Klinker

    Full Text Available The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

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


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


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

  9. Solid support resins and affinity purification mass spectrometry. (United States)

    Havis, Spencer; Moree, Wilna J; Mali, Sujina; Bark, Steven J


    Co-affinity purification-mass spectrometry (CoAP-MS) is a primary technology for elucidating the protein-protein interactions that form the basis of all biological processes. A critical component of CoAP-MS is the affinity purification (AP) of the bait protein, usually by immobilization of an antibody to a solid-phase resin. This Minireview discusses common resins, reagents, tagging methods, and their consideration for successful AP of tagged proteins. We discuss our experiences with different solid supports, their impact in AP experiments, and propose areas where chemistry can advance this important technology.

  10. Affinity purification of recombinant human plasminogen activator ...

    African Journals Online (AJOL)

    Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk. Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen ...

  11. Antibody Fragments and Their Purification by Protein L Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Gustav Rodrigo


    Full Text Available Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteria—suggesting its consideration as a key unit operation in antibody fragment processing.

  12. Peptide affinity reagents for AAV capsid recognition and purification. (United States)

    Pulicherla, N; Asokan, A


    We report the discovery of AAV capsid-binding peptides identified through phage panning. The heptapeptide motif GYVSRHP selectively recognized AAV serotype 8 capsids and blocked transduction in vitro. Recombinant AAV8 vectors were purified directly from crude cell lysate and supernatant through sequential application of peptide affinity and anion exchange chromatography. Peptide affinity reagents may serve as useful alternatives to monoclonal antibodies in AAV capsid recognition, and offer readily scalable solutions for purification of clinical grade AAV vectors.

  13. Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis. (United States)

    Ahirwar, Rajesh; Nahar, Pradip


    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. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Frank eSainsbury


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

  15. Purification and affinity labeling of dihydropyridine receptor from rabbit skeletal muscle membranes

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    Kanngiesser, U.; Nalik, P.; Pongs, O.


    Undegraded dihydropyridine (DHP)-receptor (putatively a voltage-gated Ca/sup 2 +/ channel) has been purified as a 340-kDa protein complex to approx.80% homogeneity (2.4 nmol of DHP-receptor per mg of protein) from rabbit skeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-press treatment. The DHP-receptor complex was solubilized in 1% digitonin followed by a two step-chromatographic purification procedure. The equilibrium dissociation constant of (/sup 3/H) (+) -PN200-110 binding (K/sub d/; 0.9 nM) was not significantly changed by solubilization or purification. The purified DHP-receptor is composed of two subunits with apparent molecular masses of 148 kDa and 195 kDa migrating in polyacrylamide gels under nonreducing conditions as a single moiety of approx.300 kDa. The 195-kDa subunit was affinity-labeled with (/sup 3/H)azidopine in both transverse-tubule membranes and purified DHP-receptor preparations. The subunit can be degraded by high-energy irradiation to a 26-kDa peptide and by proteolysis to a 32-kDa peptide. Thus, it is probably due to proteolytic cleavage and/or photolysis that neither purification nor affinity-labeling studies have previously identified a DHP-receptor subunit of comparable molecular mass (195 kDa).

  16. Expression and affinity purification of recombinant proteins from plants (United States)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun


    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).

  17. The Expanding Toolkit of Translating Ribosome Affinity Purification. (United States)

    Dougherty, Joseph D


    Translating ribosome affinity purification is a method initially developed for profiling mRNA from genetically defined cell types in complex tissues. It has been applied both to identify target molecules in cell types that are important for controlling a variety of behaviors in the brain, and to understand the molecular consequences on those cells due to experimental manipulations, ranging from drugs of abuse to disease-causing mutations. Since its inception, a variety of methodological advances are opening new avenues of investigation. These advances include a variety of new methods for targeting cells for translating ribosome affinity purification by features such as their projections or activity, additional tags and mouse reagents increasing the flexibility of the system, and new modifications of the method specifically focused on studying the regulation of translation. The latter includes methods to assess cell type-specific regulation of translation in specific subcellular compartments. Here, I provide a summary of these recent advances and resources, highlighting both new experimental opportunities and areas for future technical development. Copyright © 2017 the authors 0270-6474/17/3712079-09$15.00/0.

  18. Affinity purification of soluble lysosomal proteins for mass spectrometric identification. (United States)

    Jaquinod, Sylvie Kieffer-; Chapel, Agnès; Garin, Jérôme; Journet, Agnøs


    This chapter describes the process of production, purification, separation, and mass spectrometry identification of soluble lysosomal proteins. The rationale for purification of these proteins resides in their characteristic sugar, the mannose-6-phosphate (M6P), which allows an easy purification by affinity chromatography on immobilized M6P receptor (MPR). The secretion of M6P proteins (essentially soluble lysosomal proteins) from cells in culture is induced by adding a weak base in the culture medium. Secreted proteins are ammonium sulfate precipitated, dialyzed, and loaded onto the immobilized MPR column. After specific elution and collection of the M6P proteins, these are resolved by either bidimensional or monodimensional gel electrophoresis (designated as 2-DE or 1-DE, respectively). Mass spectrometry analysis is performed on spots excised from the 2-DE gel, or on discrete bands covering altogether the whole length of the 1-DE gel lane: these spots or bands are in-gel digested with trypsin and protein identification is obtained, thanks to peptide mass fingerprints [provided by analysis of the digests by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS)] or peptide amino acid sequences (provided by analysis of the digests by the coupling between liquid chromatography and tandem mass spectrometry, LC-MS/MS).

  19. Protein purification with polymeric affinity membranes containing functionalized poly(acid) brushes. (United States)

    Jain, Parul; Vyas, Mukesh Kumar; Geiger, James H; Baker, Gregory L; Bruening, Merlin L


    Porous nylon membranes modified with poly(acid) brushes and their derivatives can rapidly purify proteins via ion-exchange and metal-ion affinity interactions. Membranes containing poly(2-(methacryloyloxy)ethyl succinate) (poly(MES)) brushes bind 118 +/- 8 mg of lysozyme per cm(3) of membrane and facilitate purification of lysozyme from chicken egg white. Moreover, functionalization of the poly(MES) brushes with nitrilotriacetate (NTA)-Ni(2+) complexes yields membranes that bind poly(histidine)-tagged (His-tagged) ubiquitin with a capacity of 85 +/- 2 mg of protein per cm(3) of membrane. Most importantly, the membranes modified with poly(MES)-NTA-Ni(2+) allow isolation of His-tagged cellular retinaldehyde-binding protein directly from a cell extract in membranes containing functionalized poly(MES) brushes are attractive candidates for rapid, high-capacity purification of His-tagged proteins from cell extracts.

  20. ELISA reagent coverage evaluation by affinity purification tandem mass spectrometry. (United States)

    Henry, Scott M; Sutlief, Elissa; Salas-Solano, Oscar; Valliere-Douglass, John


    Host cell proteins (HCPs) must be adequately removed from recombinant therapeutics by downstream processing to ensure patient safety, product quality, and regulatory compliance. HCP process clearance is typically monitored by enzyme-linked immunosorbent assay (ELISA) using a polyclonal reagent. Recently, mass spectrometry (MS) has been used to identify specific HCP process impurities and monitor their clearance. Despite this capability, ELISA remains the preferred analytical approach due to its simplicity and throughput. There are, however, inherent difficulties reconciling the protein-centric results of MS characterization with ELISA, or providing assurance that ELISA has acceptable coverage against all process-specific HCP impurities that could pose safety or efficacy risks. Here, we describe efficient determination of ELISA reagent coverage by proteomic analysis following affinity purification with a polyclonal anti-HCP reagent (AP-MS). The resulting HCP identifications can be compared with the actual downstream process impurities for a given process to enable a highly focused assessment of ELISA reagent suitability. We illustrate the utility of this approach by performing coverage evaluation of an anti-HCP polyclonal against both an HCP immunogen and the downstream HCP impurities identified in a therapeutic monoclonal antibody after Protein A purification. The overall goal is to strategically implement affinity-based mass spectrometry as part of a holistic framework for evaluating HCP process clearance, ELISA reagent coverage, and process clearance risks. We envision coverage analysis by AP-MS will further enable a framework for HCP impurity analysis driven by characterization of actual product-specific process impurities, complimenting analytical methods centered on consideration of the total host cell proteome.

  1. Rapid Buffer and Ligand Screening for Affinity Chromatography by Multiplexed Surface Plasmon Resonance Imaging

    NARCIS (Netherlands)

    Geuijen, K.P.M.; Wijk-Basten, van Danielle E.J.W.; Egging, Davis F.; Schasfoort, Richard B.M.; Eppink, M.H.M.


    Protein purifications are often based on the principle of affinity chromatography, where the protein of interest selectively binds to an immobilized ligand. The development of affinity purification requires selecting proper wash and elution conditions. In recent years, miniaturization of the

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

    Energy Technology Data Exchange (ETDEWEB)

    Giannone, Richard J [ORNL; McDonald, W Hayes [ORNL; Hurst, Gregory {Greg} B [ORNL; Huang, Ying [ORNL; Wu, Jun [ORNL; Liu, Yie [National Institute on Aging, Baltimore; Wang, Yisong [ORNL


    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.

  3. A rapid one step purification procedure for murine IgD based on the specific affinity of Bandeiraea (Griffonia) simplicifolia-1 for N-linked carbohydrates on IgD. (United States)

    Oppenheim, J D; Amin, A R; Thorbecke, G J


    The alpha-D-galactopyranosyl binding lectin from the seeds of Bandeiraea simplicifolia (a.k.a. Griffonia simplicifolia) termed BS-I, strongly reacts with murine IgD and with no other protein in ascites including all other classes of immunoglobulins as determined by immunoprecipitation, hemagglutination inhibition and affinity binding. Based on this finding, murine IgD could be rapidly purified directly from whole ascitic fluid by passage over affinity beads of BS-I linked to Sepharose 4B and subsequent elution by a buffer containing 0.1 M D-galactose. The sugar eluted product is 95-99% pure as determined by SDS-PAGE and represents 90-95% of the total IgD in the initial ascites by ELISA assay. Both monomeric and dimeric murine IgD may be purified by this procedure. Human IgD is unreactive with this lectin. Treatment of purified IgD with endoglycosidases that remove either O- or N-linked glycosides indicates that BS-I binds to IgD only via N-linked carbohydrate chains.

  4. Development of an aptamer-based affinity purification method for vascular endothelial growth factor

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    Maren Lönne


    Full Text Available Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.

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

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


    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.

  6. Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant. (United States)

    Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

    To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function. Published by Elsevier Masson SAS.

  7. Robotic high-throughput purification of affinity-tagged recombinant proteins. (United States)

    Wiesler, Simone C; Weinzierl, Robert O J


    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.

  8. Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry. (United States)

    Huang, He; Alvarez, Sophie; Bindbeutel, Rebecca; Shen, Zhouxin; Naldrett, Michael J; Evans, Bradley S; Briggs, Steven P; Hicks, Leslie M; Kay, Steve A; Nusinow, Dmitri A


    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

  9. Peptide affinity reagents for AAV capsid recognition and purification


    Pulicherla, N; Asokan, A


    We report the discovery of AAV capsid-binding peptides identified through phage panning. The heptapeptide motif GYVSRHP selectively recognized AAV serotype 8 capsids and blocked transduction in vitro. Recombinant AAV8 vectors were purified directly from crude cell lysate and supernatant through sequential application of peptide affinity and anion exchange chromatography. Peptide affinity reagents may serve as useful alternatives to monoclonal antibodies in AAV capsid recognition, and offer re...

  10. Efficient purification of unique antibodies using peptide affinity-matrix columns

    DEFF Research Database (Denmark)

    Jensen, Liselotte Brix; Riise, Erik; Nielsen, Leif Kofoed


    -99. Several peptide epitopes were identified and all of them recognised specifically MK16. One peptide, ER6.1, was selected and linked to beaded agarose and demonstrated excellent performance as a peptide affinity chromatography matrix. This epitope matrix was efficient in the purification of MK16 Fab...

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  12. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation. (United States)

    Arnold, Lindsay; Chen, Rachel


    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.

  13. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans


    Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively af......, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displaced by the immunizing but not by unrelated peptides....

  14. Affinity purification combined with mass spectrometry to identify herpes simplex virus protein-protein interactions. (United States)

    Meckes, David G


    The identification and characterization of herpes simplex virus protein interaction complexes are fundamental to understanding the molecular mechanisms governing the replication and pathogenesis of the virus. Recent advances in affinity-based methods, mass spectrometry configurations, and bioinformatics tools have greatly increased the quantity and quality of protein-protein interaction datasets. In this chapter, detailed and reliable methods that can easily be implemented are presented for the identification of protein-protein interactions using cryogenic cell lysis, affinity purification, trypsin digestion, and mass spectrometry.

  15. A dual-tagging system for the affinity purification of mammalian protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Giannone, Richard J [ORNL; McDonald, W Hayes [ORNL; Hurst, Gregory {Greg} B [ORNL; Huang, Ying [ORNL; Wu, Jun [ORNL; Liu, Yie [National Institute on Aging, Baltimore; Wang, Yisong [ORNL


    One popular method to elucidate protein-protein interactions involves the native co-purification of an affinity tagged protein and its interacting partners, which are subsequently identified through mass spectrometry (MS) (1). Although straightforward, reproducible, and broadly employed, this strategy is hampered by the efficacy of protein recoveries both in terms of sensitivity and specificity. This is especially pertinent to methodologies that employ a single-step of purification, where suboptimal enrichment of the bait protein and its partners over background can lead to masking of their signals. Although improvements to MS instrumentation generally increase peptide detection sensitivities, the problem of specificity, i.e. distinguishing specific from non-specific interacting proteins, remains. Thus ultimately, the limiting factor in the identification of specific interacting proteins lies with the purification itself. An effort to resolve this specificity issue has been made with the introduction of the Tandem Affinity Purification (TAP) tag. This construct consists of an IgG-binding domain and calmodulin binding peptide domain separated by a tobacco etch virus (TEV) protease cleavage site (2). The TAP method was originally developed in yeast and has best demonstrated its utility in the systematic identification of numerous multiprotein complexes in the yeast proteome (3). Although modifications to the original TAP have been successful in examining the protein networks of mammalian cells (4-7), the strategy offers a relatively low yield of bait and specific interacting proteins (8), and the success rate are usually on case-by-case basis. In addition, problems inherent to any protein tagging strategy remain, such as variable exposure of the affinity tag, disruption of the bait protein's ability to fold properly, steric exclusion of interacting partners, and/or ectopic overexpression of the fusion protein, which can lead to complications in both the

  16. Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag. (United States)

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


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

  17. SFINX: Straightforward Filtering Index for Affinity Purification-Mass Spectrometry Data Analysis. (United States)

    Titeca, Kevin; Meysman, Pieter; Gevaert, Kris; Tavernier, Jan; Laukens, Kris; Martens, Lennart; Eyckerman, Sven


    Affinity purification-mass spectrometry is one of the most common techniques for the analysis of protein-protein interactions, but inferring bona fide interactions from the resulting data sets remains notoriously difficult. We introduce SFINX, a Straightforward Filtering INdeX that identifies true-positive protein interactions in a fast, user-friendly, and highly accurate way. SFINX outperforms alternative techniques on two benchmark data sets and is available via the Web interface at

  18. Purification of plant-derived antibodies through direct immobilization of affinity ligands on cellulose. (United States)

    Hussack, Greg; Grohs, Brittany M; Almquist, Kurt C; McLean, Michael D; Ghosh, Raja; Hall, J Christopher


    Plants possess enormous potential as factories for the large scale production of therapeutic reagents such as recombinant proteins and antibodies. A major factor limiting commercial advances of plant-derived pharmaceuticals is the cost and inefficiency of purification. As a model system, we have developed a simple yet robust method for immobilizing affinity capture ligands onto solid supports by interfacing the secreted expression and coupling of a chimeric fusion protein in Pichia pastoris to microcrystalline cellulose in a single step. The fusion protein, which consisted of antibody-binding proteins L and G fused to a cellulose-binding domain (LG-CBD), was tethered directly onto cellulose resins added to P. pastoris cultures and subsequently used for antibody purification. Both the antibody-binding protein L and protein G domains were functional, as demonstrated by the ability of cellulose-immobilized LG-CBD to purify both a scFv antibody fragment from yeast and a human IgG1 monoclonal antibody from transgenic tobacco. Furthermore, combining two P. pastoris strains expressing LG-CBD and scFv with CP-102 cellulose in a single culture allowed for easy recovery of biologically active scFv. Direct immobilization of affinity purification ligands, such as LG-CBD, onto inexpensive support matrices such as cellulose is an effective method for the generation of functional, single-use antibody purification reagents. Straightforward preparation of purification reagents will help make antibody purification from genetically modified crop plants feasible and address one of the major bottlenecks facing commercialization of plant-derived pharmaceuticals.

  19. A dual protease approach for expression and affinity purification of recombinant proteins. (United States)

    Raran-Kurussi, Sreejith; Waugh, David S


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

  20. Chromatin Affinity Purification and Quantitative Mass Spectrometry Defining the Interactome of Histone Modification Patterns* (United States)

    Nikolov, Miroslav; Stützer, Alexandra; Mosch, Kerstin; Krasauskas, Andrius; Soeroes, Szabolcs; Stark, Holger; Urlaub, Henning; Fischle, Wolfgang


    DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated. PMID:21836164

  1. Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification

    Directory of Open Access Journals (Sweden)

    Chen Guo


    Full Text Available Abstract Background PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation. Results Fusion protein PhaR-intein-target protein was expressed in recombinant Escherichia coli. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target protein was released from the particles and purified after centrifugation. As target proteins, enhanced green fluorescent protein (EGFP, maltose binding protein (MBP and β-galactosidase (lacZ, were successfully purified using the PhaR based protein purification method. Conclusion The successful purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based in vitro purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple protein purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial protein purification systems.

  2. Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags. (United States)

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


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

  3. Affinity purification of recombinant proteins using a novel silica-binding peptide as a fusion tag. (United States)

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


    We recently reported that silica is deposited on the coat of Bacillus cereus spores as a layer of nanometer-sized particles (Hirota et al. 2010 J Bacteriol 192: 111-116). Gene disruption analysis revealed that the spore coat protein CotB1 mediates the accumulation of silica (our unpublished results). Here, we report that B. cereus CotB1 (171 amino acids [aa]) and its C-terminal 14-aa region (corresponding to residues 158-171, designated CotB1p) show strong affinity for silica particles, with dissociation constants at pH 8.0 of 2.09 and 1.24 nM, respectively. Using CotB1 and CotB1p as silica-binding tags, we developed a silica-based affinity purification method in which silica particles are used as an adsorbent for CotB1/CotB1p fusion proteins. Small ubiquitin-like modifier (SUMO) technology was employed to release the target proteins from the adsorbed fusion proteins. SUMO-protease-mediated site-specific cleavage at the C-terminus of the fused SUMO sequence released the tagless target proteins into the liquid phase while leaving the tag region still bound to the solid phase. Using the fluorescent protein mCherry as a model, our purification method achieved 85 % recovery, with a purity of 95 % and yields of 0.60 ± 0.06 and 1.13 ± 0.13 mg per 10-mL bacterial culture for the CotB1-SUMO-mCherry and CotB1p-SUMO-mCherry fusions, respectively. CotB1p, a short 14-aa peptide, which demonstrates high affinity for silica, could be a promising fusion tag for both affinity purification and enzyme immobilization on silica supports.

  4. Partial Purification of the 5-Hydroxytryptamine-Reuptake System from Human Blood Platelets Using a Citalopram-Derived Affinity Resin

    NARCIS (Netherlands)

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


    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

  5. Observations on different resin strategies for affinity purification mass spectrometry of a tagged protein. (United States)

    Mali, Sujina; Moree, Wilna J; Mitchell, Morgan; Widger, William; Bark, Steven J


    Co-affinity purification mass spectrometry (CoAP-MS) is a highly effective method for identifying protein complexes from a biological sample and inferring important interactions, but the impact of the solid support is usually not considered in design of such experiments. Affinity purification (AP) experiments typically utilize a bait protein expressing a peptide tag such as FLAG, c-Myc, HA or V5 and high affinity antibodies to these peptide sequences to facilitate isolation of a bait protein to co-purify interacting proteins. We observed significant variability for isolation of tagged bait proteins between Protein A/G Agarose, Protein G Dynabeads, and AminoLink resins. While previous research identified the importance of tag sequence and their location, crosslinking procedures, reagents, dilution, and detergent concentrations, the effect of the resin itself has not been considered. Our data suggest the type of solid support is important and, under the conditions of our experiments, AminoLink resin provided a more robust solid-support platform for AP-MS. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

    DEFF Research Database (Denmark)

    Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan


    GABA(B) receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter gamma-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABA(B1), are thought to involve a differential set of associated proteins. Here, we describe two mouse...... lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice...

  7. Single-step affinity purification of recombinant proteins using the silica-binding Si-tag as a fusion partner. (United States)

    Ikeda, Takeshi; Ninomiya, Ken-ichi; Hirota, Ryuichi; Kuroda, Akio


    We previously reported that a silica-binding protein, designated Si-tag, can be used as a fusion tag to immobilize functional proteins on silica surfaces. In this study, by taking advantage of the strong affinity of Si-tag for silica, we developed a single-step purification method for Si-tagged fusion proteins. We utilized unmodified bare silica particles as a specific adsorbent and a high concentration of MgCl(2) solution as an elution buffer. A fusion protein of Si-tag and immunoglobulin-binding staphylococcal protein A, designated Si-tagged protein A, was recovered with a purity of 87+/-3% and yield of 84+/-4% from a crude extract of recombinant Escherichia coli. The simplicity of our method enables rapid, cost-effective purification of Si-tagged fusion proteins. We also discuss the mechanism of binding and dissociation of Si-tag and silica surfaces, and we suggest that the unusual basicity and disordered structure of the Si-tag polypeptide play important roles in the binding to silica. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  8. Intelligent Mixing of Proteomes for Elimination of False Positives in Affinity Purification-Mass Spectrometry. (United States)

    Eyckerman, Sven; Impens, Francis; Van Quickelberghe, Emmy; Samyn, Noortje; Vandemoortele, Giel; De Sutter, Delphine; Tavernier, Jan; Gevaert, Kris


    Protein complexes are essential in all organizational and functional aspects of the cell. Different strategies currently exist for analyzing such protein complexes by mass spectrometry, including affinity purification (AP-MS) and proximal labeling-based strategies. However, the high sensitivity of current mass spectrometers typically results in extensive protein lists mainly consisting of nonspecifically copurified proteins. Finding the true positive interactors in these lists remains highly challenging. Here, we report a powerful design based on differential labeling with stable isotopes combined with nonequal mixing of control and experimental samples to discover bona fide interaction partners in AP-MS experiments. We apply this intelligent mixing of proteomes (iMixPro) concept to overexpression experiments for RAF1, RNF41, and TANK and also to engineered cell lines expressing epitope-tagged endogenous PTPN14, JIP3, and IQGAP1. For all baits, we confirmed known interactions and found a number of novel interactions. The results for RNF41 and TANK were compared to a classical affinity purification experiment, which demonstrated the efficiency and specificity of the iMixPro approach.

  9. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies (United States)

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


    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.

  10. Parallel Exploration of Interaction Space by BioID and Affinity Purification Coupled to Mass Spectrometry. (United States)

    Hesketh, Geoffrey G; Youn, Ji-Young; Samavarchi-Tehrani, Payman; Raught, Brian; Gingras, Anne-Claude


    Complete understanding of cellular function requires knowledge of the composition and dynamics of protein interaction networks, the importance of which spans all molecular cell biology fields. Mass spectrometry-based proteomics approaches are instrumental in this process, with affinity purification coupled to mass spectrometry (AP-MS) now widely used for defining interaction landscapes. Traditional AP-MS methods are well suited to providing information regarding the temporal aspects of soluble protein-protein interactions, but the requirement to maintain protein-protein interactions during cell lysis and AP means that both weak-affinity interactions and spatial information is lost. A more recently developed method called BioID employs the expression of bait proteins fused to a nonspecific biotin ligase, BirA*, that induces in vivo biotinylation of proximal proteins. Coupling this method to biotin affinity enrichment and mass spectrometry negates many of the solubility and interaction strength issues inherent in traditional AP-MS methods, and provides unparalleled spatial context for protein interactions. Here we describe the parallel implementation of both BioID and FLAG AP-MS allowing simultaneous exploration of both spatial and temporal aspects of protein interaction networks.

  11. Nickel-Salen supported paramagnetic nanoparticles for 6-His-target recombinant protein affinity purification. (United States)

    Rashid, Zahra; Ghahremanzadeh, Ramin; Nejadmoghaddam, Mohammad-Reza; Nazari, Mahboobeh; Shokri, Mohammad-Reza; Naeimi, Hossein; Zarnani, Amir-Hassan


    In this research, a simple, efficient, inexpensive, rapid and high yield method for the purification of 6×histidine-tagged recombinant protein was developed. For this purpose, manganese ferrite magnetic nanoparticles (MNPs) were synthesized through a co-precipitation method and then they were conveniently surface-modified with tetraethyl orthosilicate (TEOS) in order to prevent oxidation and form high density of hydroxyl groups. Next, the salen ligand was prepared from condensation reaction of salicylaldehyde and 3-aminopropyl (trimethoxy) silane (APTMS) in 1:1 molar ratio; followed by complexation with Ni(OAc) 2 .4H 2 O. Finally, the prepared Ni(II)-salen complex conjugated to silica coated MNPs and MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles were obtained. The functionalized nanoparticles were spherical with an average diameter around 70nm. The obtained MNPs had a saturation magnetization about 54 emu/g and had super paramagnetic character. These MNPs were used efficiently to enrich recombinant histidine-tagged (His-tagged) protein-A from bacterial cell lysate. In about 45min, highly pure His-tagged recombinant protein was obtained, as judged by SDS-PAGE analysis and silver staining. The amount of target protein in flow through and washing fractions was minimal denoting the high efficiency of purification process. The average capacity of the matrix was found to be high and about 180±15mgg -1 (protein/MnFe 2 O 4 @SiO 2 @Ni-Salen complex). Collectively, purification process with MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles is rapid, efficient, selective and whole purification can be carried out in only a single tube without the need for expensive systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification

    DEFF Research Database (Denmark)

    Mullari, Meeli; Lyon, David; Jensen, Lars Juhl


    RNA-binding proteins (RBPs) allow cells to carry out pre-RNA processing and post-transcriptional regulation of gene expression, and aberrations in RBP functions have been linked to many diseases, including neurological disorders and cancer. Human cells encode thousands of RNA-binding proteins...... with unique RNA-binding properties. These properties are regulated through modularity of a large variety of RNA-binding domains, rendering RNA-protein interactions difficult to study. Recently, the introduction of proteomics methods has provided novel insights into RNA-binding proteins at a systems level....... However, determining the exact protein sequence regions that interact with RNA remains challenging and laborious, especially considering that many RBPs lack canonical RNA-binding domains. Here we describe a streamlined proteomic workflow called peptide cross-linking and affinity purification (p...

  13. Identification of Tyrosine Phosphorylated Proteins by SH2 Domain Affinity Purification and Mass Spectrometry. (United States)

    Buhs, Sophia; Gerull, Helwe; Nollau, Peter


    Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry. In combination with SH2 profiling, a broadly applicable platform for the characterization of phosphotyrosine profiles in cell extracts, our pull down strategy enables researchers by now to identify proteins in signaling cascades which are differentially phosphorylated and selectively recognized by distinct SH2 domains.

  14. Mapping Protein-Protein Interactions Using Affinity Purification and Mass Spectrometry. (United States)

    Lee, Chin-Mei; Adamchek, Christopher; Feke, Ann; Nusinow, Dmitri A; Gendron, Joshua M


    The mapping of protein-protein interaction (PPI) networks and their dynamics are crucial steps to deciphering the function of a protein and its role in cellular pathways, making it critical to have comprehensive knowledge of a protein's interactome. Advances in affinity purification and mass spectrometry technology (AP-MS) have provided a powerful and unbiased method to capture higher-order protein complexes and decipher dynamic PPIs. However, the unbiased calling of nonspecific interactions and the ability to detect transient interactions remains challenging when using AP-MS, thereby hampering the detection of biologically meaningful complexes. Additionally, there are plant-specific challenges with AP-MS, such as a lack of protein-specific antibodies, which must be overcome to successfully identify PPIs. Here we discuss and describe a protocol designed to bypass the traditional challenges of AP-MS and provide a roadmap to identify bona fide PPIs in plants.

  15. Scoring Large-Scale Affinity Purification Mass Spectrometry Datasets with MiST. (United States)

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


    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. Copyright © 2015 John Wiley & Sons, Inc.

  16. Affinity Purification of O-Acetylserine(thiollyase from Chlorella sorokiniana by Recombinant Proteins from Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Giovanna Salbitani


    Full Text Available In the unicellular green alga Chlorella sorokiniana (211/8 k, the protein O-acetylserine(thiollyase (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.

  17. Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain. (United States)

    Mahmood, Rubab


    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.

  18. Accurate Protein Complex Retrieval by Affinity Enrichment Mass Spectrometry (AE-MS) Rather than Affinity Purification Mass Spectrometry (AP-MS)


    Keilhauer, E.; Hein, M; Mann, M


    Protein?protein interactions are fundamental to the understanding of biological processes. Affinity purification coupled to mass spectrometry (AP-MS) is one of the most promising methods for their investigation. Previously, complexes were purified as much as possible, frequently followed by identification of individual gel bands. However, todays mass spectrometers are highly sensitive, and powerful quantitative proteomics strategies are available to distinguish true interactors from backgroun...

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

    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


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

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

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


    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. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Klotz, K.L.


    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.

  2. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography. (United States)

    Salehi, Nasrin; Peng, Ching-An


    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. © 2016 American Institute of Chemical Engineers.

  3. Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

    Directory of Open Access Journals (Sweden)

    Shangyong Li


    Full Text Available Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the 4-aminophenylboronic acid (4-APBA as affinity ligand, which was coupled with Sepharose 6B via cyanuric chloride as spacer. The molecular docking analysis showed that the boron atom was interacting with the hydroxyl group of Ser176 residue, whereas the hydroxyl group of the boronic moiety is oriented toward Leu175 and His177 residues. In addition to the covalent bond between the boron atom and hydroxyl group of Ser176, the spacer between boronic acid derivatives and medium beads contributes to the formation of an enzyme-medium complex. With this synthesized medium, we developed and optimized a one-step purification procedure and applied it for the affinity purification of metalloproteases from three commercial enzyme products. The native metalloproteases were purified to high homogeneity with more than 95% purity. The novel purification method developed in this work provides new opportunities for scientific, industrial and pharmaceutical projects.

  4. Efficient expression of codon-adapted affinity tagged super folder green fluorescent protein for synchronous protein localization and affinity purification studies in Tetrahymena thermophila. (United States)

    Yilmaz, Gürkan; Arslanyolu, Muhittin


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

  5. Affinity chromatography with pseudobiospecific ligands on high-performance supports for purification of proteins of biotechnological interest

    Directory of Open Access Journals (Sweden)

    N.B. Iannucci


    Full Text Available High-performance affinity matrices were obtained by attaching pseudobiospecific ligands to hollow-fibre membranes. The neutral protease contained in FlavourzymeTM was purified to homogeneity with Yellow 4R-HE affinity hollow-fibre membranes. Immobilisation of Red HE-3B allowed purification of a milk-clotting enzyme obtained by solid-state culture of Mucor bacilliformis. Copper immobilisation through iminodiacetic acid allowed fractionation of Biocon Bioconcentrated PlusTM to separate the pectinesterase-containing fraction. The productivity of the developed processes - 1900, 94 and 750 U/ml.min, respectively - was 10- to 15-fold higher than that achieved with the same ligands immobilised on agarose-based soft gels, mainly due to the shortening of the purification processes.

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

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


    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).

  7. Purification of papain by metal affinity partitioning in aqueous two-phase polyethylene glycol/sodium sulfate systems. (United States)

    Jiang, Zhi-Guo; Zhang, Hai-De; Wang, Wei-Tao


    A simple and inexpensive aqueous two-phase affinity partitioning system using metal ligands was introduced to improve the selectivity of commercial papain extraction. Polyethylene glycol 4000 was first activated using epichlorohydrin, then it was covalently linked to iminodiacetic acid. Finally, the specific metal ligand Cu(2+) was attached to the polyethylene glycol-iminodiacetic acid. The chelated Cu(2+) content was measured by atomic absorption spectrometry as 0.88 mol/mol (polyethylene glycol). The effects on the purification at different conditions, including polyethylene glycol molecular weight (2000, 4000, and 6000), concentration of phase-forming components (polyethylene glycol 12-20% w/w and sodium sulfate 12-20%, w/w), metal ligand type, and concentration, system pH and the commercial papain loading on papain extraction, were systematically studied. Under optimum conditions of the system, i.e. 18% w/w sodium sulfate, 18% w/w polyethylene glycol 4000, 1% w/w polyethylene glycol-iminodiacetic acid-Cu(2+) and pH 7, a maximum yield of 90.3% and a degree of purification of 3.6-fold were obtained. Compared to aqueous two phase extraction without ligands, affinity partitioning was found to be an effective technique for the purification of commercial papain with higher extraction efficiency and degree of purification. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. HB tag modules for PCR-based gene tagging and tandem affinity purification in Saccharomyces cerevisiae. (United States)

    Tagwerker, Christian; Zhang, Hongwei; Wang, Xiaorong; Larsen, Liza S Z; Lathrop, Richard H; Hatfield, G Wesley; Auer, Bernhard; Huang, Lan; Kaiser, Peter


    We have recently developed the HB tag as a useful tool for tandem-affinity purification under native as well as fully denaturing conditions. The HB tag and its derivatives consist of a hexahistidine tag and a bacterially-derived in vivo biotinylation signal peptide, which support sequential purification by Ni2+ -chelate chromatography and binding to immobilized streptavidin. To facilitate tagging of budding yeast proteins with HB tags, we have created a series of plasmids with various selectable markers. These plasmids allow single-step PCR-based tagging and expression under control of the endogenous promoters or the inducible GAL1 promoter. HB tagging of several budding yeast ORFs demonstrated efficient biotinylation of the HB tag in vivo by endogenous yeast biotin ligases. No adverse effects of the HB tag on protein function were observed. The HB tagging plasmids presented here are related to previously reported epitope-tagging plasmids, allowing PCR-based tagging with the same locus-specific primer sets that are used for other widely used epitope-tagging strategies. The Sequences for the described plasmids were submitted to GenBank under Accession Numbers DQ407918-pFA6a-HBH-kanMX6 DQ407927-pFA6a-RGS18H-kanMX6 DQ407919-pFA6a-HBH-hphMX4 DQ407928-pFA6a-RGS18H-hphMX4 DQ407920-pFA6a-HBH-TRP1 DQ407929-pFA6a-RGS18H-TRP1 DQ407921-pFA6a-HTB-kanMX6 DQ407930-pFA6a-kanMX6-PGAL1-HBH DQ407922-pFA6a-HTB-hphMX4 DQ407931-pFA6a-TRP1-PGAL1-HBH DQ407923-pFA6a-HTB-TRP1 DQ407924-pFA6a-BIO-kanMX6 DQ407925-pFA6a-BIO-hphMX4 DQ407926-pFA6a-BIO-TRP1. Copyright 2006 John Wiley & Sons, Ltd.

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

    Energy Technology Data Exchange (ETDEWEB)

    Biessen, E.A.L; Horn, A.S.; Robillard, G.T. (Univ. of Groningen (Netherlands))


    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.

  10. Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography. (United States)

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


    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography. (United States)

    Dong, Xiao-Yan; Chen, Li-Jun; Sun, Yan


    This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and beta-cyclodextrin (beta-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein-CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing beta-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6-0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.

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

    Directory of Open Access Journals (Sweden)

    Buhrman Jason S


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

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

    Directory of Open Access Journals (Sweden)

    Yangchao Dong


    Full Text Available 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.

  14. Comprehensive Characterization of Minichromosome Maintenance Complex (MCM) Protein Interactions Using Affinity and Proximity Purifications Coupled to Mass Spectrometry. (United States)

    Dubois, Marie-Line; Bastin, Charlotte; Lévesque, Dominique; Boisvert, François-Michel


    The extensive identification of protein-protein interactions under different conditions is an important challenge to understand the cellular functions of proteins. Here we use and compare different approaches including affinity purification and purification by proximity coupled to mass spectrometry to identify protein complexes. We explore the complete interactome of the minichromosome maintenance (MCM) complex by using both approaches for all of the different MCM proteins. Overall, our analysis identified unique and shared interaction partners and proteins enriched for distinct biological processes including DNA replication, DNA repair, and cell cycle regulation. Furthermore, we mapped the changes in protein interactions of the MCM complex in response to DNA damage, identifying a new role for this complex in DNA repair. In summary, we demonstrate the complementarity of these approaches for the characterization of protein interactions within the MCM complex.

  15. Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography

    Directory of Open Access Journals (Sweden)

    Geisler Markus


    Full Text Available In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993 et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 &mgr;M and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

  16. Discovery of protein complexes with core-attachment structures from Tandem Affinity Purification (TAP) data. (United States)

    Wu, Min; Li, Xiao-Li; Kwoh, Chee-Keong; Ng, See-Kiong; Wong, Limsoon


    Many cellular functions involve protein complexes that are formed by multiple interacting proteins. Tandem Affinity Purification (TAP) is a popular experimental method for detecting such multi-protein interactions. However, current computational methods that predict protein complexes from TAP data require converting the co-complex relationships in TAP data into binary interactions. The resulting pairwise protein-protein interaction (PPI) network is then mined for densely connected regions that are identified as putative protein complexes. Converting the TAP data into PPI data not only introduces errors but also loses useful information about the underlying multi-protein relationships that can be exploited to detect the internal organization (i.e., core-attachment structures) of protein complexes. In this article, we propose a method called CACHET that detects protein complexes with Core-AttaCHment structures directly from bipartitETAP data. CACHET models the TAP data as a bipartite graph in which the two vertex sets are the baits and the preys, respectively. The edges between the two vertex sets represent bait-prey relationships. CACHET first focuses on detecting high-quality protein-complex cores from the bipartite graph. To minimize the effects of false positive interactions, the bait-prey relationships are indexed with reliability scores. Only non-redundant, reliable bicliques computed from the TAP bipartite graph are regarded as protein-complex cores. CACHET constructs protein complexes by including attachment proteins into the cores. We applied CACHET on large-scale TAP datasets and found that CACHET outperformed existing methods in terms of prediction accuracy (i.e., F-measure and functional homogeneity of predicted complexes). In addition, the protein complexes predicted by CACHET are equipped with core-attachment structures that provide useful biological insights into the inherent functional organization of protein complexes. Our supplementary material can

  17. sfinx: an R package for the elimination of false positives from affinity purification-mass spectrometry datasets. (United States)

    Titeca, Kevin; Meysman, Pieter; Laukens, Kris; Martens, Lennart; Tavernier, Jan; Eyckerman, Sven


    We describe sfinx, an R package providing access to the straightforward filtering index (SFINX) for the separation of true positive from false positive protein interactions in affinity purification - mass spectrometry datasets. This package maintains the reliability and user-friendliness of the SFINX web site interface but is faster, unlimited in input size, and can be run locally within R. The sfinx R package is available for download at the comprehensive R archive network (CRAN) under the Apache License 2.0. or Supplementary data are available at Bioinformatics online.

  18. A simple one pot purification of bacterial amylase from fermented broth based on affinity toward starch-functionalized magnetic nanoparticle. (United States)

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


    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.

  19. Purification and liposomal reconstitution of the oligopeptide transport activity in rat renal cortex using ceftibuten-affinity chromatography. (United States)

    Iseki, K; Naasani, I; Kikuchi, T; Sugawara, M; Kobayashi, M; Kohri, N; Miyazaki, K


    The carrier protein(s) responsible for the transport of ceftibuten, a peptide-like dianionic cefem, in rat renal brush-border membrane were solubilized and purified by a ceftibuten-ligand specific affinity chromatography technique. The proteoliposomes reconstituted from the solubilized brush-border membrane proteins by dialysis had H+-sensitive uptake of ceftibuten and trans-stimulative effect by cephalexin. A specific uptake activity for ceftibuten was found in the 3.5 M-eluted fraction but not the flowthrough and the 0.5 M-eluted fraction of the affinity chromatography. Analyzing this active fraction by SDS/PAGE after reconstituting into liposomes gave two major proteins (approx. molecular masses of 130 and 107 kDa). The purification protocol presented in this study permitted an efficient isolation of the carrier proteins responsible for the transport of ceftibuten and other peptide-like compounds.

  20. Rapid purification of high activity Taq DNA polymerase expressed in ...

    African Journals Online (AJOL)

    A simplified method is described here for the preparation of a thermostable Taq DNA polymerase enzyme from Escherichia coli (E. coli) strain DH5a carrying the pTTQ18 expression vector transformed with the Taq polymerase gene. Standard purifications were done with 1 litre batch cultures of E. coli cells and produced ...

  1. A theoretical and experimental approach toward the development of affinity adsorbents for GFP and GFP-fusion proteins purification. (United States)

    Fernandes, Cláudia S M; Pina, Ana Sofia; Dias, Ana M G C; Branco, Ricardo J F; Roque, Ana Cecília Afonso


    The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena, but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward the optimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands based on a first-generation lead structure were rationally designed through molecular modeling protocols. The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugi reaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6 emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yield and purity was found for ligands A5C5 and A5C6. These were tested for purifying a model GFP-fusion protein, where ligand A5C5 yielded higher protein recovery and purity. The molecular interactions between the lead ligands and GFP were further assessed by molecular dynamics simulations, showing a wide range of potential hydrophobic and hydrogen-bond interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography. (United States)

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


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

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

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


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

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

    Directory of Open Access Journals (Sweden)

    Colin P De Souza

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

  5. Accurate protein complex retrieval by affinity enrichment mass spectrometry (AE-MS) rather than affinity purification mass spectrometry (AP-MS). (United States)

    Keilhauer, Eva C; Hein, Marco Y; Mann, Matthias


    Protein-protein interactions are fundamental to the understanding of biological processes. Affinity purification coupled to mass spectrometry (AP-MS) is one of the most promising methods for their investigation. Previously, complexes were purified as much as possible, frequently followed by identification of individual gel bands. However, todays mass spectrometers are highly sensitive, and powerful quantitative proteomics strategies are available to distinguish true interactors from background binders. Here we describe a high performance affinity enrichment-mass spectrometry method for investigating protein-protein interactions, in which no attempt at purifying complexes to homogeneity is made. Instead, we developed analysis methods that take advantage of specific enrichment of interactors in the context of a large amount of unspecific background binders. We perform single-step affinity enrichment of endogenously expressed GFP-tagged proteins and their interactors in budding yeast, followed by single-run, intensity-based label-free quantitative LC-MS/MS analysis. Each pull-down contains around 2000 background binders, which are reinterpreted from troubling contaminants to crucial elements in a novel data analysis strategy. First the background serves for accurate normalization. Second, interacting proteins are not identified by comparison to a single untagged control strain, but instead to the other tagged strains. Third, potential interactors are further validated by their intensity profiles across all samples. We demonstrate the power of our AE-MS method using several well-known and challenging yeast complexes of various abundances. AE-MS is not only highly efficient and robust, but also cost effective, broadly applicable, and can be performed in any laboratory with access to high-resolution mass spectrometers. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry. (United States)

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


    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

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  8. Tandem Affinity Purification Approach Coupled to Mass Spectrometry to Identify Post-translational Modifications of Histones Associated with Chromatin-Binding Proteins. (United States)

    Beyer, Sophie; Robin, Philippe; Ait-Si-Ali, Slimane


    Protein purification by tandem affinity purification (TAP)-tag coupled to mass spectrometry analysis is usually used to reveal protein complex composition. Here we describe a TAP-tag purification of chromatin-bound proteins along with associated nucleosomes, which allow exhaustive identification of protein partners. Moreover, this method allows exhaustive identification of the post-translational modifications (PTMs) of the associated histones. Thus, in addition to partner characterization, this approach reveals the associated epigenetic landscape that can shed light on the function and properties of the studied chromatin-bound protein.

  9. Monoclonal antibody affinity purification of a 78 kDa membrane ...

    Indian Academy of Sciences (India)

    Surface iodination and subcellular fractionation of the promastigotes indicated that the protein was localized on the cell surface. The 78 kDa protein was found to inhibit the binding of promastigotes to macrophages significantly, suggesting that it may play a role in the process of infection. Thus, here we report the purification ...

  10. Purification of a Mycoplasma pneumoniae adhesin by monoclonal antibody affinity chromatography.


    Leith, D K; Baseman, J B


    A 165,000-dalton surface protein of Mycoplasma pneumoniae, designated protein P1, appears to be the major attachment ligand of the pathogen. We employed monoclonal antibody affinity chromatography to obtain purified protein P1.

  11. A multiplexed microfluidic toolbox for the rapid optimization of affinity-driven partition in aqueous two phase systems. (United States)

    Bras, Eduardo J S; Soares, Ruben R G; Azevedo, Ana M; Fernandes, Pedro; Arévalo-Rodríguez, Miguel; Chu, Virginia; Conde, João P; Aires-Barros, M Raquel


    Antibodies and other protein products such as interferons and cytokines are biopharmaceuticals of critical importance which, in order to be safely administered, have to be thoroughly purified in a cost effective and efficient manner. The use of aqueous two-phase extraction (ATPE) is a viable option for this purification, but these systems are difficult to model and optimization procedures require lengthy and expensive screening processes. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic channel-based toolbox is presented. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, using less than 20μL of each phase-forming solution per experiment, while a second microfluidic structure allows the integration of multi-step extraction protocols based on the results obtained with the first device. In this paper, this microfluidic toolbox was used to demonstrate the potential of LYTAG fusion proteins used as affinity tags to optimize the partitioning of antibodies in ATPE processes, where a maximum partition coefficient (K) of 9.2 in a PEG 3350/phosphate system was obtained for the antibody extraction in the presence of the LYTAG-Z dual ligand. This represents an increase of approx. 3.7 fold when compared with the same conditions without the affinity molecule (K=2.5). Overall, this miniaturized and versatile approach allowed the rapid optimization of molecule partition followed by a proof-of-concept demonstration of an integrated back extraction procedure, both of which are critical procedures towards obtaining high purity biopharmaceuticals using ATPE. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

    Energy Technology Data Exchange (ETDEWEB)

    Akduman, Begüm [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Uygun, Murat [Koçarlı Vocational and Training School, Adnan Menderes University, Aydın (Turkey); Uygun, Deniz Aktaş, E-mail: [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Akgöl, Sinan [Biochemistry Department, Ege University, İzmir (Turkey); Denizli, Adil [Chemistry Department, Hacettepe University, Ankara (Turkey)


    In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0 mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH 5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0 M NaCI at pH 8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. - Highlights: • Poly(HEMA–GMA) cryogels were synthesized by radical cryocopolymerization technique. • Prepared cryogels were functionalized with IDA, then Zn(II) ions were chelated to the cryogel. • Zn(II) chelated poly

  13. Easy and Rapid Purification of Highly Active Nisin

    Directory of Open Access Journals (Sweden)

    André Abts


    Full Text Available Nisin is an antimicrobial peptide produced and secreted by several L. lactis strains and is specifically active against Gram-positive bacteria. In previous studies, nisin was purified via cation exchange chromatography at low pH employing a single-step elution using 1 M NaCl. Here, we describe an optimized purification protocol using a five-step NaCl elution to remove contaminants. The obtained nisin is devoid of impurities and shows high bactericidal activity against the nisin-sensitive L. lactis strain NZ9000. Purified nisin exhibits an IC50 of ~3 nM, which is a tenfold improvement as compared to nisin obtained via the one-step elution procedure.

  14. Efficient separation of mannan-protein mixtures by ionic liquid aqueous two-phase system, comparison with lectin affinity purification. (United States)

    Čížová, Alžbeta; Korcová, Jana; Farkaš, Pavol; Bystrický, Slavomír


    Analysis of carbohydrates from complex biological samples often requires their isolation from proteins and other contaminants to avoid interference. An effective separation of mannan-protein mixtures by 1-butyl-3-methylimidazolium bromide/K2HPO4 ionic liquid aqueous two-phase system (IL-APTS) is reported. Extraction efficiency of bovine serum albumin (BSA) ranged from 92% to 97% while extraction efficiency of mannan reached values from 95% to about 100% depending on phase and/or model sample composition. On the contrary, lower efficiency of BSA removal (73-84%) was recorded for lectin affinity purification with concanavalin A-triazine bead cellulose (Con A-TBC); the low mannan-binding capacity was limiting factor here. The size exclusion chromatography pattern of model mannan-BSA samples after both IL-APTS and Con A-TBC treatments were consistent with the spectrophotometric component analysis. In case of biological experiment, the ionic liquid separation technique was superior in pre-purification of 2-aminobenzamide-labelled mannan from cell culture medium prior to HPLC-FLD analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Purification of the nicotinic acetylcholine receptor protein by affinity chromatography using a regioselectively modified and reversibly immobilized alpha-toxin from Naja nigricollis

    NARCIS (Netherlands)

    Ringler, P; Kessler, P; Menez, A; Brisson, A


    A new method of affinity chromatography purification of the detergent-solubilized nicotinic acetylcholine receptor protein (nAChR) is presented, based on the reversible coupling of a chemically monomodified alpha-toxin from Naja nigricollis to a resin. The alpha-toxin was monothiolated on the

  16. Analytical workflow for rapid screening and purification of bioactives from venom proteomes

    NARCIS (Netherlands)

    Otvos, R.A.; Heus, F.A.M.; Vonk, F.J.; Halff, J.; Bruynzeel, B.; Paliukhovich, I.; Smit, A.B.; Niessen, W.M.A.; Kool, J.


    Animal venoms are important sources for finding new pharmaceutical lead molecules. We used an analytical platform for initial rapid screening and identification of bioactive compounds from these venoms followed by fast and straightforward LC-MS only guided purification to obtain bioactives for

  17. Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases

    DEFF Research Database (Denmark)

    Vandecaetsbeek, Ilse; Christensen, Søren Brøgger; Liu, Huizhen


    protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated...

  18. Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Eszter Szarka


    Full Text Available Background: In rheumatoid arthritis (RA, anti-citrullinated protein/peptide antibodies (ACPAs are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: KD values of affinity-purified ACPA IgGs varied between 10−6 and 10−8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

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

    NARCIS (Netherlands)

    J. Groen (Jan); N. Juntti; J.S. Teppema; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert); G.F. Rimmelzwaan (Guus)


    textabstractImmuno 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

  20. Spotlite: web application and augmented algorithms for predicting co-complexed proteins from affinity purification--mass spectrometry data. (United States)

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


    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 ( ). 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.

  1. Studies on the affinity chromatography purification of anti-patulin polyclonal antibodies by enzyme linked immunosorbent assay and electrophoresis. (United States)

    Mhadhbi, H; Benrejeb, S; Martel, A


    Patulin is a mycotoxin produced by fungal species that frequently grow on fruit and vegetables. It presents risks, particularly for children consuming compotes and fruit juices. Thus, it is important to have methods such as immunoassays to screen a large number of samples. In the relevant literature, previous studies on the production of antibodies against patulin derivatives described qualitative tests for a patulin derivative or showed slight responses. The present study reinvestigated the production of polyclonal antibodies against patulin and their purification since crude antiserum could react non-specifically in immunoassays. Patulin-hemiglutarate was synthesized and conjugated to bovine serum albumin as the immunogen for the immunization of five New Zealand white rabbits. The immunoglobulin G (IgG) fraction was isolated twice by affinity chromatography using Sepharose-LS gel and recombinant G-protein. Classic affinity chromatography using Sepharose-LS gel was unable to eliminate serum albumin from the IgG fraction and the use of recombinant G-protein was efficient to isolate the purified IgG. Titres and specificity were determined by indirect competitive enzyme-linked immunosorbent assay. Patulin-hemiglutarate-ovalbumin gave complete displacement, while patulin displaced 30% of bound antibodies. Thus, a fraction of the antibodies are specific for free patulin. The non-specific binding increased with patulin concentrations. The electrophilic properties of patulin might also induce intermolecular cross-links in vitro that hinder the possibility of responses displacement when free patulin is used.

  2. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization. (United States)

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


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

  3. Protein A and protein A/G coupled magnetic SiO2microspheres for affinity purification of immunoglobulin G. (United States)

    Salimi, Kouroush; Usta, Duygu Deniz; Koçer, İlkay; Çelik, Eda; Tuncel, Ali


    Protein A carrying magnetic, monodisperse SiO 2 microspheres [Mag(SiO 2 )] with bimodal pore size distribution including both mesoporous and macroporous compartments were proposed as an affinity sorbent for IgG purification. Protein A was tightly bound onto the aldehyde functionalized-Mag(SiO 2 ) microspheres. The mesoporous compartment provided high surface area for protein A binding and IgG adsorption while the macropores made easier the intraparticular diffusion of protein A and IgG. The selection of relatively larger microspheres with high saturation magnetization allowed faster magnetic separation of affinity sorbent from the IgG isolation medium, less than 1min. With these properties, the proposed sorbent is an alternative to the common sorbents in the form of core-shell type, magnetic silica nanoparticles with more limited surface area and slower magnetic response. By using protein A attached-Mag(SiO 2 ) microspheres with the concentrations lower than 50mg/mL, IgG isolation from rabbit serum was performed with a purity higher than 95%, with an isolation yield comparable to commercial magnetic resins, and in shorter isolation periods. IgG could be also quantitatively isolated from rabbit serum with the sorbent concentrations higher than 50mg/mL. Successive IgG isolation runs indicated that no significant protein A leaching occurred from the magnetic matrix. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS. (United States)

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


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

  5. Efficient purification of unique antibodies using peptide affinity-matrix columns

    DEFF Research Database (Denmark)

    Jensen, Liselotte Brix; Riise, Erik; Nielsen, Leif Kofoed


    Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85...... fragments and had no affinity for other antibodies. Using this peptide matrix MK16 IgG could be purified from cell culture supernatants thereby separating MK16 IgG from bovine IgG normally present in the enriched growth media used for such cells. Investigations of the fine specificity of the ER6.1 peptide...... demonstrated that it recognised a unique epitope within the heavy chain CDR3 region of the MK16 antibody. Thus, variants of MK16 antibody, which had retained the specificity and affinity of the original antibody but had slightly different amino acid composition in the CDR3 region, were not recognised by the ER...

  6. Resolving protein interactions and complexes by affinity purification followed by label-based quantitative mass spectrometry. (United States)

    Trinkle-Mulcahy, Laura


    Label-based quantitative mass spectrometry analysis of affinity purified complexes, with its built-in negative controls and relative ease of use, is an increasingly popular choice for defining protein-protein interactions and multiprotein complexes. This approach, which differentially labels proteins/peptides from two or more populations and combines them prior to analysis, permits direct comparison of a protein pulldown (e.g. affinity purified tagged protein) to that of a control pulldown (e.g. affinity purified tag alone) in a single mass spectrometry (MS) run, thus avoiding the variability inherent in separate runs. The use of quantitative techniques has been driven in large part by significant improvements in the resolution and sensitivity of high-end mass spectrometers. Importantly, the availability of commercial reagents and open source identification/quantification software has made these powerful techniques accessible to nonspecialists. Benefits and drawbacks of the most popular labeling-based approaches are discussed here, and key steps/strategies for the use of labeling in quantitative immunoprecipitation experiments detailed. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Metal chelate affinity precipitation of RNA and purification of plasmid DNA (United States)

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


    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.

  8. A rapid and economic in-house DNA purification method using glass syringe filters.

    Directory of Open Access Journals (Sweden)

    Yun-Cheol Kim

    Full Text Available BACKGROUND: Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations. METHODOLOGY/PRINCIPAL FINDINGS: We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit. CONCLUSIONS/SIGNIFICANCE: This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.

  9. An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography

    Directory of Open Access Journals (Sweden)

    Büssow Konrad


    Full Text Available Abstract Background Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner. Results We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag or glutathione S-transferase (GST-tag from bacterial expression systems. Proteins are expressed in 96-well microplates and are purified by a fully automated procedure on a pipetting robot. Up to 90 microgram purified protein can be obtained from 1 ml microplate cultures. The procedure is readily reproducible and 96 proteins can be purified in approximately three hours. It avoids clearing of crude cellular lysates and the use of magnetic affinity beads and is therefore less expensive than comparable commercial systems. We have used this method to compare purification of a set of human proteins via His-tag or GST-tag. Proteins were expressed as fusions to an N-terminal tandem His- and GST-tag and were purified by metal chelating or glutathione affinity chromatography. The purity of the obtained protein samples was similar, yet His-tag purification resulted in higher yields for some proteins. Conclusion A fully automated, robust and cost effective method was developed for the purification of proteins that can be used to quickly characterise expression clones in high throughput and to produce large numbers of proteins for functional studies. His-tag affinity purification was found to be more efficient than purification via GST-tag for some proteins.

  10. Transient conformational modification of immunoglobulin G during purification by protein A affinity chromatography. (United States)

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


    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. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography. (United States)

    Konami, Y; Yamamoto, K; Osawa, T


    A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

  12. Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco1 (United States)

    Fujita, Satoshi; Uchimura, Seiichi; Noguchi, Masahiro; Demura, Taku


    Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures. PMID:26747285

  13. Engineering split intein DnaE from Nostoc punctiforme for rapid protein purification. (United States)

    Ramirez, Miguel; Valdes, Najla; Guan, Dongli; Chen, Zhilei


    We report the engineering of a DnaE intein able to catalyze rapid C-terminal cleavage in the absence of N-terminal cleavage. A single mutation in DnaE intein from Nostoc punctiforme PCC73102 (NpuDnaE), Asp118Gly, was introduced based on sequence alignment with a previously engineered C-terminal cleaving intein mini-MtuRecA. This mutation was able to both suppress N-terminal cleavage and significantly elevate C-terminal cleavage efficiency. Molecular modeling suggests that in NpuDnaE Asp118 forms a hydrogen bond with the penultimate Asn, preventing its spontaneous cyclization prior to N-terminal cleavage. Mutation of Asp118 to Gly essentially abolishes this restriction leading to subsequent C-terminal cleavage in the absence of N-terminal cleavage. The Gly118 NpuDnaE mutant exhibits rapid thio-dependent C-terminal cleavage kinetics with 80% completion within 3 h at room temperature. We used this newly engineered intein to develop both column-free and chromatography-based protein purification methods utilizing the elastin-like-polypeptide and chitin-binding protein as removable purification tags, respectively. We demonstrate rapid target protein purification to electrophoretic purity at yields up to 84 mg per liter of Escherichia coli culture.

  14. Isolation of protein complexes from the model legume Medicago truncatula by tandem affinity purification in hairy root cultures. (United States)

    Goossens, Jonas; De Geyter, Nathan; Walton, Alan; Eeckhout, Dominique; Mertens, Jan; Pollier, Jacob; Fiallos-Jurado, Jennifer; De Keyser, Annick; De Clercq, Rebecca; Van Leene, Jelle; Gevaert, Kris; De Jaeger, Geert; Goormachtig, Sofie; Goossens, Alain


    Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most powerful techniques to isolate protein complexes and elucidate protein interaction networks. Here, we describe the development of a TAP-MS strategy for the model legume Medicago truncatula, which is widely studied for its ability to produce valuable natural products and to engage in endosymbiotic interactions. As biological material, transgenic hairy roots, generated through Agrobacterium rhizogenes-mediated transformation of M. truncatula seedlings, were used. As proof of concept, proteins involved in the cell cycle, transcript processing and jasmonate signalling were chosen as bait proteins, resulting in a list of putative interactors, many of which confirm the interologue concept of protein interactions, and which can contribute to biological information about the functioning of these bait proteins in planta. Subsequently, binary protein-protein interactions among baits and preys, and among preys were confirmed by a systematic yeast two-hybrid screen. Together, by establishing a M. truncatula TAP-MS platform, we extended the molecular toolbox of this model species. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  15. Development of a novel affinity chromatography resin for platform purification of bispecific antibodies with modified protein a binding avidity. (United States)

    Tustian, Andrew D; Laurin, Linus; Ihre, Henrik; Tran, Travis; Stairs, Robert; Bak, Hanne


    There is strong interest in the production of bispecific monoclonal antibodies that can simultaneously bind two distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Regeneron's bispecific technology, based upon a standard IgG, consists of a heterodimer of two different heavy chains, and a common light chain. Co-expression of two heavy chains leads to the formation of two parental IgG impurities, the removal of which is facilitated by a dipeptide substitution in the Fc portion of one of the heavy chains that ablates Fc Protein A binding. Therefore the affinity capture (Protein A) step of the purification process must perform both bulk capture and high resolution of these mAb impurities, a task current commercially available resins are not designed for. Resolution can be further impaired by the ability of Protein A to bind some antibodies in the variable region of the heavy chain (V H ). This paper details development of a novel Protein A resin. This resin combines an alkali stable ligand with a base matrix exhibiting excellent mass transfer properties to allow high capacity single step capture and resolution of bispecific antibodies with high yields. The developed resin, named MabSelect SuRe™ pcc, is implemented in GMP production processes for several bispecific antibodies. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

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

    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


    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. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. From pathways to networks: connecting dots by establishing protein-protein interaction networks in signaling pathways using affinity purification and mass spectrometry. (United States)

    Li, Xu; Wang, Wenqi; Chen, Junjie


    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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    Directory of Open Access Journals (Sweden)

    Safar Farajnia


    Full Text Available Purpose: Recombinant tumor necrosis factor-alpha (TNF-α has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.

  19. Affinity purification of hen egg lysozyme using sephadex G75 | Islam ...

    African Journals Online (AJOL)

    We found lysozyme binds Sephadex G75, a dextran-based matrix routinely used for Gel-filtration chromatography, in a pH dependent manner. The binding is rapid and specific in a buffer containing 25 mM NaCl at pH 8.0, and requires only 0.1 ml of swollen Sephadex G75 suspension per mg of lysozyme. The bound ...

  20. Use of specific polysaccharide-immobilized monodisperse poly(glycidyl methacrylate) core-silica shell microspheres for affinity purification of lectins. (United States)

    Antonyuk, Volodymyr; Grama, Silvia; Plichta, Zdeněk; Magorivska, Iryna; Horak, Daniel; Stoika, Rostyslav


    Immobilization of polysaccharides (yeast mannan and gum arabic) on the macroporous poly(glycidyl methacrylate) monodisperse microspheres coated with silica (SiO2 )-containing amino groups on the surface was used to prepare affinity sorbents for lectin purification. The efficiency of isolating mannose specific Pisum sativum lectin was demonstrated on sorbent with immobilized yeast mannan and that of galactose specific Glycine hispida lectin on sorbent with immobilized gum arabic. The microspheres with immobilized polysaccharides can be used for selecting an affinity sorbent for purification of other mannose- and galactose-specific lectins. In contrast to yeast mannan, the gum arabic immobilized on the microspheres possesses much narrower specificity and is suitable for purification of only those galactose specific lectins which interact well with l-rhamnose or l-arabinose. The synthesized macroporous particles are capable of immobilizing 50 mg of polysaccharide per 1 g of the matrix, which is 10 times higher than the capacity of epoxy-activated Sepharose 6B. That makes it possible to obtain the same lectin quantity using a column of 10 times smaller volume. Another advantage of novel affinity sorbents comparing corresponding Sepharose gels is the possibility of sorbent drying after use. Copyright © 2014 John Wiley & Sons, Ltd.

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

    Directory of Open Access Journals (Sweden)

    He Huang


    Full Text Available 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.

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

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


    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).

  3. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    Directory of Open Access Journals (Sweden)

    Wenping Gong

    Full Text Available 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.


    Directory of Open Access Journals (Sweden)

    J. Hamedi


    Full Text Available Large proportion of microbial population in the world is unculturable. Extraction of total DNA from soil is usually a crucial step considering to the difficulties of study the uncultivable microorganisms. Humic acid is considered as the main inhibitory agent in the environmental DNA studies. Here, we introduced a rapid and efficient method for DNA extraction and purification from soil. Yield of DNA extraction by the presented method was 130 ng/µl. Three conventional methods of DNA extraction including liquid nitrogen incursion, bead beating and sonication were performed as control methods. Yield of DNA extraction by these methods were 110, 90 and 50 ng/µl, respectively. A rapid and efficient one step DNA purification method was introduced instead of hazardous conventional phenol-chloroform methods. Humic acid removal percentage by the introduced method was 95.8 % that is comparable with 97 % gained by the conventional gel extraction method and yield of DNA after purification was 84 % and 73 %, respectively. This study could be useful in molecular ecology and metagenomics study as a fast and reliable method.

  5. Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling.

    Directory of Open Access Journals (Sweden)

    Dawson Fogen

    Full Text Available To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C, respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M that are similar to SAVSBPM18. Although SBP(A18C binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation-a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C tags in excess, two SBP(A18C tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.

  6. Production, rapid purification and catalytic characterization of extracellular phytase from Aspergillus ficuum. (United States)

    Ullah, A H


    A rapid purification scheme utilizing three chromatographic steps resulted in 6 fold purification of Aspergillus ficuum phytase (myo-inositol-hexakisphosphate 3-phosphohydrolase, EC At pH 5.0 and 60 degrees C the enzyme performed acceptably for 2.0 hr with only 30% diminished catalytic rate at the end. Substrate concentration exceeding 2mM was inhibitory. The inorganic orthophosphate, the product and a weak inhibitor, exhibited a Ki of 1.9 x 10(-3)M. The extracellular phytase has the potential for industrial use since it can be over produced, easily purified, remain catalytically active for a longer period and is not subjected to severe product inhibition.

  7. Purification

    DEFF Research Database (Denmark)

    Andersen, Astrid Oberborbeck


    of categories can be understood as practices of purification. However, a purely technical grip on water is never possible. Unruly elements, like weather, contamination, urban dwellers, and competing interests, interfere and make processes of intervention unstable. Water is never completely cleaned, and, equally......In Arequipa, Peru’s second largest city, engineers work hard to control water flows and provide different sectors with clean and sufficient water. In 2011, only 10 percent of the totality of water used daily by Arequipa’s then close to 1 million people—in households, tourism, industry, and mining......—was treated before it was returned to the river where it continues its flow downstream towards cultivated fields and, finally, into the Pacific Ocean. It takes specialized knowledge and manifold technologies to manage water and sustain life in Arequipa, and engineers are central actors for making water flow...

  8. Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry. (United States)

    Zhang, B; Palcic, M M; Mo, H; Goldstein, I J; Hindsgaul, O


    The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin ( approximately 25 microg) onto porous glass beads in a tubing column (9.8 microl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc containing oligosaccharides with K(d) values near 10 microM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.

  9. Inference of a Geminivirus-Host Protein-Protein Interaction Network through Affinity Purification and Mass Spectrometry Analysis. (United States)

    Wang, Liping; Ding, Xue; Xiao, Jiajing; Jiménez-Gόngora, Tamara; Liu, Renyi; Lozano-Durán, Rosa


    Viruses reshape the intracellular environment of their hosts, largely through protein-protein interactions, to co-opt processes necessary for viral infection and interference with antiviral defences. Due to genome size constraints and the concomitant limited coding capacity of viruses, viral proteins are generally multifunctional and have evolved to target diverse host proteins. Inference of the virus-host interaction network can be instrumental for understanding how viruses manipulate the host machinery and how re-wiring of specific pathways can contribute to disease. Here, we use affinity purification and mass spectrometry analysis (AP-MS) to define the global landscape of interactions between the geminivirus Tomato yellow leaf curl virus (TYLCV) and its host Nicotiana benthamiana. For this purpose, we expressed tagged versions of each of TYLCV-encoded proteins (C1/Rep, C2/TrAP, C3/REn, C4, V2, and CP) in planta in the presence of the virus. Using a quantitative scoring system, 728 high-confidence plant interactors were identified, and the interaction network of each viral protein was inferred; TYLCV-targeted proteins are more connected than average, and connect with other proteins through shorter paths, which would allow the virus to exert large effects with few interactions. Comparative analyses of divergence patterns between N. benthamiana and potato, a non-host Solanaceae, showed evolutionary constraints on TYLCV-targeted proteins. Our results provide a comprehensive overview of plant proteins targeted by TYLCV during the viral infection, which may contribute to uncovering the underlying molecular mechanisms of plant viral diseases and provide novel potential targets for anti-viral strategies and crop engineering. Interestingly, some of the TYLCV-interacting proteins appear to be convergently targeted by other pathogen effectors, which suggests a central role for these proteins in plant-pathogen interactions, and pinpoints them as potential targets to

  10. Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae. (United States)

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè


    Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Simple and Effective Affinity Purification Procedures for Mass Spectrometry-Based Identification of Protein-Protein Interactions in Cell Signaling Pathways. (United States)

    Kwan, Julian H M; Emili, Andrew


    Identification of protein-protein interactions can be a critical step in understanding the function and regulation of a particular protein and for exploring intracellular signaling cascades. Affinity purification coupled to mass spectrometry (APMS) is a powerful method for isolating and characterizing protein complexes. This approach involves the tagging and subsequent enrichment of a protein of interest along with any stably associated proteins that bind to it, followed by the identification of the interacting proteins using mass spectrometry. The protocol described here offers a quick and simple method for routine sample preparation for APMS analysis of suitably tagged human cell lines.

  12. Rapid and simple purification of lysozyme from the egg shell membrane. (United States)

    Kozuka, Miyuki; Murao, Sato; Yamane, Takuya; Inoue, Tsutomu; Ohkubo, Iwao; Ariga, Hiroyoshi


    Lysozyme (EC is a hydrolytic enzyme that cleaves the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, a major bacterial cell wall polymer. In the food industry, lysozyme is used as an additive mainly in the production of wine and beer. Lysozyme was found to be localized in the egg shell membrane. In this study, we found that lysozyme was easily purified from the egg shell membrane and that the enzyme also had antibacterial activity. Furthermore, we found that the antibacterial activity of purified lysozyme from the egg shell membrane was lower than that of purified lysozyme from the egg white at alkaline pH. The method for rapid purification of lysozyme developed in this study should contribute to the food industry.

  13. Rapid Purification of Salmonella DNA in Minced Meat and Detection by Real-time PCR

    DEFF Research Database (Denmark)

    Jenikova, G.; Jensen, Annette Nygaard; Demnerova, K.


    of DNeasy was found to be 6-8 CFU in just 19 end-point fluorescence (C-t) values, while this was 22 C-t for a combination of DNeasy and BactXtractor. Extraction by DNeasy resulted in C-t cells per 25 g, when the samples were inoculated with Salmonella......Four rapid and simple DNA purification and sample treatment protocols were evaluated for detection of Salmonella enterica in spiked minced meat, using a fluorogenic 5' nuclease (TaqMan) PCR assay in an ABI-Prism 7700 Sequence Detector. The detection limit with the single separation treatment...... before the overnight preenrichment. The method is currently being adapted to a BioRobot 3000 platform. However, the use of paramagnetic beads (DNA Direct) resulted in poor and variable detection limit....

  14. A new epitope tag from hepatitis B virus preS1 for immunodetection, localization and affinity purification of recombinant proteins. (United States)

    Oh, Mee Sook; Kim, Keun Soo; Jang, Young Kug; Maeng, Cheol Young; Min, Soon Hong; Jang, Myeong Hee; Yoon, Sun Ok; Kim, Jung Hee; Hong, Hyo Jeong


    Previously, a murine monoclonal antibody (mAb) KR127 (IgG2a/kappa) that binds specifically to the preS1 of hepatitis B virus (HBV) was generated and the fine epitope was mapped to amino acids (aa) 37-45 (NSNNPDWDF). In this current study, the epitope in combination with KR127 was tested for protein tagging. Initially, to evaluate the importance of each residue of the KR127 epitope in antibody binding, alanine substitution mutants of the epitope were constructed and characterized for KR127 binding by immunoblot analysis and competition ELISA. The results showed that substitution of Ser(38) by alanine (S38A) increased the affinity to KR127. The mutated epitope (NANNPDWDF), designated S1 tag, was fused to the amino (N)- or carboxyl (C)-terminus of three human recombinant proteins, soluble B lymphocyte stimulator (sBLyS), the N-terminal domain of thrombopoietin (nTPO), and a mitochondrial ribosomal protein (CGI-113) for expression in mammalian cells, while it was fused to the N- or C-terminus of two proteins, a single-chain antibody fragment (ScFv) and the carboxyl-terminal domain (PAc) of the protective antigen of Bacillus anthracis for expression in Escherichia coli. The immunodetection, immunoprecipitation, and affinity purification of the expressed S1-tagged proteins by KR127 were successfully demonstrated. In addition, a KR127 mutant (AP2) with higher affinity, K(d) (0.9 nM), for the S1 tag compared to that (20 nM) of KR127 was obtained by mutational analysis of the heavy chain CDR3 (HCDR3) of KR127. The AP2 antibody was 4-fold more sensitive in detecting the S1-tagged protein than KR127. The S1 tag-KR127 or AP2 combination could be universally used for monitoring protein expression, localizing proteins, and protein purification, as well as studying protein interactions.

  15. Large-scale overproduction, functional purification and ligand affinities of the His-tagged human histamine H1 receptor.

    NARCIS (Netherlands)

    Ratnala, V.R.; Swarts, H.G.P.; Oostrum, J. van; Leurs, R.; Groot, H.J.M. de; Bakker, R.; Grip, W.J. de


    This report describes an efficient strategy for amplified functional purification of the human H1 receptor after heterologous expression in Sf9 cells. The cDNA encoding a C-terminally histidine-tagged (10xHis) human histamine H1 receptor was used to generate recombinant baculovirus in a Spodoptera

  16. [Identification of the interacting proteins with S100A8 or S100A9 by affinity purification and mass spectrometry]. (United States)

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


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

  17. Rapid Screening for α-Glucosidase Inhibitors from Gymnema sylvestre by Affinity Ultrafiltration–HPLC-MS

    Directory of Open Access Journals (Sweden)

    Mingquan Guo


    Full Text Available Gymnema sylvestre R. Br. (Asclepiadaceae has been known to posses potential anti-diabetic activity, and the gymnemic acids were reported as the main bioactive components in this plant species. However, the specific components responsible for the hypoglycemic effect still remain unknown. In the present study, the in vitro study revealed that the extract of G. sylvestre exhibited significant inhibitory activity against α-glucosidase with IC50 at 68.70 ± 1.22 μg/mL compared to acarbose (positive control at 59.03 ± 2.30 μg/mL, which further indicated the potential anti-diabetic activity. To this end, a method based on affinity ultrafiltration coupled with liquid chromatography mass spectrometry (UF-HPLC-MS was established to rapidly screen and identify the α-glucosidase inhibitors from G. sylvestre. In this way, 9 compounds with higher enrichment factors (EFs were identified according to their MS/MS spectra. Finally, the structure-activity relationships revealed that glycosylation could decrease the potential antisweet activity of sapogenins, and other components except gymnemic acids in G. sylvestre could also be good α-glucosidase inhibitors due to their synergistic effects. Taken together, the proposed method combing α-glucosidase and UF-HPLC-MS presents high efficiency for rapidly screening and identifying potential inhibitors of α-glucosidase from complex natural products, and could be further explored as a valuable high-throughput screening (HTS platform in the early anti-diabetic drug discovery stage.

  18. 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. (United States)

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


    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. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Magnetic poly(2-hydroxyethyl methacrylate) microspheres for affinity purification of monospecific anti-p46 kDa/Myo1C antibodies for early diagnosis of multiple sclerosis patients

    Czech Academy of Sciences Publication Activity Database

    Horák, Daniel; Hlídková, Helena; Kit, Y.; Antonyuk, V.; Myronovsky, S.; Stoika, R.


    Roč. 37, č. 2 (2017), s. 1-10, č. článku BSR20160526. ISSN 0144-8463 R&D Projects: GA MŠk(CZ) LH14318 Institutional support: RVO:61389013 Keywords : poly(2-hydroxyethyl methacrylate) * magnetic microspheres * affinity purification Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 2.906, year: 2016

  20. Affinity purification of native glycodelin from amniotic fluid for biological investigations and development of a glycodelin ELISA for clinical studies

    DEFF Research Database (Denmark)

    Sørensen, Steen; Myrhøj, Vibeke; Nguyen, Thanh Ha


    for functional studies because the carbohydrate part can be lacking or be insufficient in recombinant glycodelin from prokaryotic and eukaryotic cell systems. METHODS AND RESULTS: Native glycodelin was purified from amniotic fluid by a series of affinity chromatography steps and had many glycosylated forms...

  1. Streptavidin-Binding Peptide (SBP-tagged SMC2 allows single-step affinity fluorescence, blotting or purification of the condensin complex

    Directory of Open Access Journals (Sweden)

    Graham Alison N


    Full Text Available Abstract Background Cell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function. Often protein purification, cellular localisation and Western blot analyses can be multi-step processes, where protein is lost, activity is destroyed or effective antibodies have not yet been generated. Aim To develop a method that simplifies the critical protein analytical steps of the laboratory researcher, leading to easy, efficient and rapid protein purification, cellular localisation and quantification. Results We have tagged the SMC2 subunit of the condensin complex with the Streptavidin-Binding Peptide (SBP, optimising and demonstrating the efficacious use of this tag for performing these protein analytical steps. Based on silver staining, and Western analysis, SBP delivered an outstanding specificity and purity of the condensin complex. We also developed a rapid and highly specific procedure to localise SBP-tagged proteins in cells in a single step procedure thus bypassing the need for using antibodies. Furthermore we have shown that the SBP tag can be used for isolating tagged proteins from chemically cross-linked cell populations for capturing DNA-protein interactions. Conclusions The small 38-amino acid synthetic SBP offers the potential to successfully perform all four critical analytical procedures as a single step and should have a general utility for the study of many proteins and protein complexes.

  2. Tricalcium phosphate nanoparticles enable rapid purification, increase transduction kinetics, and modify the tropism of mammalian viruses. (United States)

    Dreesen, Imke A J; Lüchinger, Norman A; Stark, Wendelin J; Fussenegger, Martin


    Adenoviral, adeno-associated viral, and retroviral particles are chosen as gene delivery shuttles in more than 50% of all gene therapy clinical trials. Bulk availability of clinical-grade viral particles and their efficiency to transduce the therapeutic cargo into specific target cells remain the most critical bottlenecks in gene therapy applications to date. Capitalizing on the flame-spray technology for the reproducible economic large-scale production of amorphous tricalcium phosphate nanoparticulate powders (ATCP), we designed a scalable ready-to-use gravity-flow column set-up for the straightforward concentration and purification of transgenic adenoviral, adeno-associated viral, and lentiviral particles. Specific elution buffers enabled rapid release of viral particles from the ATCP matrix of the column and provided high-titer virus preparations in an unsurpassed period of time. The interaction of ATCP with adenoviral, adeno-associated viral, and lentiviral particles in solution increased the transduction kinetics of several mammalian cell lines in culture. The nanoparticles were also able to modify the tropism of murine leukemia virus (MLV) towards transduction of human cells. Based on these findings, we believe that the use of flame-spray tricalcium phosphate nanoparticles will lead to important progress in the development of future gene therapy initiatives.

  3. Rapid purification and characterization of γ-glutamyl-transpeptidase from shiitake mushroom (Lentinus edodes). (United States)

    Li, Jianrong; Huang, Ju; Yin, Jie; Wu, Ning; Song, Jun; Zhang, Lei; Jiang, Tianjia


    γ-Glutamyl-transpeptidase (GGT) is one of the important enzymes in the pathway of odor formation in shiitake mushroom (Lentinus edodes). Rapid purification and characterization of GGT from shiitake mushroom were studied in this work. The GGT was purified 179-fold after 3 primary steps: precipitation by ammonium sulfate, isolation by Phenyl Sepharose 6 FF, and desalting by Sephadex G-25. The enzyme, consisting of a small and a large subunit with Mr 28 KDa and 60 KDa, respectively, is composed of 17 kinds of amino acids with the ratio of basic and acidic residues 1: 1.84, and its secondary structure was also determined by Fourier transform infrared spectroscopy. The properties of GGT were studied with γ-glutamyl-p-nitroanilide as the substrate. The results showed Km value of 2.601μM, optimal temperature of 40 °C, and isoelectric point of 6.4. In addition, the activity of GGT was promoted by Na⁺, K⁺, and Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Zn²⁺, and Fe³⁺. In this work, the γ-glutamyl-transpeptidase (GGT) from shiitake mushroom was purified with a simple scheme. Through the characterization of GGT, the relationship between endogenous formaldehyde and odor formation is to be clarified and assist in finding means of formaldehyde control in shiitake mushroom. © 2012 Institute of Food Technologists®

  4. Thermally moderated hollow fiber sorbent modules in rapidly cycled pressure swing adsorption mode for hydrogen purification

    KAUST Repository

    Lively, Ryan P.


    We describe thermally moderated multi-layered pseudo-monolithic hollow fiber sorbents entities, which can be packed into compact modules to provide small-footprint, efficient H2 purification/CO2 removal systems for use in on-site steam methane reformer product gas separations. Dual-layer hollow fibers are created via dry-jet, wet-quench spinning with an inner "active" core of cellulose acetate (porous binder) and zeolite NaY (69 wt% zeolite NaY) and an external sheath layer of pure cellulose acetate. The co-spun sheath layer reduces the surface porosity of the fiber and was used as a smooth coating surface for a poly(vinyl-alcohol) post-treatment, which reduced the gas permeance through the fiber sorbent by at least 7 orders of magnitude, essentially creating an impermeable sheath layer. The interstitial volume between the individual fibers was filled with a thermally-moderating paraffin wax. CO2 breakthrough experiments on the hollow fiber sorbent modules with and without paraffin wax revealed that the "passively" cooled paraffin wax module had 12.5% longer breakthrough times than the "non-isothermal" module. The latent heat of fusion/melting of the wax offsets the released latent heat of sorption/desorption of the zeolites. One-hundred rapidly cycled pressure swing adsorption cycles were performed on the "passively" cooled hollow fiber sorbents using 25 vol% CO2/75 vol% He (H2 surrogate) at 60 °C and 113 psia, resulting in a product purity of 99.2% and a product recovery of 88.1% thus achieving process conditions and product quality comparable to conventional pellet processes. Isothermal and non-isothermal dynamic modeling of the hollow fiber sorbent module and a traditional packed bed using gPROMS® indicated that the fiber sorbents have sharper fronts (232% sharper) and longer adsorbate breakthrough times (66% longer), further confirming the applicability of the new fiber sorbent approach for H2 purification. © 2012, Hydrogen Energy Publications, LLC

  5. Rapid analysis of the interactions between drugs and human serum albumin (HSA) using high-performance affinity chromatography (HPAC). (United States)

    Kim, Hee Seung; Wainer, Irving W


    This study used a combination of zonal elution and frontal affinity chromatography on immobilized human serum albumin (HSA) high-performance affinity chromatography (HPAC) column to examine the association constants of various compounds that have been studied by equilibrium dialysis or ultra filtration. A standard plot was generated from retention factors of reference compounds using zonal elution chromatography against association constants of reference compounds using frontal affinity chromatography. The linear relationship was established (r2=0.9993) between retention factors and association constants of reference compounds. This standard plot was later used for rapid determination of association constants of various drugs which show low to medium binding affinity to HSA. Association constants of those drugs from this study were compared to that of more generally used methods (i.e., equilibrium dialysis or ultra filtration) from literature and resulted in a relatively high correlation (r2=0.945) value. This combination of zonal elution and frontal affinity chromatography method for determining association constants showed several advantages against traditional methods. Depending on drugs of interest, an association constant of drug to HSA can be measured as fast as 1.5 min. Other notable advantages include an ease of automation and its ability to distinguish association constants of chiral compounds at the same time. The same approach could be used for studying interaction of other drugs and proteins and should further improve overall drug screening process.

  6. A rapid Orthopoxvirus purification protocol suitable for high-containment laboratories. (United States)

    Hughes, Laura; Wilkins, Kimberly; Goldsmith, Cynthia S; Smith, Scott; Hudson, Paul; Patel, Nishi; Karem, Kevin; Damon, Inger; Li, Yu; Olson, Victoria A; Satheshkumar, P S


    Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols. Classical virus purification techniques are difficult to execute in a BSL-3 or BSL-4 laboratory because of the work practices used in these environments. Orthopoxviruses are a family of viruses that, in some cases, requires work in a high-containment laboratory and due to size do not lend themselves to simpler purification methods. Current CDC purification techniques of orthopoxviruses uses 1,1,2-trichlorotrifluoroethane, commonly known as Genetron®. Genetron® is a chlorofluorocarbon (CFC) that has been shown to be detrimental to the ozone and has been phased out and the limited amount of product makes it no longer a feasible option for poxvirus purification purposes. Here we demonstrate a new Orthopoxvirus purification method that is suitable for high-containment laboratories and produces virus that is not only comparable to previous purification methods, but improves on purity and yield. Published by Elsevier B.V.

  7. Deciphering the ubiquitin proteome: Limits and advantages of high throughput global affinity purification-mass spectrometry approaches. (United States)

    Polge, Cécile; Uttenweiler-Joseph, Sandrine; Leulmi, Roza; Heng, Anne-Elisabeth; Burlet-Schiltz, Odile; Attaix, Didier; Taillandier, Daniel


    Ubiquitination is a posttranslational modification of proteins that involves the covalent attachment of ubiquitin, either as a single moiety or as polymers. This process controls almost every cellular metabolic pathway through a variety of combinations of linkages. Mass spectrometry now allows high throughput approaches for the identification of the thousands of ubiquitinated proteins and of their ubiquitination sites. Despite major technological improvements in mass spectrometry in terms of sensitivity, resolution and acquisition speed, the use of efficient purification methods of ubiquitinated proteins prior to mass spectrometry analysis is critical to achieve an efficient characterization of the ubiquitome. This critical step is achieved using different approaches that possess advantages and pitfalls. Here, we discuss the limits that can be encountered when deciphering the ubiquitome. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Engineering and rapid selection of a low-affinity glucose/galactose-binding protein for a glucose biosensor. (United States)

    Amiss, Terry J; Sherman, Douglas B; Nycz, Colleen M; Andaluz, Sandra A; Pitner, J Bruce


    Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/D-galactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.2 microM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7-33 mM). To accomplish this a focused library was constructed using structure-based site-saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H-bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site-specifically at E149C with the fluorophore IANBD (N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylene-diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000-fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H-bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure-based site-saturation mutagenesis and periplasmic expression screening to discover low-affinity GGBP mutants having potential utility for measuring glucose in humans.

  9. Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography-tandem mass spectrometry to affinity purification followed by enzyme immunoassay. (United States)

    Armstrong, Michael; Liu, Andrew H.; Harbeck, Ronald; Reisdorph, Rick; Rabinovitch, Nathan; Reisdorph, Nichole


    A new analytical method suitable for high throughput measurements of LTE4 in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/ tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500 pg/mL in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE4 from healthy adults and children are reported. PMID:19726242

  10. A tandem laboratory scale protein purification process using Protein A affinity and anion exchange chromatography operated in a weak partitioning mode. (United States)

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


    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. Copyright © 2013 Wiley Periodicals, Inc.

  11. Preparation of hybrid molecularly imprinted polymer with double-templates for rapid simultaneous purification of theophylline and chlorogenic acid in green tea. (United States)

    Tang, Weiyang; Li, Guizhen; Row, Kyung Ho; Zhu, Tao


    A novel double-templates technique was adopted for solid-phase extraction packing agent, and the obtained hybrid molecularly imprinted polymers with double-templates (theophylline and chlorogenic acid) were characterized by fourier transform infrared and field emission scanning electron microscope. The molecular recognition ability and binding capability for theophylline and chlorogenic acid of polymers was evaluated by static absorption and dynamic adsorption curves. A rapid and accurate approach was established for simultaneous purification of theophylline and chlorogenic acid in green tea by coupling hybrid molecularly imprinted solid-phase extraction with high performance liquid chromatography. With optimization of SPE procedure, a reliable analytical method was developed for highly recognition towards theophylline and chlorogenic acid in green tea with satisfactory extraction recoveries (theophylline: 96.7% and chlorogenic acid: 95.8%). The limit of detection and limit of quantity of the method were 0.01 μg/mL and 0.03 μg/mL for theophylline, 0.05 μg/mL and 0.17 μg/mL for chlorogenic acid, respectively. The recoveries of proposed method at three spiked levels analysis were 98.7-100.8% and 98.3-100.2%, respectively, with the relative standard deviation less than 1.9%. Hybrid molecularly imprinted polymers with double-templates showed good performance for two kinds of targets, and the proposed approach with high affinity of hybrid molecularly imprinted polymers might offer a novel method for the purification of complex samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

    Directory of Open Access Journals (Sweden)

    Gildo Almeida da Silva


    Full Text Available The purpose of this work was to study a rapid yeast DNA extraction by boiling and freeze-thawing processes without using chemical reagents or any purification procedures, to obtain a high grade PCR-product. A specific DNA fragment of the 18S region of Dekkera bruxellensis and Saccharomyces cerevisiae was chosen. The described boiling and freeze-thawing protocols generated the PCR-grade product preparations and could be used to process many samples. The amplification of the fragments could be observed after 30 and 35 cycles. These processes of extraction without using any kind of chemical reagents, especial water, and purification procedures proved to be efficient, reproducible, simple, fast, and inexpensive.

  13. Rapid development of bromodomain probes using flow synthesis methods and frontal affinity chromatography


    Ingham, Richard; Guetzoyan, Lucie; Nikbin, Nikzad; Ley, Steven V.


    This poster illustrates aspects of a project involving the development of new probes for bromodomain 9 protein. Flow chemistry technologies and remote monitoring techniques were used for the synthesis, and Frontal Affinity Chromatography assays were used for analysis of the products.

  14. RNase one gene isolation, expression, and affinity purification models research experimental progression and culminates with guided inquiry-based experiments. (United States)

    Bailey, Cheryl P


    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 of materials and methods and the semester culminates in a poster session. Experimental plans take into account the expense and time required to move from gene isolation to enzyme assays. This combination of instructor-guided and student-designed experiments is a manageable foray into guided inquiry-based learning in a biochemistry laboratory course, while providing a cohesive story and context for individual experiments. Copyright © 2009 International Union of Biochemistry and Molecular Biology, Inc.

  15. An affinity-based aqueous two-phase mixed micellar system and its purification of Yeast 3',5'-bisphosphate nucleotidase. (United States)

    Liu, Yao; Sun, Mei-Hao; Shao, Shi-Kuan; Deng, Gang


    Aqueous two-phase micellar system (ATPMS), as an alternative liquid-liquid extraction technique, has been extensively exploited for the precise separation or large-scale concentration of biomolecules. In this article, a novel affinity-based ATPMS composed of mixed micelles was constructed by introducing a Copper-chelated Triton X-114 (TX-Cu(II)) into an aqueous solution of hydrophobically modified ethylene oxide polymer (HM-EO). Phase diagram of the HM-EO/TX-Cu(II) system was measured, and the partitioning behavior of model proteins (YND, BSA, lysozyme) were studied by using this new system. The addition of HM-EO can result in formation of the micellar network in the micelle-rich phase, making the phase separation easier and stabler. In addition, the extractive performance of ATPMS was enhanced due to the existence of the mixed micelles composed by HM-EO and Cu(II)-chelated TX. It was found in the partitioning experiments that the hexahistidine-tagged Yeast 3',5'-bisphosphate nucleotidase (YND) was selectively extracted into the micelle-rich phase, while the histidine-poor proteins (BSA and lysozyme) remained in the micelle-poor phase. Finally, HM-EO/TX-Cu(II) was used directly to process the fermentation broth. The target protein, YND could be recovered from the cell lysate with a recovery yield of 49.23% and purification factor of 2.63. The results indicated that the new affinity-based HM-EO/TX-Cu(II) system had high partitioning performance which is promising for effectively separation of the histidine-tagged proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties. (United States)

    Chafik, Abdelbasset; Essamadi, Abdelkhalid; Çelik, Safinur Yildirim; Mavi, Ahmet


    Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al3+, Cd2+ and Mg2+, whereas Ca2+, Co2+ and Ni2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The Km and Vmax of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with Ki value of 14.43μM, the IC50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Rapid purification of diastereoisomers from Piper kadsura using supercritical fluid chromatography with chiral stationary phases. (United States)

    Xin, Huaxia; Dai, Zhuoshun; Cai, Jianfeng; Ke, Yanxiong; Shi, Hui; Fu, Qing; Jin, Yu; Liang, Xinmiao


    Supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an advanced solution for the separation of achiral compounds in Piper kadsura. Analogues and stereoisomers are abundant in natural products, but there are obstacles in separation using conventional method. In this paper, four lignan diastereoisomers, (-)-Galbelgin, (-)-Ganschisandrin, Galgravin and (-)-Veraguensin, from Piper kadsura were separated and purified by chiral SFC. Purification strategy was designed, considering of the compound enrichment, sample purity and purification throughput. Two-step achiral purification method on chiral preparative columns with stacked automated injections was developed. Unconventional mobile phase modifier dichloromethane (DCM) was applied to improve the sample solubility. Four diastereoisomers was prepared at the respective weight of 103.1mg, 10.0mg, 152.3mg and 178.6mg from 710mg extract with the purity of greater than 98%. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Reinvestigation of aminoacyl-tRNA synthetase core complex by affinity purification-mass spectrometry reveals TARSL2 as a potential member of the complex.

    Directory of Open Access Journals (Sweden)

    Kyutae Kim

    Full Text Available Twenty different aminoacyl-tRNA synthetases (ARSs link each amino acid to their cognate tRNAs. Individual ARSs are also associated with various non-canonical activities involved in neuronal diseases, cancer and autoimmune diseases. Among them, eight ARSs (D, EP, I, K, L, M, Q and RARS, together with three ARS-interacting multifunctional proteins (AIMPs, are currently known to assemble the multi-synthetase complex (MSC. However, the cellular function and global topology of MSC remain unclear. In order to understand the complex interaction within MSC, we conducted affinity purification-mass spectrometry (AP-MS using each of AIMP1, AIMP2 and KARS as a bait protein. Mass spectrometric data were funneled into SAINT software to distinguish true interactions from background contaminants. A total of 40, 134, 101 proteins in each bait scored over 0.9 of SAINT probability in HEK 293T cells. Complex-forming ARSs, such as DARS, EPRS, IARS, Kars, LARS, MARS, QARS and RARS, were constantly found to interact with each bait. Variants such as, AIMP2-DX2 and AIMP1 isoform 2 were found with specific peptides in KARS precipitates. Relative enrichment analysis of the mass spectrometric data demonstrated that TARSL2 (threonyl-tRNA synthetase like-2 was highly enriched with the ARS-core complex. The interaction was further confirmed by coimmunoprecipitation of TARSL2 with other ARS core-complex components. We suggest TARSL2 as a new component of ARS core-complex.

  19. Identifying novel protein complexes in cancer cells using epitope-tagging of endogenous human genes and affinity-purification mass spectrometry. (United States)

    Song, Jing; Hao, Yujun; Du, Zhanwen; Wang, Zhenghe; Ewing, Rob M


    Affinity-purification mass spectrometry (AP-MS) is the preeminent technique for identification of eukaryotic protein complexes in vivo. AP-MS workflows typically express epitope-tagged bait proteins, immunopurify, and then identify associated protein complexes using mass spectrometry. However, challenges of existing strategies include the construction of expression vectors for large open reading frames and the possibility that overexpression of bait proteins may result in expression of nonphysiological levels of the bait protein with concomitant perturbation of endogenous protein complexes. To address these issues, we use human cell lines with epitope-tagged endogenous genes as AP-MS substrates to develop a platform that we call "knock-in AP-MS", thereby avoiding the challenges of expression vector construction and ensuring that expression of tagged proteins is driven by endogenous regulatory mechanisms. Using three different bait genes (MRE11A, DNMT1 and APC), we show that cell lines expressing epitope-tagged endogenous genes make good substrates for sensitive and reproducible identification of protein interactions using AP-MS. In particular, we identify novel interactors of the important oncoprotein Adenomatous Polyposis Coli (APC), including an interaction with Flightless-1 homologue (FLII) that is enriched in nuclear fractions.

  20. Quantitative Assessment of the Effects of Trypsin Digestion Methods on Affinity Purification-Mass Spectrometry-based Protein-Protein Interaction Analysis. (United States)

    Zhang, Yueqing; Sun, Hong; Zhang, Jing; Brasier, Allan R; Zhao, Yingxin


    Affinity purification-mass spectrometry (AP-MS) has become the method of choice for discovering protein-protein interactions (PPIs) under native conditions. The success of AP-MS depends on the efficiency of trypsin digestion and the recovery of the tryptic peptides for MS analysis. Several different protocols have been used for trypsin digestion of protein complexes in AP-MS studies, but no systematic studies have been conducted on the impact of trypsin digestion conditions on the identification of PPIs. Here, we used NFκB/RelA and Bromodomain-containing protein 4 (BRD4) as baits and test five distinct trypsin digestion methods (two using "on-beads," three using "elution-digestion" protocols). Although the performance of the trypsin digestion protocols change slightly depending on the different baits, antibodies and cell lines used, we found that elution-digestion methods consistently outperformed on-beads digestion methods. The high-abundance interactors can be identified universally by all five methods, but the identification of low-abundance RelA interactors is significantly affected by the choice of trypsin digestion method. We also found that different digestion protocols influence the selected reaction monitoring (SRM)-MS quantification of PPIs, suggesting that optimization of trypsin digestion conditions may be required for robust targeted analysis of PPIs.

  1. Studies on the glycoprotein nature of the thyrotropin receptor: interaction with lectins and purification of the bovine protein with the use of Bandeiraea (Griffonia) simplicifolia I affinity chromatography. (United States)

    Kress, B C; Spiro, R G


    The TSH receptor from Triton-solubilized bovine microsomal membranes was found to bind to a substantial extent to columns of the immobilized lectins Bandeiraea (Griffonia) simplicifolia I, Ricinus communis I, wheat germ, and Concanavalin A, whereas it was not retained by Dolichos biflorus. Elution of TSH receptor activity from these lectins could be achieved with the appropriate saccharides in all cases except Concanavalin A. The most extensive adsorption of the receptor occurred on B. simplicifolia I-agarose (84%), and the terminal alpha-D-galactosyl specificity of this interaction was substantiated by its susceptibility to alpha-galactosidase treatment. Whereas TSH itself was not bound to this immobilized lectin, a complex of this hormone with its receptor did interact and could be eluted with methyl-alpha-D-galactoside. Purification (800-fold) of the bovine TSH receptor was achieved by a combination of TSH and B. simplicifolia I affinity chromatographies. Polyacrylamide gel electrophoresis of the purified TSH receptor after radioiodination revealed three major components with apparent mol wt of 316,000, 115,000, and 54,000.

  2. 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. (United States)

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


    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].

  3. Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling. (United States)

    Li, Xu; Gao, Min; Choi, Jong Min; Kim, Beom-Jun; Zhou, Mao-Tian; Chen, Zhen; Jain, Antrix N; Jung, Sung Yun; Yuan, Jingsong; Wang, Wenqi; Wang, Yi; Chen, Junjie


    Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined c lustered, r egularly i nterspaced s hort p alindromic r epeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. ADP-ribose-specific chromatin-affinity purification for investigating genome-wide or locus-specific chromatin ADP-ribosylation. (United States)

    Bisceglie, Lavinia; Bartolomei, Giody; Hottiger, Michael O


    Protein ADP-ribosylation is a structurally heterogeneous post-translational modification (PTM) that influences the physicochemical and biological properties of the modified protein. ADP-ribosylation of chromatin changes its structural properties, thereby regulating important nuclear functions. A lack of suitable antibodies for chromatin immunoprecipitation (ChIP) has so far prevented a comprehensive analysis of DNA-associated protein ADP-ribosylation. To analyze chromatin ADP-ribosylation, we recently developed a novel ADP-ribose-specific chromatin-affinity purification (ADPr-ChAP) methodology that uses the recently identified ADP-ribose-binding domains RNF146 WWE and Af1521. In this protocol, we describe how to use this robust and versatile method for genome-wide and loci-specific localization of chromatin ADP-ribosylation. ADPr-ChAP enables bioinformatic comparisons of ADP-ribosylation with other chromatin modifications and is useful for understanding how ADP-ribosylation regulates biologically important cellular processes. ADPr-ChAP takes 1 week and requires standard skills in molecular biology and biochemistry. Although not covered in detail here, this technique can also be combined with conventional ChIP or DNA analysis to define the histone marks specifically associated with the ADP-ribosylated chromatin fractions and dissect the molecular mechanism and functional role of chromatin ADP-ribosylation.

  5. Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

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    O Rickard Nilsson

    Full Text Available Recent studies indicate that defective activity of complement factor H (FH is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

  6. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

    Directory of Open Access Journals (Sweden)

    Yin Zongyi

    Full Text Available Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods. Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets. In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  7. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells. (United States)

    Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang; Baifeng, Li


    Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  8. Grafting iminodiacetic acid on silica nanoparticles for facilitated refolding of like-charged protein and its metal-chelate affinity purification. (United States)

    Liu, Hu; Dong, Xiaoyan; Sun, Yan


    A series of highly charged nanoscale chelators were fabricated by grafting of poly(glycidyl methacrylate-iminodiacetic acid) (pGI) chains with iminodiacetic acid (IDA) chelating group on silica nanoparticles (SNPs) via atom transfer radical polymerization (ATRP). The nanoscale chelators, denoted as SNPs-pGI, possessed a nickel ion chelating capacity as high as 2800 μmol/g, 50 times higher than the IDA-modified Sepharose FF (IDA-Sepharose) resin reported in literature and offered a high affinity binding capacity for hexahistidine-tagged enhanced green fluorescence protein (6 × His-EGFP) after nickel ion loading. More importantly, the anionic SNPs-pGI of high charge densities displayed much better performance than IDA-Sepharose in facilitating the refolding of like-charged 6 × His-EGFP from inclusion bodies (IBs). For example, for 0.2mg/mL 6 × His-EGFP IB refolding, addition of 6.2 μL/mL SNPs-pGI with the highest charge density led to a refolding yield of 90%, over 43% higher than that obtained with 460 μL/mL IDA-Sepharose. It is notable that the much higher efficiency of the nanoscale chelator was obtained with a chelator consumption corresponding to only 1.4% of IDA-Sepharose. Moreover, the highly charged SNPs-pGI could efficiently facilitate the refolding of 6 × His-EGFP at higher IB concentrations (0.4 and 0.8 mg/mL). After refolding, nickel ions addition led to the recovery of the refolded 6 × His-EGFP with high yield (80%), purity (96%) and enrichment ratio (1.8). All the results suggest that the SNPs-pGI of high charge densities were promising for cost-effective recovery of His-tagged proteins expressed as IBs with the integrative like-charge facilitated refolding and metal-chelate affinity purification strategy. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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    Yuichi Ikeda

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Nicholas J Matheson

    Full Text Available 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.

  11. Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification. (United States)

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


    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.

  12. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

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    Li Li


    Full Text Available Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1 DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2 Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3 Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4 High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  13. Characterization of the RNA Required for Biosynthesis of delta-Aminolevulinic Acid from Glutamate : Purification by Anticodon-Based Affinity Chromatography and Determination That the UUC Glutamate Anticodon Is a General Requirement for Function in ALA Biosynthesis. (United States)

    Schneegurt, M A; Beale, S I


    The heme and chlorophyll precursor delta-aminolevulinic acid acid (ALA) is formed in plants and algae from glutamate in a process that requires at least three enzyme components plus a low molecular weight RNA which co-purifies with the tRNA fraction during DEAE-cellulose column chromatography. RNA that is effective in the in vitro ALA biosynthetic system was extracted from several plant and algal species that form ALA via this route. In all cases, the effective RNA contained the UUC glutamate anticodon, as determined by its specific retention on an affinity resin containing an affine ligand directed against this anticodon. Construction of the affinity resin was based on the fact that the UUC glutamate anticodon is complementary to the GAA phenylalanine anticodon. By covalently linking the 3' terminus of yeast tRNA(Phe(GAA)) to hydrazine-activated polyacrylamide gel beads, a resin carrying an affine ligand specific for the anticodon of tRNA(Glu(UUC)) was obtained. Column chromatography of plant and algal RNA extracts over this resin yielded a fraction that was highly enriched in the ability to stimulate ALA formation from glutamate when added to enzyme extracts of the unicellular green alga Chlorella vulgaris. Enhancement of ALA formation per A(260) unit added was as much as 50 times greater with the affinity-purified RNA than with the RNA before affinity purification. The affinity column selectively retained RNA which supported ALA formation upon chromatography of RNA extracts from species of the diverse algal groups Chlorophyta (Chlorella Vulgaris), Euglenophyta (Euglena gracilis), Rhodophyta (Cyanidium caldarium), and Cyanophyta (Synechocystis sp. PCC 6803), and a higher plant (spinach). Other glutamate-accepting tRNAs that were not retained by the affinity column were ineffective in supporting ALA formation. These results indicate that possession of the UUC glutamate anticodon is a general requirement for RNA to participate in the conversion of glutamate to ALA

  14. An Automated Microwave-Assisted Synthesis Purification System for Rapid Generation of Compound Libraries. (United States)

    Tu, Noah P; Searle, Philip A; Sarris, Kathy


    A novel methodology for the synthesis and purification of drug-like compound libraries has been developed through the use of a microwave reactor with an integrated high-performance liquid chromatography-mass spectrometry (HPLC-MS) system. The strategy uses a fully automated synthesizer with a microwave as energy source and robotic components for weighing and dispensing of solid reagents, handling liquid reagents, capper/crimper of microwave reaction tube assemblies, and transportation. Crude reaction products were filtered through solid-phase extraction cartridges and injected directly onto a reverse-phase chromatography column via an injection valve. For multistep synthesis, crude products were passed through scavenger resins and reintroduced for subsequent reactions. All synthetic and purification steps were conducted under full automation with no handling or isolation of intermediates, to afford the desired purified products. This approach opens the way to highly efficient generation of drug-like compounds as part of a lead discovery strategy or within a lead optimization program. © 2015 Society for Laboratory Automation and Screening.

  15. Seamless bead to microarray screening: rapid identification of the highest affinity protein ligands from large combinatorial libraries. (United States)

    Astle, John M; Simpson, Levi S; Huang, Yong; Reddy, M Muralidhar; Wilson, Rosemary; Connell, Steven; Wilson, Johnnie; Kodadek, Thomas


    Several approaches have been developed for screening combinatorial libraries or collections of synthetic molecules for agonists or antagonists of protein function, each with its own advantages and limitations. In this report, we describe an experimental platform that seamlessly couples massively parallel bead-based screening of one-bead one-compound combinatorial libraries with microarray-based quantitative comparisons of the binding affinities of the many hits isolated from the bead library. Combined with other technical improvements, this technique allows the rapid identification of the best protein ligands in combinatorial libraries containing millions of compounds without the need for labor-intensive resynthesis of the hits. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  16. Surface-Modified Cobalt Ferrite Nanoparticles for Rapid Capture, Detection, and Removal of Pathogens: a Potential Material for Water Purification. (United States)

    Bohara, Raghvendra A; Throat, Nanasaheb D; Mulla, Nayeem A; Pawar, Shivaji H


    Enteric infections resulting from the consumption of contaminated drinking water, inadequate supply of water for personal hygiene, and poor sanitation take a heavy toll worldwide, and developing countries are the major sufferers. Consumption of microbiologically contaminated water leads to diseases such as amoebiasis, cholera, shigellosis, typhoid, and viral infections leading to gastroenteritis and hepatitis B. The present investigation deals with the development of effective method to capture and eliminate microbial contamination of water and improve the quality of water and thus decreasing the contaminated waterborne infections. Over the last decade, numerous biomedical applications have emerged for magnetic nanoparticles (MNPs) specifically iron oxide nanoparticles. For the first time, we have explored functionalized cobalt ferrite nanoparticles (NPs) for capture and detection of pathogens. The captured bacterial were separated by using simple magnet. To begin with, the prepared NPs were confirmed for biocompatibility study and further used for their ability to detect the bacteria in solution. For this, standard bacterial concentrations were prepared and used to confirm the ability of these particles to capture and detect the bacteria. The effect of particle concentration, time, and pH has been studied, and the respective results have been discussed. It is observed that the presence of amine group on the surface of NPs shows nonspecific affinity and capability to capture Escherichia coli and Staphylococcus aureus. The possible underlying mechanism is discussed in the present manuscript. Based upon this, the present material can be considered for large-scale bacteria capture in water purification application.

  17. Rapid determination of total aflatoxins and ochratoxins A in meat products by immuno-affinity fluorimetry. (United States)

    Abd-Elghany, Samir Mohammed; Sallam, Khalid Ibrahim


    Total aflatoxins (AFT) and ochratoxin A (OTA) levels were estimated using the VICAM AflaTest and OchraTest immunoaffinity fluorometric method in a total of 50 meat products (25 each of beef luncheon and beef burger) purchased from different supermarkets in Mansoura city, Egypt. All the meat samples analyzed were contaminated with both AFT and OTA with mean values of 1.1 μg/kg and 5.23 μg/kg, respectively, for beef luncheon and mean values of 3.22 μg/kg and 4.55 μg/kg, respectively, for beef burger. None of the beef luncheon and burger samples analyzed exceeded the permissible limits set by FDA for AFT, but 40% of beef burgers exceeded the FAO AFT permissible limit. Similarly, 52% and 36% of beef luncheon and beef burger samples exceeded the FAO OTA permissible limit. Application of the immunoaffinity fluorometric method is an accurate, safe and rapid method for mycotoxins determination in meat products to ensure their safety for human consumption. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Rapid analysis of the interactions between drugs and human serum albumin (HSA) using high-performance affinity chromatography (HPAC)


    Kim, Hee Seung; Wainer, Irving W.


    This study used a combination of zonal elution and frontal affinity chromatography on immobilized human serum albumin (HSA) high-performance affinity chromatography (HPAC) column to examine the association constants of various compounds that have been studied by equilibrium dialysis or ultra filtration. A standard plot was generated from retention factors of reference compounds using zonal elution chromatography against association constants of reference compounds using frontal affinity chrom...

  19. Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

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    Pierre-Olivier Buclez


    Full Text Available Recombinant adeno-associated viruses (rAAV are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology.

  20. Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus (United States)

    Hou, Han Wei; Petchakup, Chayakorn; Tay, Hui Min; Tam, Zhi Yang; Dalan, Rinkoo; Chew, Daniel Ek Kwang; Li, King Ho Holden; Boehm, Bernhard O.


    Advanced management of dysmetabolic syndromes such as diabetes will benefit from a timely mechanistic insight enabling personalized medicine approaches. Herein, we present a rapid microfluidic neutrophil sorting and functional phenotyping strategy for type 2 diabetes mellitus (T2DM) patients using small blood volumes (fingerprick ~100 μL). The developed inertial microfluidics technology enables single-step neutrophil isolation (>90% purity) without immuno-labeling and sorted neutrophils are used to characterize their rolling behavior on E-selectin, a critical step in leukocyte recruitment during inflammation. The integrated microfluidics testing methodology facilitates high throughput single-cell quantification of neutrophil rolling to detect subtle differences in speed distribution. Higher rolling speed was observed in T2DM patients (P < 0.01) which strongly correlated with neutrophil activation, rolling ligand P-selectin glycoprotein ligand 1 (PSGL-1) expression, as well as established cardiovascular risk factors (cholesterol, high-sensitive C-reactive protein (CRP) and HbA1c). Rolling phenotype can be modulated by common disease risk modifiers (metformin and pravastatin). Receiver operating characteristics (ROC) and principal component analysis (PCA) revealed neutrophil rolling as an important functional phenotype in T2DM diagnostics. These results suggest a new point-of-care testing methodology, and neutrophil rolling speed as a functional biomarker for rapid profiling of dysmetabolic subjects in clinical and patient-oriented settings.

  1. Protein A affinity precipitation of human immunoglobulin G. (United States)

    Janoschek, Lars; Freiherr von Roman, Matthias; Berensmeier, Sonja


    The potential of protein A affinity precipitation as an alternative method for traditional antibody purification techniques was investigated. Recombinant produced protein A from Staphylococcus aureus (SpA) was covalently linked to the pH-responsive copolymer Eudragit(®) S-100 and used for purification of human immunoglobulin G (hIgG). The Eudragit-SpA conjugate had a static binding capacity of 93.9 ± 2.8 mg hIgG per g conjugate and a dissociation constant of 787 ± 67 nM at 7 ± 1°C. The antibody was adsorbed rapidly onto Eudragit-SpA and reached equilibrium within 5 min. An excess of hIgG binding sites, provided by the conjugate, as well as adjusted elution conditions resulted in an appropriate hIgG purification performance. In summary, Eudragit-SpA was successfully applied to capture hIgG from a protein mixture with 65% antibody yield in the elution step. Nearly 96% purity and a purification factor of 12.4 were achieved. The Eudragit-SpA conjugate showed a stable ligand density over several cycles, which enabled reusability for repeated precipitation of hIgG. According to this, pH induced affinity precipitation can be seen as a potential alternative for protein A chromatography in antibody purification processes. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography


    Scheich, Christoph; Sievert, Volker; Büssow, Konrad


    Abstract Background Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner. Results We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag) or glutathione S-transferase (GST)-tag from bacterial expression systems. Proteins ar...

  3. A Generalized Design for Affinity Chromatography Columns


    Kao, Lee-Wei; Wang, Nien-Hwa Linda


    In affinity chromatography, an adsorbent with a high selectivity for a target solute is used to isolate the target molecule from other impurities. With sufficient selectivity, the target molecule can be isolated in a highly purified and concentrated state. Common applications of affinity chromatography include Protein A chromatography for antibody purification and Immobilized Metal Affinity Chromatography (IMAC) for protein purification. The well-known design method based on constant-pattern ...

  4. Rapid screening of dioxin-contaminated soil by accelerated solvent extraction/purification followed by immunochemical detection

    Energy Technology Data Exchange (ETDEWEB)

    Nording, Malin [Umeaa University, Environmental Chemistry, Umeaa (Sweden); Swedish Defence Research Agency, Umeaa (Sweden); Nichkova, Mikaela; Gee, Shirley J.; Hammock, Bruce D. [University of California, Department of Entomology and Cancer Research Center, Davis, CA (United States); Spinnel, Erik; Persson, Ylva; Haglund, Peter [Umeaa University, Environmental Chemistry, Umeaa (Sweden)


    Since soils at industrial sites might be heavily contaminated with polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), there is a need for large-scale soil pollution surveys and, thus, for cost-efficient, high-throughput dioxin analyses. However, trace analysis of dioxins in complex matrices requires exhaustive extraction, extensive cleanup, and very sensitive detection methods. Traditionally, this has involved the use of Soxhlet extraction and multistep column cleanup, followed by gas chromatography - high-resolution mass spectrometry (GC/HRMS), but bioanalytical techniques may allow much more rapid, cost-effective screening. The study presented here explores the possibility of replacing the conventional method with a novel approach based on simultaneous accelerated solvent extraction (ASE) and purification, followed by an enzyme-linked immunosorbent assay (ELISA). Both the traditional and the novel cleanup and detection approaches were applied to contaminated soil samples, and the results were compared. ELISA and GC/HRMS results for Soxhlet-extracted samples were linearly correlated, although the ELISA method slightly underestimated the dioxin levels. To avoid an unacceptable rate of false-negative results, the use of a safety factor is recommended. It was also noted that the relative abundance of the PCDDs/PCDFs, evaluated by principal component analysis, had an impact on the ELISA performance. To minimize this effect, the results may be corrected for differences between the ELISA cross-reactivities and the corresponding toxic equivalency factor values. Finally, the GC/HRMS and ELISA results obtained following the two sample preparation methods agreed well; and the ELISA and GC/HRMS results for ASE extracts were strongly correlated (correlation coefficient, 0.90). Hence, the ASE procedure combined with ELISA analysis appears to be an efficient approach for high-throughput screening of PCDD-/PCDF-contaminated soil samples. (orig.)

  5. Data for the identification of proteins and post-translational modifications of proteins associated to histones H3 and H4 in S. cerevisiae, using tandem affinity purification coupled with mass spectrometry. (United States)

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè


    Tandem affinity purification method (TAP) allows the efficient purification of native protein complexes which incorporate a target protein fused with the TAP tag. Purified multiprotein complexes can then be subjected to diverse types of proteomic analyses. Here we describe the data acquired after applying the TAP strategy on histones H3 and H4 coupled with mass spectrometry to identify associated proteins and protein post-translational modifications in the budding yeast, Saccharomyces cerevisiae. The mass spectrometry dataset described here consists of 14 files generated from four different analyses in a 5600 Triple TOF (Sciex) by information-dependent acquisition (IDA) LC-MS/MS. The above files contain information about protein identification, protein relative abundance, and PTMs identification. The instrumental raw data from these files has been also uploaded to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier PRIDE: PXD002671 and These data are discussed and interpreted in Valero et al. (2016) [1].

  6. Magnetic Solid-phase Extraction with Fe3O4/Molecularly Imprinted Polymers Modified by Deep Eutectic Solvents and Ionic Liquids for the Rapid Purification of Alkaloid Isomers (Theobromine and Theophylline from Green Tea

    Directory of Open Access Journals (Sweden)

    Guizhen Li


    Full Text Available Different kinds of deep eutectic solvents (DES based on choline chloride (ChCl and ionic liquids (ILs based on 1-methylimidazole were used to modify Fe3O4/molecularly imprinted polymers (Fe3O4/MIPs, and the resulting materials were applied for the rapid purification of alkaloid isomers (theobromine and theophylline from green tea with magnetic solid-phase extraction (M-SPE. The M-SPE procedure was optimized using the response surface methodology (RSM to analyze the maximum conditions. The materials were characterized by Fourier transform infrared spectroscopy (FI-IR and field emission scanning electron microscopy (FE-SEM. Compared to the ILs-Fe3O4/MIPs, the DESs-Fe3O4/MIPs were developed for the stronger recognition and higher recoveries of the isomers (theophylline and theobromine from green tea, particularly DES-7-Fe3O4/MIPs. With RSM, the optimal recovery condition for theobromine and theophylline in the M-SPE were observed with ratio of methanol (80% as the washing solution, methanol/acetic acid (HAc (8:2 as the eluent at pH 3, and an eluent volume of 4 mL. The practical recoveries of theobromine and theophylline in green tea were 92.27% and 87.51%, respectively, with a corresponding actual extraction amount of 4.87 mg•g−1 and 5.07 mg•g−1. Overall, the proposed approach with the high affinity of Fe3O4/MIPs might offer a novel method for the purification of complex isomer samples.

  7. Magnetic Solid-phase Extraction with Fe₃O₄/Molecularly Imprinted Polymers Modified by Deep Eutectic Solvents and Ionic Liquids for the Rapid Purification of Alkaloid Isomers (Theobromine and Theophylline) from Green Tea. (United States)

    Li, Guizhen; Wang, Xiaoqin; Row, Kyung Ho


    Different kinds of deep eutectic solvents (DES) based on choline chloride (ChCl) and ionic liquids (ILs) based on 1-methylimidazole were used to modify Fe3O4/molecularly imprinted polymers (Fe3O4/MIPs), and the resulting materials were applied for the rapid purification of alkaloid isomers (theobromine and theophylline) from green tea with magnetic solid-phase extraction (M-SPE). The M-SPE procedure was optimized using the response surface methodology (RSM) to analyze the maximum conditions. The materials were characterized by Fourier transform infrared spectroscopy (FI-IR) and field emission scanning electron microscopy (FE-SEM). Compared to the ILs-Fe3O4/MIPs, the DESs-Fe3O4/MIPs were developed for the stronger recognition and higher recoveries of the isomers (theophylline and theobromine) from green tea, particularly DES-7-Fe3O4/MIPs. With RSM, the optimal recovery condition for theobromine and theophylline in the M-SPE were observed with ratio of methanol (80%) as the washing solution, methanol/acetic acid (HAc) (8:2) as the eluent at pH 3, and an eluent volume of 4 mL. The practical recoveries of theobromine and theophylline in green tea were 92.27% and 87.51%, respectively, with a corresponding actual extraction amount of 4.87 mg•g-1 and 5.07 mg•g-1. Overall, the proposed approach with the high affinity of Fe3O4/MIPs might offer a novel method for the purification of complex isomer samples.

  8. Purification of clenbuterol-like beta2-agonist drugs of new generation from bovine urine and hair by alpha1-acid glycoprotein affinity chromatography and determination by gas chromatography-mass spectrometry. (United States)

    Gallo, Pasquale; Brambilla, Gianfranco; Neri, Bruno; Fiori, Maurizio; Testa, Cecilia; Serpe, Luigi


    The control of illegal use of clenbuterol and other beta(2)-agonist drugs as growth promoters in the European Union countries has led to outlaw practices for synthesizing new concept molecules, showing similar biological activity but not detectable by test methods usually employed to perform the official monitoring programmes. The synthesis schemes of some beta(2)-agonist compounds, formally derived from clenbuterol, were found out by Italian detective authorities. These compounds were synthesised ex novo in our laboratories: then, both their molecular structures and biological activities were characterised. In this paper, we describe different strategies for purifying some beta(2)-agonist drugs of new concept, more hydrophobic than clenbuterol. A two-step clean up procedure, prior to gas chromatography-mass spectrometry analysis, was developed for the multi-residue determination of these beta(2)-agonists from bovine hair and urine. The purification strategy we chose was based on adsorption solid phase extraction and, subsequently, on specific molecular recognition by affinity chromatography. The affinity columns were homemade by coupling bovine alpha(1)-acid glycoprotein, a plasmatic acceptor for basic drugs, to a chromatographic support; their effectiveness for purifying new beta(2)-agonists was discussed. The data about method recoveries and repeatability were also reported.

  9. Use of Strep-tag II for rapid detection and purification of Mycobacterium tuberculosis recombinant antigens secreted by Streptomyces lividans. (United States)

    Ayala, Julio C; Pimienta, Elsa; Rodríguez, Caridad; Anné, Jozef; Vallín, Carlos; Milanés, María T; King-Batsios, Emmanuel; Huygen, Kris; Van Mellaert, Lieve


    Recent results with respect to the secretory production of bio-active Mycobacterium tuberculosis proteins in Streptomyces have stimulated the further exploitation of this host as a bacterial cell factory. However, the rapid isolation of a recombinant protein by conventional procedures can be a restrictive step. A previous attempt to isolate recombinant antigens fused to the widely used 6His-tag was found to be relatively incompatible with secretory production in the Streptomyces host. As an alternative, the eight-residue Strep-tag® II (WSHPQFEK), displaying intrinsic binding affinity towards streptavidin, was evaluated for the secretory production of two M. tuberculosis immunodominant antigens in Streptomyces lividans and their subsequent downstream processing. Therefore, the genes ag85A (Rv3804c, encoding the mycolyl-transferase Ag85A) and Rv2626c (encoding hypoxic response protein 1), were equipped with a 3'-Strep-tag® II-encoding sequence and placed under control of the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) transcriptional, translational and signal sequences. Strep-tagged Ag85A and Rv2626c proteins were detected in the spent medium of recombinant S. lividans cultures at 48h of growth, and purified using a Strep-Tactin Superflow® matrix. Recombinant Ag85A appeared as a 30-kDa protein of which the N-terminal amino acid sequence was identical to the expected one. Rv2626c was produced in two forms of 17 and 37kDa respectively, both with the same predicted N-terminal sequence, suggesting that the 37-kDa product is an Rv2626c dimer. The obtained results indicate that the Strep-tagII is proteolytically stable in Streptomyces and does not interfere with the membrane translocation of Ag85A and Rv2626c. A comparison of reactivity of serum from tuberculosis patients versus healthy persons by ELISA showed that both S. lividans-derived antigens were recognized by sera of individuals infected with M. tuberculosis, indicating that they remained

  10. Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia


    de Melo Silva, Alex Jose José; Alves, Lumar Lucena; Pakay, Julian


    This work was performed as an integrated practical of a Biomedical Science undergraduate course of Biochemistry subject, in order to demonstrate used techniques to purify of Green Fluorescent Protein (GFP). To perform the experiments the main methodology applied was the by immobilized metal affinity chromatography (IMAC).  The open reading frame for enhanced GFP was sub-cloned into the pQE30 expression vector. The subsequent production of protein tagged N-terminally with hexahistidine, facili...

  11. Affinity Reagents for Multiplexed, Rapid Diagnosis of Bacterial Infections at the Point of Care using Diagnostic Magnetic Resonance (United States)


    determinants and small molecules as probes. Our milestones are: 1. Assemble panel of clinical isolates of S. pneumoniae and representative gram...developing for DMR detection. Below, we present the results for each of the proposed specific aims. 1. Assemble panel of clinical isolates of S...albeit with different trade-offs. Specifically, anti-PPD is a high affinity antibody that can discern between mycobacteria and other bacteria, but

  12. Characterizations of heparin-binding proteins in human urine by affinity purification-mass spectrometry and defining "L-x(2,3)-A-x(0,1)-L" as a novel heparin-binding motif. (United States)

    Manissorn, Juthatip; Thongboonkerd, Visith


    Heparin-binding proteins (HBPs) are considered as potential modulators of kidney stone formation. However, HBPs had not been characterized in the urine previously. In this study, we applied affinity purification-mass spectrometry (AP-MS) using cellufine sulfate column chromatography and liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-Q-TOF MS/MS) to identify HBPs in normal human urine. Using this approach, 83 HBPs were identified, including those involved in metabolic process, cellular process, immune system, developmental process, response to stimuli, cell communication, transport, cell adhesion and others. The AP-MS data were confirmed by Western blot analysis and chemico-protein interactions analysis using STITCH tool. In addition, 59, 55 and 51 identified HBPs had the known heparin-binding motifs "XBBXnBX", "XBXnBBX" and "XBBBnX", respectively. Moreover, a novel heparin-binding motif "L-x(2,3)-A-x(0,1)-L" was found in 58 identified HBPs using PRATT tool. The sensitivity and specificity of this novel motif were 85% and 100%, respectively, by validation using 20 known HBPs and 11 non-HBPs. We report herein for the first time a large number of HBPs in normal human urine and defined "L-x(2,3)-A-x(0,1)-L" as a novel heparin-binding motif. These findings will be useful to further understand the renal physiology and may also lead to identification of novel modulators of kidney stone formation. Heparin-binding proteins (HBPs) have several important roles in various biological processes, including kidney stone formation. However, HBPs had not been characterized in the urine. Our present work using affinity purification coupled to mass spectrometry (AP-MS) is the first large-scale study on HBPs in human urine. In addition to the three known heparin-binding motifs, "XBBXnBX", "XBXnBBX", and "XBBBnX", we successfully defined the amino acid pattern "L-x(2,3)-A-x(0,1)-L" as a novel heparin-binding motif. These findings will be useful to further

  13. Use of peak decay analysis and affinity microcolumns containing silica monoliths for rapid determination of drug-protein dissociation rates. (United States)

    Yoo, Michelle J; Hage, David S


    This report examined the use of silica monoliths in affinity microcolumns containing human serum albumin (HSA) to measure the dissociation rates for various drugs from this protein. Immobilized HSA and control monolith columns with dimensions of 1 mm × 4.6 mm i.d. were prepared for this work and used with a noncompetitive peak decay method. Several drugs known to bind HSA were examined, such as warfarin, diazepam, imipramine, acetohexamide, and tolbutamide. Items that were studied and optimized in this method included the sample volume, sample concentration, and elution flow rate. It was found that flow rates up to 10 mL/min could be used in this approach. Work with HSA silica monoliths at these high flow rates made it possible to provide dissociation rate constants for drugs such as warfarin in less than 40s. The dissociation rate constants that were measured gave good agreement with values reported in the literature or that had been obtained with other solutes that had similar binding affinities for HSA. This approach is a general one that should be useful in examining the dissociation of other drugs from HSA and in providing a high-throughput method for screening drug-protein interactions. Copyright © 2010 Elsevier B.V. All rights reserved.

  14. Guiding the evolution to catch the virus: An in silico study of affinity maturation against rapidly mutating antigen (United States)

    Wang, Shenshen; Burton, Dennis; Kardar, Mehran; Chakraborty, Arup


    The immune system comprises an intricate and evolving collection of cells and molecules that enables a defense against pathogenic agents. Its workings present a rich source of physical problems that impact human health. One intriguing example is the process of affinity maturation (AM) through which an antibody (Ab)--a component of the host immune system--evolves to more efficiently bind an antigen (Ag)--a unique part of a foreign pathogen such as a virus. Sufficiently strong binding to the Ag enables recognition and neutralization. A major challenge is to contain a diversifying mixture of Ag variants, that arise in natural infection, from evading Ab neutralization. This entails a thorough understanding of AM against multiple Ag species and mutating Ag. During AM, Ab-encoding cells undergo cycles of mutation and selection, a process reminiscent of Darwinian evolution yet occurring in real time. We first cast affinity-dependent selection into an extreme value problem and show how the binding characteristics scale with Ag diversity. We then develop an agent-based residue-resolved computational model of AM which allows us to track the evolutionary trajectories of individual cells. This dynamic model not only reveals significant stochastic effects associated with the relatively small and highly dynamic population size, it also uncovers the markedly distinct maturation outcomes if designed Ag variants are presented in different temporal procedures. Insights thus obtained would guide rational design of vaccination protocols.

  15. A phytohemagglutinin from Sunn hemp seeds (Crotalaria juncea). II. Purification by a high capacity biospecific affinity adsorbent and its physicochemical properties. (United States)

    Ersson, B


    A galactose-specific lectin from seeds of Sunn Hemp (Crotalaria juncea) has been purified by fractional precipitation with ammonium sulfate followed by biospecific affinity chromatography and preparative isoelectric focusing. The adsorbent was prepared by coupling galactose to Sepharose 6B activated with divinyl sulfone. The lectin was homogeneous as judged by ultracentrifugation and by electrophoresis in cellulose acetate strips and in polyacrylamide gradient gel. Its isoelectric point is pH 8.8 and the molecular weight is about 120 000. It is a glycoprotein containing 9.8% also carbohydrate (mannose, N-acetyl-D-glucosamine, fucose, and xylose). The lectin contains 3.2 mol Ca2+, 2.2 mol Mg2+ and 0.2 mol Mn2+ per 120 000 g. No sulphur-containing amino acids were detected.

  16. Comparison of ZnS semiconductor nanoparticles capped with various functional groups as the matrix and affinity probes for rapid analysis of cyclodextrins and proteins in surface-assisted laser desorption/ionization time-of-flight mass spectrometry. (United States)

    Kailasa, Suresh Kumar; Kiran, Kamatam; Wu, Hui-Fen


    Zinc sulfide (ZnS) semiconductor nanoparticles (NPs) capped with a variety of functional groups including bare ZnS NPs, 3-mercaptopropanoic acid (ZnS-3-MPA), sodium citrate (ZnS-citrate), cysteamine (ZnS-Cys), and 2-mercaptoethane sulfonate (ZnS-2-MES) have been investigated as the matrix and affinity probes for analysis of alpha-, beta-, and gamma-cyclodextrins (CDs), ubiquitin, and insulin in biological samples by using surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF-MS). Various parameters that would influence the ionization efficiency and sensitivity of these ZnS NPs in SALDI-TOF-MS were examined including the effect of capping agents, sample pH, ion abundance, and concentration of ZnS NPs. Among these ZnS NPs, our results have demonstrated that ZnS-3-MPA exhibited the highest efficiency toward CDs, ubiquitin, and insulin for high-sensitivity detection in SALDI-TOF-MS. The detection limits were 20-55 nM for CDs, 91 nM for ubiquitin, and 85 nM for insulin. The applicability of the present method is demonstrated by detection of ubiquitin-like proteins in oyster mushroom and also in the analysis of analytes in biological samples such as human urine and plasma. To our best knowledge, this is the first time semiconductor NPs were used as the matrix and affinity probes for high-sensitivity detection of organic and biomolecules in SALDI-TOF-MS. This approach exhibits the advantages of being simple, rapid, efficient, and straightforward for direct analysis of organic and biological samples in SALDI-TOF-MS without the need for time-consuming separation processes, tedious washing steps, or further laborious purification. In addition, it also can provide a sensitive and reliable quantitative assay for small- and large-molecule analysis with the detectable mass up to 8500 Da. We believe that this novel ZnS nanoprobe is simple, efficient, lower cost (compared with Au, Ag, and Pt NPs), fast, and with the potential for high

  17. Preparation of core-shell structure Fe3 O4 @SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His-tag protein purification. (United States)

    Ni, Qian; Chen, Bing; Dong, Shaohua; Tian, Lei; Bai, Quan


    The core-shell structure Fe3 O4 /SiO2 magnetic microspheres were prepared by a sol-gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu(2+) , Ni(2+) and Zn(2+) , were chelated on the Fe3 O4 @SiO2 -IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni(2+) -chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe3 O4 @SiO2 -IDA-Ni(2+) magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His-tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd.

  18. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography. (United States)

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja


    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc. © 2014 Wiley Periodicals, Inc.

  19. Purification by ceftibuten-affinity chromatography and the functional reconstitution of oligopeptide transporter(s) in rat intestinal brush-border membrane. (United States)

    Iseki, K; Yonemura, K; Kikuchi, T; Naasani, I; Sugawara, M; Kobayashi, M; Kohri, N; Miyazaki, K


    The transport activity of ceftibuten, a dianionic peptide-like compound, was extracted from rat intestinal brush-border membrane by n-octylglucoside and reconstituted into asolectin liposomes by dialysis. The proteoliposomes prepared from the membrane extract showed an inward H+-gradient-dependent uptake of ceftibuten and glycylsarcosine. Ceftibuten-immobilized affinity chromatography of the membrane extract permitted the isolation of two polypeptides (apparent molecular mass of 117 and 127 kDa) that can recognize the dianionic peptide structure of ceftibuten. Proteoliposomes prepared from reconstituting the isolated proteins into asolectin vesicles showed an overshooting uptake of ceftibuten in the presence of an inwardly directed H+ gradient, and this uptake could be inhibited by L-valyl-L-proline. N-glycanase digestion of the isolated proteins, 117 and 127 kDa, trimmed them into 78 and 120 kDa products, respectively. The protein core size of the smaller protein was in agreement with the calculated molecular mass of approximately 79 kDa for the rat PepT1 transporter obtained by other investigators. Copyright 1998 Elsevier Science B.V.

  20. Metal Affinity-Enabled Capture and Release Antibody Reagents Generate a Multiplex Biomarker Enrichment System that Improves Detection Limits of Rapid Diagnostic Tests. (United States)

    Bauer, Westley S; Gulka, Christopher P; Silva-Baucage, Lidalee; Adams, Nicholas M; Haselton, Frederick R; Wright, David W


    Multi-antigen rapid diagnostic tests (RDTs) are highly informative, simple, mobile, and inexpensive, making them valuable point-of-care (POC) diagnostic tools. However, these RDTs suffer from several technical limitations-the most significant being the failure to detect low levels of infection. To overcome this, we have developed a magnetic bead-based multiplex biomarker enrichment strategy that combines metal affinity and immunospecific capture to purify and enrich multiple target biomarkers. Modifying antibodies to contain histidine-rich peptides enables reversible loading onto immobilized metal affinity magnetic beads, generating a novel class of antibodies coined "Capture and Release" (CaR) antibody reagents. This approach extends the specificity of immunocapture to metal affinity magnetic beads while also maintaining a common trigger for releasing multiple biomarkers. Multiplex biomarker enrichment is accomplished by adding magnetic beads equipped with CaR antibody reagents to a large sample volume to capture biomarkers of interest. Once captured, these biomarkers are magnetically purified, concentrated, and released into a RDT-compatible volume. This system was tailored to enhance a popular dual-antigen lateral flow malaria RDT that targets Plasmodium falciparum histidine-rich protein-II (HRPII) and Plasmodium lactate dehydrogenase (pLDH). A suite of pLDH CaR antibody reagents were synthesized, characterized, and the optimal CaR antibody reagent was loaded onto magnetic beads to make a multiplex magnetic capture bead that simultaneously enriches pLDH and HRPII from Plasmodium falciparum parasitized blood samples. This system achieves a 17.5-fold improvement in the dual positive HRPII/pan-pLDH detection limits enabling visual detection of both antigens at levels correlating to 5 p/μL. This front-end sample processing system serves as an efficient strategy to improve the sensitivity of RDTs without the need for modifications or remanufacturing.

  1. Simple and Rapid Quantitative Determination of Thiol-Containing Toxicants Using Silver Nanoparticles as an Affinity Probe (United States)

    Sharma, A.; Tapadia, K.


    A rapid and low-cost nano-drop spectrophotometric method using citrate-modified silver nanoparticles (Ag NPs) for the determination of thiol-containing toxicants was developed. The introduction of thioglycolic acid (TGA) and thiourea (TU) reduced the overall surface charge of Ag NPs, resulting in aggregation of Ag NPs, and a colorimetric response that was individually correlated with the concentration of TGA and TU. Under optimum experimental conditions, the maximum molar absorptivity values for TGA and TU were 1.04 × 105 and 2.13 × 105 L × mol-1 × cm-1, respectively, at λmax of 415 nm. The linear range used was 0.5-2.5 mg/L for TGA, and 0.3-1.5 mg/L for TU. The detection limits (3S) and % relative standard deviation (RSD) for the method were found to be 3 ppb, 2 ppb, and ±1.13%, ±0.96% for TGA and TU, respectively. This new chromogenic method provided a facile and sensitive scheme for the determination of TGA and TU, and could be applied for the determination of thiol-containing biomolecules. This scheme was tested for the analysis of real samples such as urine, blood, and environmental samples.

  2. Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase. (United States)

    Lenoir, Guillaume; Menguy, Thierry; Corre, Fabienne; Montigny, Cédric; Pedersen, Per A; Thinès, Denyse; le Maire, Marc; Falson, Pierre


    Large amounts of heterologous C-terminally his-tagged SERCA1a Ca(2+)-ATPase were expressed in yeast using a galactose-regulated promoter and purified by Ni(2+) affinity chromatography followed by Reactive red chromatography. Optimizing the number of galactose inductions and increasing the amount of Gal4p transcription factor improved expression. Lowering the temperature from 28 degrees C to 18 degrees C during expression enhanced the recovery of solubilized and active Ca(2+)-ATPase. In these conditions, a 4 l yeast culture produced 100 mg of Ca(2+)-ATPase, 60 and 22 mg being pelleted with the heavy and light membrane fractions respectively, representing 7 and 1.7% of total proteins. The Ca(2+)-ATPase expressed in light membranes was 100% solubilized with L-alpha-lysophosphatidylcholine (LPC), 50% with n-dodecyl beta-D-maltoside (DM) and 25% with octaethylene glycol mono-n-dodecyl ether (C(12)E(8)). Compared to LPC, DM preserved specific activity of the solubilized Ca(2+)-ATPase during the chromatographic steps. Starting from 1/6 (3.8 mg) of the total amount of Ca(2+)-ATPase expressed in light membranes, 800 microg could be routinely purified to 50% purity by metal affinity chromatography and then 200 microg to 70% with Reactive red chromatography. The purified Ca(2+)-ATPase displayed the same K(m) for calcium and ATP as the native enzyme but a reduced specific activity ranging from 4.5 to 7.3 micromol ATP hydrolyzed/min/mg Ca(2+)-ATPase. It was stable and active for several days at 4 degrees C or after removal of DM with Bio-beads and storage at -80 degrees C.

  3. Affinity purification of recombinant human plasminogen activator ...

    African Journals Online (AJOL)

    Sheng Xu1, Yuguo Yuan1,2 and Yong Cheng1,2*. 1Engineering Research Centre for Transgenic Animal Pharmaceutics in Jiangsu Province, College of Veterinary Medicine,. Yangzhou University, 2Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and. Zoonoses, Yangzhou ...

  4. Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway. (United States)

    Kutilek, Victoria D; Andrews, Christine L; Richards, Matthew P; Xu, Zangwei; Sun, Tianxiao; Chen, Yiping; Hashke, Andrew; Smotrov, Nadya; Fernandez, Rafael; Nickbarg, Elliott B; Chamberlin, Chad; Sauvagnat, Berengere; Curran, Patrick J; Boinay, Ryan; Saradjian, Peter; Allen, Samantha J; Byrne, Noel; Elsen, Nathaniel L; Ford, Rachael E; Hall, Dawn L; Kornienko, Maria; Rickert, Keith W; Sharma, Sujata; Shipman, Jennifer M; Lumb, Kevin J; Coleman, Kevin; Dandliker, Peter J; Kariv, Ilona; Beutel, Bruce


    The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway. © 2016 Society for Laboratory Automation and Screening.

  5. Application of an efficient strategy based on liquid-liquid extraction, high-speed counter-current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum. (United States)

    Chen, Tao; Liu, Yongling; Zou, Denglang; Chen, Chen; You, Jinmao; Zhou, Guoying; Sun, Jing; Li, Yulin


    This study presents an efficient strategy based on liquid-liquid extraction, high-speed counter-current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum. A new solvent system composed of petroleum ether/ethyl acetate/water (4:2:1, v/v/v) was developed for the liquid-liquid extraction of the crude extract from R. tanguticum. As a result, emodin, aloe-emodin, physcion, and chrysophanol were greatly enriched in the organic layer. In addition, an efficient method was successfully established to separate and purify the above anthraquinones by high-speed counter-current chromatography and preparative HPLC. This study supplies a new alternative method for the rapid enrichment, separation, and purification of emodin, aloe-emodin, physcione, and chrysophanol. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. A Mixed Stimuli-Responsive Magnetic and Gold Nanoparticle System for Rapid Purification, Enrichment, and Detection of Biomarkers (United States)

    Nash, Michael A.; Yager, Paul; Hoffman, Allan S.; Stayton, Patrick S.


    A new diagnostic system for the enrichment and detection of protein biomarkers from human plasma is presented. Gold nanoparticles (AuNPs) were surface-modified with a diblock copolymer synthesized using reversible addition fragmentation chain transfer (RAFT) polymerization. The diblock copolymer contained a thermally-responsive poly(N-isopropylacrylamide) (pNIPAAm) block, a cationic amine-containing block, and a semi-telechelic PEG2-biotin end group. When a mixed suspension of 23 nm pNIPAAm-modified AuNPs was heated with pNIPAAm-coated 10 nm iron oxide magnetic nanoparticles (mNPs) in human plasma, the thermally-responsive pNIPAAm directed the formation of mixed AuNP/mNP aggregates that could be separated efficiently with a magnet. Model studies showed that this mixed nanoparticle system could efficiently purify and strongly enrich the model biomarker protein streptavidin in spiked human plasma. A 10 ng/mL streptavidin sample was mixed with the biotinylated and pNIPAAm modified AuNP and magnetically separated in the mixed nanoparticle system with pNIPAAm mNPs. The aggregates were concentrated into a 50-fold smaller fluid volume at room temperature where the gold nanoparticle reagent redissolved with the streptavidin target still bound. The concentrated gold-labeled streptavidin could be subsequently analyzed directly using lateral flow immunochromatography. This rapid capture and enrichment module thus utilizes the mixed stimuli-responsive nanoparticle system to achieve direct concentration of a gold-labeled biomarker that can be directly analyzed using lateral flow or other rapid diagnostic strategies. PMID:21070026

  7. Targeting synaptic pathology with a novel affinity mass spectrometry approach

    National Research Council Canada - National Science Library

    Brinkmalm, Ann; Brinkmalm, Gunnar; Honer, William G; Moreno, Julie A; Jakobsson, Joel; Mallucci, Giovanna R; Zetterberg, Henrik; Blennow, Kaj; Öhrfelt, Annika


    .... This method combines affinity purification and mass spectrometry and can be applied directly for studies of SNARE complex proteins in multiple species or modified to target other key elements in neuronal function...

  8. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.

    NARCIS (Netherlands)

    E. de Boer (Ernie); P. Rodriguez (Patrick); E. Bonte (Edgar); J. Krijgsveld (Jeroen); E. Katsantoni (Eleni); A.J.R. Heck (Albert); F.G. Grosveld (Frank); J. Strouboulis (John)


    textabstractProteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated

  9. One step purification of biological active human interleukin-2 protein ...

    African Journals Online (AJOL)

    Pharmacological importance of recombinant human interleukin-2 protein has increased the demand to establish effective, reliable and cost effective chromatography method for its production and purification on large scale. One step mimetic ligand affinity chromatography method for purification of mutated human ...

  10. One-step purification of E. coli elongation factor Tu

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.; Degn, B


    The tuf A gene, encoding the E. coli elongation factor Tu, was cloned in the pGEX gene fusion system. Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose. This allows purification of EF-Tu in a one...

  11. Expression and purification of recombinant Shiga toxin 2B from ...

    African Journals Online (AJOL)

    The two step purification trains were used to purify native Stx2B. First step purification was Ni-immobilized metal ion affinity chromatography (IMAC) column, followed by second step using HaloLink resin. The native Stx2B was obtained after column cleavage of halo-tag using HaloTEV protease. Maximum protein expression ...

  12. Hamiltonian purification

    Energy Technology Data Exchange (ETDEWEB)

    Orsucci, Davide [Scuola Normale Superiore, I-56126 Pisa (Italy); Burgarth, Daniel [Department of Mathematics, Aberystwyth University, Aberystwyth SY23 3BZ (United Kingdom); Facchi, Paolo; Pascazio, Saverio [Dipartimento di Fisica and MECENAS, Università di Bari, I-70126 Bari (Italy); INFN, Sezione di Bari, I-70126 Bari (Italy); Nakazato, Hiromichi; Yuasa, Kazuya [Department of Physics, Waseda University, Tokyo 169-8555 (Japan); Giovannetti, Vittorio [NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, I-56126 Pisa (Italy)


    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.

  13. HaloTag-based purification of functional human kinases from mammalian cells. (United States)

    Ohana, Rachel Friedman; Hurst, Robin; Vidugiriene, Jolanta; Slater, Michael R; Wood, Keith V; Urh, Marjeta


    Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. Platelet affinity for burro aorta collagen

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, M.D.


    Despite ingenious concepts, there are no unequivocal clues as to what, when, and how some undefined biochemical factor(s) or constituent(s) that localizes in the arterial wall can precipitate a thromboatheromatous lesion or arterial disease. The present study focused on the extraction, partial purification, and characterization of a collagen-active platelet stimulator from the aortas of aged burros. The aggregator moiety in the aorta extracts invariably had a higher affinity for platelets in citrated platelet-rich plasma of human beings than for platelets of homologous burros. The platelet-aggregating factor(s) in the aorta extract was retained by incubation with ..cap alpha..-chymotrypsin. Platelet-aggregating activity was rapidly abolished after incubation with collagenase, as determined by platelet-aggregometry tests. Evidence based on light microscope and polysaccharide histochemical reactions indicates a probability that the intracellular amorphous matrix (PAS-positive) and filamentous components (PTAH-positive) expelled from smooth muscle cells disrupted during homogenization of the aorta may be a principal source of a precursor collagen species which is a potent inducer of platelet aggregation.

  15. Tuning the Protein Corona of Hydrogel Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity Reagents. (United States)

    O'Brien, Jeffrey; Shea, Kenneth J


    Nanomaterials, when introduced into a complex, protein-rich environment, rapidly acquire a protein corona. The type and amount of proteins that constitute the corona depend significantly on the synthetic identity of the nanomaterial. For example, hydrogel nanoparticles (NPs) such as poly(N-isopropylacrylamide) (NIPAm) have little affinity for plasma proteins; in contrast, carboxylated poly(styrene) NPs acquire a dense protein corona. This range of protein adsorption suggests that the protein corona might be "tuned" by controlling the chemical composition of the NP. In this Account, we demonstrate that small libraries of synthetic polymer NPs incorporating a diverse pool of functional monomers can be screened for candidates with high affinity and selectivity to targeted biomacromolecules. Through directed synthetic evolution of NP compositions, one can tailor the protein corona to create synthetic organic hydrogel polymer NPs with high affinity and specificity to peptide toxins, enzymes, and other functional proteins, as well as to specific domains of large proteins. In addition, many NIPAm NPs undergo a change in morphology as a function of temperature. This transformation often correlates with a significant change in NP-biomacromolecule affinity, resulting in a temperature-dependent protein corona. This temperature dependence has been used to develop NP hydrogels with autonomous affinity switching for the protection of proteins from thermal stress and as a method of biomacromolecule purification through a selective thermally induced catch and release. In addition to temperature, changes in pH or buffer can also alter a NP protein corona composition, a property that has been exploited for protein purification. Finally, synthetic polymer nanoparticles with low nanomolar affinity for a peptide toxin were shown to capture and neutralize the toxin in the bloodstream of living mice. While the development of synthetic polymer alternatives to protein affinity reagents is

  16. Polonium purification

    Energy Technology Data Exchange (ETDEWEB)

    Baker, J.D.


    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.

  17. Rapid one-step purification of single-cells encapsulated in alginate microcapsules from oil to aqueous phase using a hydrophobic filter paper: implications for single-cell experiments. (United States)

    Lee, Do-Hyun; Jang, Miran; Park, Je-Kyun


    By virtue of the biocompatibility and physical properties of hydrogel, picoliter-sized hydrogel microcapsules have been considered to be a biometric signature containing several features similar to that of encapsulated single cells, including phenotype, viability, and intracellular content. To maximize the experimental potential of encapsulating cells in hydrogel microcapsules, a method that enables efficient hydrogel microcapsule purification from oil is necessary. Current methods based on centrifugation for the conventional stepwise rinsing of oil, are slow and laborious and decrease the monodispersity and yield of the recovered hydrogel microcapsules. To remedy these shortcomings we have developed a simple one-step method to purify alginate microcapsules, containing a single live cell, from oil to aqueous phase. This method employs oil impregnation using a commercially available hydrophobic filter paper without multistep centrifugal purification and complicated microchannel networks. The oil-suspended alginate microcapsules encapsulating single cells from mammalian cancer cell lines (MCF-7, HepG2, and U937) and microorganisms (Chlorella vulgaris) were successfully exchanged to cell culture media by quick (~10 min) depletion of the surrounding oil phase without coalescence of neighboring microcapsules. Cell proliferation and high integrity of the microcapsules were also demonstrated by long-term incubation of microcapsules containing a single live cell. We expect that this method for the simple and rapid purification of encapsulated single-cell microcapsules will attain widespread adoption, assisting cell biologists and clinicians in the development of single-cell experiments. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. A rapid and high-precision method for sulfur isotope δ(34)S determination with a multiple-collector inductively coupled plasma mass spectrometer: matrix effect correction and applications for water samples without chemical purification. (United States)

    Lin, An-Jun; Yang, Tao; Jiang, Shao-Yong


    Previous studies have indicated that prior chemical purification of samples, although complex and time-consuming, is essential in obtaining precise and accurate results for sulfur isotope ratios using multiple-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). In this study, we introduce a new, rapid and precise MC-ICP-MS method for sulfur isotope determination from water samples without chemical purification. The analytical work was performed on an MC-ICP-MS instrument with medium mass resolution (m/Δm ~ 3000). Standard-sample bracketing (SSB) was used to correct samples throughout the analytical sessions. Reference materials included an Alfa-S (ammonium sulfate) standard solution, ammonium sulfate provided by the lab of the authors and fresh seawater from the South China Sea. A range of matrix-matched Alfa-S standard solutions and ammonium sulfate solutions was used to investigate the matrix (salinity) effect (matrix was added in the form of NaCl). A seawater sample was used to confirm the reliability of the method. Using matrix-matched (salinity-matched) Alfa-S as the working standard, the measured δ(34)S value of AS (-6.73 ± 0.09‰) was consistent with the reference value (-6.78 ± 0.07‰) within the uncertainty, suggesting that this method could be recommended for the measurement of water samples without prior chemical purification. The δ(34)S value determination for the unpurified seawater also yielded excellent results (21.03 ± 0.18‰) that are consistent with the reference value (20.99‰), thus confirming the feasibility of the technique. The data and the results indicate that it is feasible to use MC-ICP-MS and matrix-matched working standards to measure the sulfur isotopic compositions of water samples directly without chemical purification. In comparison with the existing MC-ICP-MS techniques, the new method is better for directly measuring δ(34)S values in water samples with complex matrices; therefore, it can

  19. Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti{sup 4+}-SPE enrichment for mass spectrometric analysis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ying [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032 (China); Peng, Ye; Bin, Zhichao [Department of Chemistry, Fudan University, Shanghai 200032 (China); Wang, Huijie [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Lu, Haojie, E-mail: [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Department of Chemistry, Fudan University, Shanghai 200032 (China); Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032 (China)


    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{sup 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{sup 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. - Graphical abstract: A selective enrichment method for the N-glycome is reported. N-glycans were chemically labeled with a phosphate derivatization reagent (AMS), then the phospho-containing glycans were enriched using Ti{sup 4+}-microspheres. - Highlights: • A highly specific N-glycans purification method based on phosphate derivatization combined with Ti{sup 4+}-SPE was developed

  20. Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory. (United States)

    Anderson, Alexander J.


    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. Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry (United States)

    Ladwig, Paula M.; Barnidge, David R.; Willrich, Maria A. V.


    As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure.

  2. Purification of mammalian DNA repair protein XRCC1

    Energy Technology Data Exchange (ETDEWEB)

    Chen, I. [Univ. of California, Berkeley, CA (United States)


    Malfunctioning DNA repair systems lead to cancer mutations, and cell death. XRCC1 (X-ray Repair Cross Complementing) is a human DNA repair gene that has been found to fully correct the x-ray repair defect in Chinese hamster ovary (CHO) cell mutant EM9. The corresponding protein (XRCC1) encoded by this gene has been linked to a DNA repair pathway known as base excision repair, and affects the activity of DNA ligase III. Previously, an XRCC1 cDNA minigene (consisting of the uninterrupted coding sequence for XRCC1 protein followed by a decahistidine tag) was constructed and cloned into vector pET-16b for the purpose of: (1) overproduction of XRCC1 in both prokaryotic and eukaryotic cells; and (2) to facilitate rapid purification of XRCC1 from these systems. A vector is basically a DNA carrier that allows recombinant protein to be cloned and overexpressed in host cells. In this study, XRCC1 protein was overexpressed in E. coli and purified by immobilized metal affinity chromatography. Currently, the XRCC1 minigene is being inserted into a new vector [pET-26b(+)] in hopes to increase overexpression and improve purification. Once purified XRCC1 can be crystallized for structural studies, or studied in vitro for its biological function.

  3. Data of expression and purification of recombinant Taq DNA polymerase

    Directory of Open Access Journals (Sweden)

    Na Fang


    Full Text Available Polymerase chain reaction (PCR technique is widely used in many experimental conditions, and Taq DNA polymerase is critical in PCR process. In this article, the Taq DNA polymerase expression plasmid is reconstructed and the protein product is obtained by rapid purification, (“Rapid purification of high-activity Taq DNA polymerase” (Pluthero, 1993 [1], “Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli” (Desai and Pfaffle, 1995 [2]. Here we present the production data from protein expression and provide the analysis results of the production from two different vectors. Meanwhile, the purification data is also provided to show the purity of the protein product.

  4. Antibody-based affinity cryo-EM grid. (United States)

    Yu, Guimei; Li, Kunpeng; Jiang, Wen


    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Purification of N-Acetylgalactosaminidase by Isoelectric Focusing. (United States)


    obtained from Joan Quay at NBL. Dr . Bong Park, a Visiting Scientist, undertook the culture of the organism and studied the early steps in purification of...matrix% The affinity agent used was originally developed by Harpaz, Flowers and Sharon (1974) for the purification of coffee bean aipha-galactosidase...collaboration with Dr % Myron Leon of the Department of Immunology and Microbiology. These radioimmunoassays had a larger than desirable background when type

  6. Purification of glycocalicin from human plasma. (United States)

    HadjKacem, Basma; Mkaouar, Héla; Ben Amor, Ikram; Gargouri, Jalel; Gargouri, Ali


    Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Lindbladian purification (United States)

    Arenz, Christian; Burgarth, Daniel; Giovannetti, Vittorio; Nakazato, Hiromichi; Yuasa, Kazuya


    In a recent work (Burgarth et al 2014, Nat. Commun. 5 5173), it was shown that a series of frequent measurements can project the dynamics of a quantum system onto a subspace in which the dynamics can be more complex. In this subspace, even full controllability can be achieved, although the controllability over the system before the projection is very poor since the control Hamiltonians commute with each other. We can also think of the opposite: any Hamiltonians of a quantum system, which are in general noncommutative with each other, can be made commutative by embedding them in an extended Hilbert space, thus the dynamics in the extended space becomes trivial and simple. This idea of making noncommutative Hamiltonians commutative is called ‘Hamiltonian purification.’ The original noncommutative Hamiltonians are recovered by projecting the system back onto the original Hilbert space through frequent measurements. Here, we generalise this idea to open-system dynamics by presenting a simple construction to make Lindbladians, as well as Hamiltonians, commutative on a larger space with an auxiliary system. We show that the original dynamics can be recovered through frequently measuring the auxiliary system in a non-selective way. Moreover, we provide a universal pair of Lindbladians that describe an ‘accessible’ open quantum system for generic system sizes. This allows us to conclude that through a series of frequent non-selective measurements a nonaccessible open quantum system generally becomes accessible. This sheds further light on the role of measurement backaction on the control of quantum systems.

  8. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

    DEFF Research Database (Denmark)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua


    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

  9. Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography. (United States)

    Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas


    In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10(5) U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

  10. Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris. (United States)

    Li, Sumei; Hong, Tao; Wang, Kun; Lu, Yinghong; Zhou, Min


    Most proteins occur and function in complexes rather than as isolated entities in membranes. In most cases macromolecules with multiple subunits are purified from endogenous sources. In this study, an endogenous membrane-protein complex was obtained from Pichia pastoris, which can be grown at high densities to significantly improve the membrane protein yield. We successfully isolated the membrane-bound Vo complex of V-ATPase from P. pastoris using a fusion FLAG tag attached to the C-terminus of subunit a to generate the vph-tag strain, which was used for dissociation and purification. After FLAG affinity and size exclusion chromatography purification, the production quantity and purity of the membrane-bound Vo complex was 20 μg l-1 and >98%, respectively. The subunits of the endogenous membrane-bound Vo complex observed in P. pastoris were similar to those obtained from S. cerevisiae, as demonstrated by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Therefore, successful dissociation and purification of the membrane-bound Vo complex at a high purity and sufficient quantity was achieved via a rapid and simple procedure that can be used to obtain the endogenous membrane-protein complexes from P. pastoris. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. On affine rigidity

    Directory of Open Access Journals (Sweden)

    Steven J. Gortler


    Full Text Available We study the properties of affine rigidity of a hypergraph and prove a variety of fundamental results. First, we show that affine rigidity is a generic property (i.e., depends only on the hypergraph, not the particular embedding. Then we prove that a graph is generically neighborhood affinely rigid in d-dimensional space if it is (d+1-vertex-connected. We also show neighborhood affine rigidity of a graph implies universal rigidity of its squared graph.  Our results, and affine rigidity more generally, have natural applications in point registration and localization, as well as connections to manifold learning.

  12. Sodium purification in Rapsodie; La purification du sodium a Rapsodie

    Energy Technology Data Exchange (ETDEWEB)

    Giraud, B. [Commissariat a l' Energie Atomique, Dir. des Piles Atomiques, Cadarache (France). Centre d' Etudes Nucleaires


    This report is one of a series of publications presenting the main results of tests carried out during the start-up of the first french fast neutron reactor: Rapsodie. The article presents the sodium purification techniques used in the reactor cooling circuits both from the constructional point of view and with respect to results obtained during the first years working. (author) [French] Ce rapport fait partie d'une serie de publications presentant l'essentiel des resultats des essais effectues a l'occasion du demarrage du premier reacteur francais a neutrons rapides: RAPSODIE. Cet article expose les techniques de la purification du sodium utilise dans les circuits de refroidissement du reacteur tant au point de vue de leur realisation technologique, que des resultats obtenus pendant la premiere annee de fonctionnement. (auteur)

  13. Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

    Directory of Open Access Journals (Sweden)

    Qiang Wang

    Full Text Available Recent successes of adeno-associated virus (AAV–based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE, we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37 and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9. The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors’ in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

  14. Purification of transthyretin as nutritional biomarker of selenium status. (United States)

    Mahn, Andrea; Lienqueo, María Elena; Quilodrán, Claudia; Olivera-Nappa, Alvaro


    Transthyretin has been proposed as nutritional biomarker of selenium intake. Previous transthyretin purification methods used different procedures to isolate transthyretin either from plasma or from pathological urine of humans. In general, the procedure for purification of transthyretin is laborious and expensive, and extensive sample recycling is necessary for purification in appreciable amounts. This work proposes a new, promissory, and cheap two-step process to purify transthyretin from blood plasma, composed by a first aqueous two-phase system fractionation followed by affinity chromatography, using thyroxine-immobilized on epoxy-activated Sepharose CL-6B. The aqueous two-phase system fractionation was demonstrated to perform better than commercial immunoaffinity-based kits for albumin depletion in blood plasma samples and is an effective first step for transthyretin purification. Thyroxine affinity chromatography was designed to bind transthyretin with high affinity, and was demonstrated to be useful to purify transthyretin, but was unable to completely resolve transthyretin from thyroxine-binding globulin and serum albumin, although the relative amount of albumin was lowered in the eluates. This purification process could be used in nutritional diagnosis tools or as a first step in structural and functional studies. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. MAP Tag: A Novel Tagging System for Protein Purification and Detection. (United States)

    Fujii, Yuki; Kaneko, Mika K; Kato, Yukinari


    Protein purification is an essential procedure in fields such as biochemistry, molecular biology, and biophysics. Acquiring target proteins with high quality and purity is still difficult, although several tag systems have been established for protein purification. Affinity tag systems are excellent because they possess high affinity and specificity for acquiring the target proteins. Nevertheless, further affinity tag systems are needed to compensate for several disadvantages of the presently available affinity tag systems. Herein, we developed a novel affinity tag system designated as the MAP tag system. This system is composed of a rat anti-mouse podoplanin monoclonal antibody (clone PMab-1) and MAP tag (GDGMVPPGIEDK) derived from the platelet aggregation-stimulating domain of mouse podoplanin. PMab-1 possesses high affinity and specificity for the MAP tag, and the PMab-1/MAP tag complex dissociates in the presence of the epitope peptide, indicating that the MAP tag system is suitable for protein purification. We successfully purified several proteins, including a nuclear protein, soluble proteins, and a membrane protein using the MAP tag system. The MAP tag system is very useful not only for protein purification but also in protein detection systems such as western blot and flow cytometric analyses. Taken together, these findings indicate that the MAP tag system could be a powerful tool for protein purification and detection.

  16. Affine Grassmann codes

    DEFF Research Database (Denmark)

    Høholdt, Tom; Beelen, Peter; Ghorpade, Sudhir Ramakant


    We consider a new class of linear codes, called affine Grassmann codes. These can be viewed as a variant of generalized Reed-Muller codes and are closely related to Grassmann codes.We determine the length, dimension, and the minimum distance of any affine Grassmann code. Moreover, we show...

  17. Continuous affine processes

    DEFF Research Database (Denmark)

    Buchardt, Kristian


    Affine processes possess the property that expectations of exponential affine transformations are given by a set of Riccati differential equations, which is the main feature of this popular class of processes. In this paper we generalise these results for expectations of more general transformati......Affine processes possess the property that expectations of exponential affine transformations are given by a set of Riccati differential equations, which is the main feature of this popular class of processes. In this paper we generalise these results for expectations of more general...... transformations. This is of interest in, e.g. doubly stochastic Markov models, in particular in life insurance. When using affine processes for modelling the transition rates and interest rate, the results presented allow for easy calculation of transition probabilities and expected present values....

  18. Extension of the selection of protein chromatography and the rate model to affinity chromatography. (United States)

    Sandoval, G; Shene, C; Andrews, B A; Asenjo, J A


    The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time. Copyright © 2010 John Wiley & Sons, Ltd.

  19. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe


    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  20. Affine and Projective Geometry

    CERN Document Server

    Bennett, M K


    An important new perspective on AFFINE AND PROJECTIVE GEOMETRY. This innovative book treats math majors and math education students to a fresh look at affine and projective geometry from algebraic, synthetic, and lattice theoretic points of view. Affine and Projective Geometry comes complete with ninety illustrations, and numerous examples and exercises, covering material for two semesters of upper-level undergraduate mathematics. The first part of the book deals with the correlation between synthetic geometry and linear algebra. In the second part, geometry is used to introduce lattice theory

  1. Non-Chromatographic Purification of Endohedral Metallofullerenes. (United States)

    Wang, Zhiyong; Omachi, Haruka; Shinohara, Hisanori


    The purification of endohedral metallofullerenes by high performance liquid chromatography is very time-consuming and expensive. A number of rapid and inexpensive non-chromatographic methods have thus been developed for large-scale purification of metallofullerenes. In this review, we summarize recent advances in non-chromatographic purification methods of metallofullerenes. Lewis acid-based complexation is one of the most efficient and powerful methods for separation of metallofullerenes from empty fullerenes. The first oxidation potential of metallofullerenes is a critical factor that affects the separation efficiency of the Lewis acid-based method. Supramolecular methods are effective for separation of fullerenes and metallofullerenes that are different in size and shape. Chemical/electrochemical reduction and exohedral functionalization are also utilized to separate and purify metallofullerenes on a large scale.

  2. Robust Affine Invariant Descriptors

    Directory of Open Access Journals (Sweden)

    Jianwei Yang


    Full Text Available An approach is developed for the extraction of affine invariant descriptors by cutting object into slices. Gray values associated with every pixel in each slice are summed up to construct affine invariant descriptors. As a result, these descriptors are very robust to additive noise. In order to establish slices of correspondence between an object and its affine transformed version, general contour (GC of the object is constructed by performing projection along lines with different polar angles. Consequently, affine in-variant division curves are derived. A slice is formed by points fall in the region enclosed by two adjacent division curves. To test and evaluate the proposed method, several experiments have been conducted. Experimental results show that the proposed method is very robust to noise.

  3. The Borexino purification system (United States)

    Benziger, Jay


    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.


    Directory of Open Access Journals (Sweden)

    О. V. Sviatenko


    Full Text Available Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding specificities and affinities of these proteins differ between species and antibody subclass. Protein А has high affinity to human IgG1, IgG2, IgG4, mouse IgG2a, IgG2b, IgG3, goat and sheep IgG2, dog, cat, guinea pig, rabbit IgG. Protein G binds strongly to human, mouse, cow, goat, sheep and rabbit IgG. Protein L has ability of strong binding to immunoglobulin kappa-chains of human, mouse, rat and pig. Expediency of application of affinity chromatography with usage of sorbents on the basis of immobilized proteins A, G, L are shown for isolation and purification of antibodies different classes. Previously mentioned method is used as an alternative to conventional methods of protein purification, such as ion-exchange, hydrophobic interactions, metal affinity chromatography, ethanol precipitation due to simplicity in usage, possibility of one-step purification process, obtaining of proteins high level purity, multiuse at maintenance of proper storage and usage conditions. Affinity sorbents on the basis of immobilized proteins A, G, L are used not only for antibodies purification, but also for extraction of different antibodies fractions from blood serum.

  5. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels (United States)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua; Chait, Brian T.; Rout, Michael P.; Jensen, Torben Heick; LaCava, John


    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous-level tagged proteins. Isolations of triple-FLAG and GFP-tagged fusion proteins involved in RNA metabolism are presented. PMID:22668517

  6. Induced affine inflation (United States)

    Azri, Hemza; Demir, Durmuş


    Induced gravity, metrical gravity in which gravitational constant arises from vacuum expectation value of a heavy scalar, is known to suffer from Jordan frame vs Einstein frame ambiguity, especially in inflationary dynamics. Induced gravity in affine geometry, as we show here, leads to an emergent metric and gravity scale, with no Einstein-Jordan ambiguity. While gravity is induced by the vacuum expectation value of the scalar field, nonzero vacuum energy facilitates generation of the metric. Our analysis shows that induced gravity results in a relatively large tensor-to-scalar ratio in both metrical and affine gravity setups. However, the fact remains that the induced affine gravity provides an ambiguity-free framework.

  7. A simple method for purification of herpesvirus DNA

    DEFF Research Database (Denmark)

    Christensen, Laurids Siig; Normann, Preben


    A rapid and reliable method for purification of herpesvirus DNA from cell cultures is described. The method is based on the isolation of virus particles and/or nucleocapsids by differential centrifugation and exploits the solubilizing and denaturing capabilities of cesium trifluoroacetate during...... isopycnic centrifugation, so that phenol/chloroform extractions can be omitted. The method was used for the purification of DNA from several members of the Alfaherpesvirinae subfamily....

  8. Expression and purification of the cystic fibrosis transmembrane conductance regulator protein in Saccharomyces cerevisiae. (United States)

    O'Ryan, Liam; Rimington, Tracy; Cant, Natasha; Ford, Robert C


    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein(1-3). Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed (4-9). Proteins involved in the processing of recombinant CFTR in yeast have been described(6-9) .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)(10). The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for

  9. Quantum affine algebras (United States)

    Chari, Vyjayanthi; Pressley, Andrew


    We classify the finite-dimensional irreducible representations of the quantum affine algebraU_q (hat sl_2 ) in terms of highest weights (this result has a straightforward generalization for arbitrary quantum affine algebras). We also give an explicit construction of all such representations by means of an evaluation homomorphismU_q (hat sl_2 ) to U_q (sl_2 ), first introduced by M. Jimbo. This is used to compute the trigonometric R-matrices associated to finite-dimensional representations ofU_q (hat sl_2 ).

  10. Teaching molecular biology to undergraduate biology students: An illustration of protein expression and purification*. (United States)

    Sommer, César Adolfo; Silva, Flávio Henrique; Novo, Maria Teresa Marques


    Practical classes on protein expression and purification were given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering." The heterologous expression of the green fluorescent protein (GFP)* of Aequorea victoria is an interesting system for didactic purposes because it can be viewed easily during experiments. The students were provided with basic information about the molecular features and applications of the GFP in molecular biology, the available heterologous expression systems, and the theoretical and experimental details of GFP expression in Escherichia coli and its purification. E. coli BL21-competent cells were transformed with the pET28a expression vector containing the GFP gene fused to a histidine (His) tag. During the induction of a transformed clone by isopropylthiogalactoside, a time course for GFP expression was analyzed by SDS-PAGE, and the expression was also visualized by the increasing green fluorescence of the bacterial culture. After cellular disruption, protein purification was illustrated by affinity chromatography of the His-tagged protein in a nickel column. Eluted fractions containing imidazole in increasing concentrations were analyzed visually and also by SDS-PAGE, demonstrating the role of imidazole in protein recovery by competition with nonspecific proteins and the His-tagged protein. The results obtained and the experimental factors involved in protein expression, solubilization, and folding were discussed following the laboratory experiments. These practical classes allowed several current approaches to molecular biology to be demonstrated rapidly and helped underscore some of the topics taught during the course. Copyright © 2004 International Union of Biochemistry and Molecular Biology, Inc.

  11. Affine stochastic mortality

    NARCIS (Netherlands)

    Schrager, D.F.


    We propose a new model for stochastic mortality. The model is based on the literature on affine term structure models. It satisfies three important requirements for application in practice: analytical tractibility, clear interpretation of the factors and compatibility with financial option pricing

  12. Affine Sphere Relativity (United States)

    Minguzzi, E.


    We investigate spacetimes whose light cones could be anisotropic. We prove the equivalence of the structures: (a) Lorentz-Finsler manifold for which the mean Cartan torsion vanishes, (b) Lorentz-Finsler manifold for which the indicatrix (observer space) at each point is a convex hyperbolic affine sphere centered on the zero section, and (c) pair given by a spacetime volume and a sharp convex cone distribution. The equivalence suggests to describe (affine sphere) spacetimes with this structure, so that no algebraic-metrical concept enters the definition. As a result, this work shows how the metric features of spacetime emerge from elementary concepts such as measure and order. Non-relativistic spacetimes are obtained replacing proper spheres with improper spheres, so the distinction does not call for group theoretical elements. In physical terms, in affine sphere spacetimes the light cone distribution and the spacetime measure determine the motion of massive and massless particles (hence the dispersion relation). Furthermore, it is shown that, more generally, for Lorentz-Finsler theories non-differentiable at the cone, the lightlike geodesics and the transport of the particle momentum over them are well defined, though the curve parametrization could be undefined. Causality theory is also well behaved. Several results for affine sphere spacetimes are presented. Some results in Finsler geometry, for instance in the characterization of Randers spaces, are also included.

  13. Purification and fluorescent labeling of the human serotonin transporter

    DEFF Research Database (Denmark)

    Rasmussen, Søren G F; Gether, Ulrik


    To establish a purification procedure for the human serotonin transporter (hSERT) we expressed in Sf9 insect cells an epitope-tagged version of the transporter containing a FLAG epitope at the N-terminus and a polyhistidine tail at the C-terminus (FLAG-hSERT-12H). For purification, the transporter...... was solubilized in digitonin followed by nickel affinity and subsequent concanavalin A chromatography. Using this procedure we were able to obtain an overall purification of 700-fold and a yield of approximately 0.1 mg/L of cell culture. The purified transporter displayed pharmacological properties similar...... quenching experiments revealed that the aqueous quencher iodide was able to cause marked quenching of the fluorescence of the IANBD labeled transporter with a K(SV) of 3.4 +/- 0.10 M(-)(1). In a mutant transporter with five cysteines mutated (5CysKO) we observed a significant reduction in this quenching (K...

  14. Bromelain: an overview of industrial application and purification strategies. (United States)

    Arshad, Zatul Iffah Mohd; Amid, Azura; Yusof, Faridah; Jaswir, Irwandi; Ahmad, Kausar; Loke, Show Pau


    This review highlights the use of bromelain in various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially in many applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant bromelain for novel applications in the future.

  15. Engineering a recyclable elastin-like polypeptide capturing scaffold for non-chromatographic protein purification. (United States)

    Liu, Fang; Chen, Wilfred


    Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification. © 2013 American Institute of Chemical Engineers.

  16. Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome (United States)

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


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

  17. Surface sieving coordinated IMAC material for purification of His-tagged proteins. (United States)

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


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

  18. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data

    NARCIS (Netherlands)

    Mellacheruvu, D.; Wright, Z.; Couzens, A.L.; Low, T.Y.|info:eu-repo/dai/nl/411298437; Halim, V.A.|info:eu-repo/dai/nl/326157441; Heck, A.J.R.|info:eu-repo/dai/nl/105189332; Mohammed, S.|info:eu-repo/dai/nl/30483632X; Nesvizhskii, A.


    Affinity purification coupled with mass spectrometry (AP-MS) is 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

  19. Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain. (United States)

    Munshi, R; Linden, J


    A1 adenosine receptors and guanine nucleotide-binding proteins (G proteins) solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate have been co-purified from bovine cerebral cortex. A portion of solubilized receptors which displays high affinity GTP-sensitive agonist binding (40-50%) adheres tightly to agonist affinity columns composed of N6-aminobenzyladenosine-agarose. A1 adenosine receptors and G proteins are rapidly and selectively coeluted from agonist columns by the addition of 8-p-sulfophenyltheophylline, but only in combination with Mg2+-GTP or N-ethylmaleimide, agents which lower the affinity of receptors for agonists. Purified receptors and G protein alpha-subunits can be detected with the potent A1-selective antagonist radioligand, [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentylxanthine (125I-BW-A844U) and [35S]guanosine 5'-3-O-(thio)triphosphate [( 35S]GTP gamma S), respectively. Pretreatment of solubilized receptors with 0.1 mM N-ethylmaleimide or 0.1 mM R-phenylisopropyladenosine abolishes adsorption of receptors and G proteins to affinity columns. Following removal of 8-p-sulfophenyltheophylline and GTP, purified receptors bind agonists (2 sites) and antagonists (1 site) with affinities similar to crude soluble receptors and typical of A1 receptors. Some receptors may be denatured as a result of purification since only 23% of the radioligand binding sites which adhere to the affinity column can be detected in the eluate. The Bmax of purified receptors, 820 +/- 100 pmol/mg protein (n = 3) is 1800-fold higher than crude soluble receptors. The specific activity of [35S]GTP gamma S binding sites in affinity column eluates is 4640 pmol/mg protein. Assuming a 1:1 stoichiometry, this specific activity indicates that receptor-G protein complexes are greater than 50% pure following affinity chromatography. The photoaffinity labeled purified receptor was identified by polyacrylamide gel electrophoresis as a single band with a

  20. Affinity driven social networks (United States)

    Ruyú, B.; Kuperman, M. N.


    In this work we present a model for evolving networks, where the driven force is related to the social affinity between individuals of a population. In the model, a set of individuals initially arranged on a regular ordered network and thus linked with their closest neighbors are allowed to rearrange their connections according to a dynamics closely related to that of the stable marriage problem. We show that the behavior of some topological properties of the resulting networks follows a non trivial pattern.

  1. Water purification in Borexino

    Energy Technology Data Exchange (ETDEWEB)

    Giammarchi, M. [Infn Milano (Italy); Balata, M.; Ioannucci, L.; Nisi, S. [Laboratori Nazionali del Gran Sasso (Italy); Goretti, A.; Ianni, A. [Princeton University (United States); Miramonti, L. [Dip. di Fisica dell' Università di Milano e Infn (Italy)


    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.

  2. Water purification in Borexino (United States)

    Giammarchi, M.; Balata, M.; Goretti, A.; Ianni, A.; Ioannucci, L.; Miramonti, L.; Nisi, S.


    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.

  3. Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions. (United States)

    Mateo, C; Fernandez-Lorente, G; Pessela, B C; Vian, A; Carrascosa, A V; Garcia, J L; Fernandez-Lafuente, R; Guisan, J M


    New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve

  4. Twisted Quantum Affine Algebras (United States)

    Chari, Vyjayanthi; Pressley, Andrew

    We give a highest weight classification of the finite-dimensional irreducible representations of twisted quantum affine algebras. As in the untwisted case, such representations are in one-to-one correspondence with n-tuples of monic polynomials in one variable. But whereas in the untwisted case n is the rank of the underlying finite-dimensional complex simple Lie algebra ?, in the twisted case n is the rank of the subalgebra of ? fixed by the diagram automorphism. The way in which such an n-tuple determines a representation is also more complicated than in the untwisted case.

  5. Monodisperse REPO4 (RE = Yb, Gd, Y) hollow microspheres covered with nanothorns as affinity probes for selectively capturing and labeling phosphopeptides. (United States)

    Cheng, Gong; Zhang, Ji-Lin; Liu, Yan-Lin; Sun, De-Hui; Ni, Jia-Zuan


    Rare-earth phosphate microspheres with unique structures were developed as affinity probes for the selective capture and tagging of phosphopeptides. Prickly REPO(4) (RE = Yb, Gd, Y) monodisperse microspheres, that have hollow structures, low densities, high specific surface areas, and large adsorptive capacities were prepared by an ion-exchange method. The elemental compositions and crystal structures of these affinity probes were confirmed by energy-dispersive spectroscopy (EDS), powder X-ray diffraction (XRD), and Fourier-transform infrared (FTIR) spectroscopy. The morphologies of these compounds were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and nitrogen-adsorption isotherms. The potential ability of these microspheres for selectively capturing and labeling target biological molecules was evaluated by using protein-digestion analysis and a real sample as well as by comparison with the widely used TiO(2) affinity microspheres. These results show that these porous rare-earth phosphate microspheres are highly promising probes for the rapid purification and recognition of phosphopeptides. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Purification of the neurotensin receptor from bovine brain

    Energy Technology Data Exchange (ETDEWEB)

    Mills, A.; Demoliou-Mason, C.D.; Barnard, E.A.


    The neurotensin receptor protein, solubilized with digitonin/asolectin from bovine cerebral cortex membranes, was purified to apparent homogeneity by affinity chromatography using immobilized neurotensin. The product exhibits saturable and specific binding of (3,11-tyrosyl-3,5-/sup 3/H) neurotensin with an apparent affinity (K/sub d/ = 5.5 nM) comparable to that measured in intact membranes and crude soluble extracts. The affinity-purified material, after reduction with 100 mM dithiothreitol, in denaturing gel electrophoresis showed a single polypeptide of M/sub r/ 72,000. Under nonreducing conditions the apparent M/sub r/, however, was 50,000, suggesting the presence of intramolecular disulfide bonds. The purified neurotensin receptor was judged to be homogenous, in that (i) only a single polypeptide was detectable; and (ii) the overall purification was 30,000-50,000-fold, giving a specific neurotensin-binding activity close to the theoretical maximum.

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

    DEFF Research Database (Denmark)

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


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

  8. Purification of biomaterials by phase partitioning (United States)

    Harris, J. M.


    A technique which is particularly suited to microgravity environments and which is potentially more powerful than electrophoresis is phase partitioning. Phase partitioning is purification by partitioning between the two immiscible aqueous layers formed by solution of the polymers poly(ethylene glycol) and dextran in water. This technique proved to be very useful for separations in one-g but is limited for cells because the cells are more dense than the phase solutions thus tend to sediment to the bottom of the container before reaching equilibrium with the preferred phase. There are three phases to work in this area: synthesis of new polymers for affinity phase partitioning; development of automated apparatus for ground-based separations; and design of apparatus for performing simple phase partitioning space experiments, including examination of mechanisms for separating phases in the absence of gravity.

  9. Removal of PCR error products and unincorporated primers by metal-chelate affinity chromatography.

    Directory of Open Access Journals (Sweden)

    Indhu Kanakaraj

    Full Text Available Immobilized Metal Affinity Chromatography (IMAC has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and "histidine tags" genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu(2+-iminodiacetic acid (IDA agarose spin column, 94-99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu(2+-IDA agarose can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs.

  10. Portable neon purification system

    Energy Technology Data Exchange (ETDEWEB)

    Richardson, R.A.; Schmitt, R.L.


    This paper describes the principle design features of a portable neon purification system and the results of the system performance testing. Neon gas replaces air in the Ring Imaging Cherenkov detector without using vacuum, in experiment E781(SELEX) at Fermilab. The portable neon purification system purifies neon gas by, first purging air with CO{sub 2}, freezing the CO{sub 2}, then cryoadsorbing the remaining contaminants. The freezer removes carbon dioxide from a neon gas mixture down to a maximum concentration of 500 parts-per-million (ppm). The charcoal bed adsorber removes nitrogen from neon gas down to a maximum concentration of 100 ppm. The original RICH vessel was designed to hold vacuum but its photomultiplier tube plates were not.

  11. Electron beam silicon purification

    Energy Technology Data Exchange (ETDEWEB)

    Kravtsov, Anatoly [SIA ' ' KEPP EU' ' , Riga (Latvia); Kravtsov, Alexey [' ' KEPP-service' ' Ltd., Moscow (Russian Federation)


    Purification of heavily doped electronic grade silicon by evaporation of N-type impurities with electron beam heating was investigated in process with a batch weight up to 50 kilos. Effective temperature of the melt, an indicative parameter suitable for purification process characterization was calculated and appeared to be stable for different load weight processes. Purified material was successfully approbated in standard CZ processes of three different companies. Each company used its standard process and obtained CZ monocrystals applicable for photovoltaic application. These facts enable process to be successfully scaled up to commercial volumes (150-300 kg) and yield solar grade silicon. (copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  12. High oxygen affinity hemoglobins. (United States)

    Mangin, O


    High oxygen affinity hemoglobins are responsible for rare and heterogeneous autosomic dominant genetic diseases. They cause pure erythrocytosis, sometimes accountable for hyperviscosity and thrombosis, or hemolysis. Differential diagnoses must be first ruled out. The diagnosis is based on the identification of a decreased P50, and their possible characterization by cation exchange-high performance liquid chromatography and capillary electrophoresis. Finally, genetic studies of the responsible globin chain gene will confirm the mutation. The prognosis mainly relies on the P50 decrease rate and on the hemoglobin cooperativity impairment. Disease management should be personalized, and it should primarily depend on smoking cessation and physical activity. Phlebotomy and platelet aggregation inhibitors' prescriptions can be discussed. There is no contraindication to flights, high-altitude conditions, or pregnancy. Nevertheless, blood donation must be prohibited. Copyright © 2016 Société Nationale Française de Médecine Interne (SNFMI). Published by Elsevier SAS. All rights reserved.

  13. Quantum affine algebras

    Energy Technology Data Exchange (ETDEWEB)

    Chari, V. (Tata Inst. of Fundamental Research, Bombay (India). School of Mathematics); Pressley, A. (King' s Coll., London (United Kingdom). Dept. of Mathematics)


    A quantum group is a Hopf algebra U{sub q}(a), depending on a parameter q element of C, which 'tends to' the universal enveloping algebra U(a) of a Lie algebra a as q tends to 1. In this paper, we develop a highest weight theory for the finite-dimensional representations of U{sub q}(a) when a is the affine algebra sl{sub 2}, assuming that q is not a root of unity. We also give a concrete construction of all finite-dimensional irreducible representations of U{sub q}(sl{sub 2}). Many, but not all, of the results extend without difficulty to the case of U{sub q}(g) with g any finite-dimensional complex simple Lie algebra. (orig./HSI).

  14. Adjoint affine fusion and tadpoles

    Energy Technology Data Exchange (ETDEWEB)

    Urichuk, Andrew, E-mail: [Physics and Astronomy Department, University of Lethbridge, Lethbridge, Alberta T1K 3M4 (Canada); Walton, Mark A., E-mail: [Physics and Astronomy Department, University of Lethbridge, Lethbridge, Alberta T1K 3M4 (Canada); International School for Advanced Studies (SISSA), via Bonomea 265, 34136 Trieste (Italy)


    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  15. Influence of a water purification unit on the contamination level of salmonella in outcoming water and sludge


    Jacob, Benoit; Korsak Koulagenko, Nicolas; Grooven, Bénédicte; Flament, Etienne; Daube, Georges


    Foodborne pathogens occasionally harboured in the gastro-intestinal tract of some domestic animals may be retrieved in slaughterhouses waste water and in sludge of water purification units. Salmonella, athogen common to man and Animals, is often used as a biological risk indicator. The aim of the present study was to assess effectiveness of a recent water purification unit by rapid and semi-quantitative detection of this micro-organism. The water purification unit collects waste water ...

  16. Purification and characterization of osteopontin from human milk. (United States)

    Sørensen, Steen; Justesen, Steen Just; Johnsen, Anders H


    Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further separated into its different molecular forms by means of a two-step procedure, involving size exclusion chromatography and reverse phase chromatography. A rabbit polyclonal antibody was raised to purified intact OPN and high M(r) OPN components; the immunoreactivity of both forms was almost equal when investigated by enzyme immunoassay (EIA). The procedures facilitate the purification of intact OPN and OPN fragments for purposes of standardization, preparation of monospecific antibodies, and functional studies.


    Skeggs, Leonard T.; Kahn, Joseph R.; Shumway, Norman P.


    The enzymatic conversion of hypertensin I to hypertensin II is described together with the subsequent purification of the product by means of counter-current distribution. Improved methods are also presented for the preparation of renin and its substrate, as well as in methods for the reaction of these materials and the purification of the resulting hypertensin I. PMID:13295488


    African Journals Online (AJOL)

    Prof. Adipala Ekwamu

    osmotic shock, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. Initially, a DEAE column was used leading to 28% yield and 77 times fold purification, followed by. Sephacryl gel filtration column giving 25% yield and 72 times fold purification; indicating loss of enzyme in ...

  19. Air/Water Purification (United States)


    After 18 years of research into air/water pollution at Stennis Space Center, Dr. B. C. Wolverton formed his own company, Wolverton Environmental Services, Inc., to provide technology and consultation in air and water treatment. Common houseplants are used to absorb potentially harmful materials from bathrooms and kitchens. The plants are fertilized, air is purified, and wastewater is converted to clean water. More than 100 U.S. communities have adopted Wolverton's earlier water hyacinth and artificial marsh applications. Catfish farmers are currently evaluating the artificial marsh technology as a purification system.

  20. Water Purification Product (United States)


    Ecomaster, an affiliate of BioServe Space Technologies, this PentaPure technology has been used to purify water for our nation's Space Shuttle missions since 1981. WTC-Ecomaster of Mirneapolis, Minnesota manufactures water purification systems under the brand name PentaPure (TM). BioServe researcher Dr. George Marchin, of Kansas State University, first demonstrated the superiority of this technology and licensed it to WTC. Marchin continues to perform microgravity research in the development of new technologies for the benefit of life on Earth.

  1. High quality protein microarray using in situ protein purification

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D


    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  2. Trematode hemoglobins show exceptionally high oxygen affinity. (United States)

    Kiger, L; Rashid, A K; Griffon, N; Haque, M; Moens, L; Gibson, Q H; Poyart, C; Marden, M C


    Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.

  3. Purification of SUMO-1 modified IκBα and complex formation with NF-κB. (United States)

    Lens, Zoé; Dewitte, Frédérique; Van Lint, Carine; de Launoit, Yvan; Villeret, Vincent; Verger, Alexis


    Covalent modification of proteins with SUMO (Small Ubiquitin-like MOdifier) affects many cellular processes, including transcriptional regulation, DNA repair and signal transduction. Although hundreds of SUMO targets have been identified, many biological outcomes of protein sumoylation remain poorly understood. In particular, biochemical and structural analysis can only be easily conducted if highly pure sumoylated substrates are available. Purification of sumoylated substrates in vitro or in bacteria have been previously reported but separating the sumoylated protein from the undesired unmodified fraction is often technically challenging, inefficient and time consuming. Here we develop a new vector system for in vivo sumoylation in Escherichia coli which improves purification of sumoylated proteins. We describe the purification of IκBα, its sumoylation, the subsequent separation and purification of the modified and the unmodified forms and the purification of the complex IκBα-SUMO-1/NF-κB. After a first GST affinity chromatography and GST-tag removal, a unique metal-ion affinity chromatography using a 6xHis-SUMO-1 tag results in mgs of highly pure SUMO-1 modified IκBα. Our pure SUMO-1 modified IκB/NF-κB complex could be a useful tool to identify new interaction partner specific of the SUMO-1 modified IκBα form. This approach may be extended to other SUMO substrates not isolable by classical chromatography techniques. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture. (United States)

    Doherty, Margaret; Bones, Jonathan; McLoughlin, Niaobh; Telford, Jayne E; Harmon, Bryan; DeFelippis, Michael R; Rudd, Pauline M


    Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Effect of charcoal on water purification


    Suzuki, Hirotaka; Kawahigashi, Tatsuo


    [Abstract] A natural basin system purifies water through self-purification, but the water pollution load of a river might exceed its self-purification capacity. Charcoal, which is used for other uses aside from heating, such as air purification, was evaluated experimentally for water quality purification. The experiment described herein is based on simple water quality measurements. Some experimentally obtained results are discussed.

  6. Purification and characterization of osteopontin from human milk

    DEFF Research Database (Denmark)

    Sørensen, Steen; Justesen, Steen Just; Johnsen, Anders H


    Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible...... biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further...

  7. Mathematical analysis of frontal affinity chromatography in particle and membrane configurations. (United States)

    Tejeda-Mansir, A; Montesinos, R M; Guzmán, R


    The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular

  8. The utility of affine variables and affine coherent states (United States)

    Klauder, John R.


    Affine coherent states are generated by affine kinematical variables much like canonical coherent states are generated by canonical kinematical variables. Although all classical and quantum formalisms normally entail canonical variables, it is shown that affine variables can serve equally well for many classical and quantum studies. This general purpose analysis provides tools to discuss two major applications: (1) the completely successful quantization of a nonrenormalizable scalar quantum field theory by affine techniques, in complete contrast to canonical techniques which only offer triviality; and (2) a formulation of the kinematical portion of quantum gravity that favors affine kinematical variables over canonical kinematical variables, and which generates a framework in which a favorable analysis of the constrained dynamical issues can take place. All this is possible because of the close connection between the affine and the canonical stories, while the few distinctions can be used to advantage when appropriate. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’.

  9. The silica-binding Si-tag functions as an affinity tag even under denaturing conditions. (United States)

    Ikeda, Takeshi; Motomura, Kei; Agou, Yuuya; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio


    We recently reported a one-step affinity purification method using a silica-binding protein, designated Si-tag, as a fusion partner and silica particles as the specific adsorbents (Ikeda et al., Protein Expr. Purif. 71 [2010] 91-95) [13]. In this study, we demonstrate that the Si-tag also binds to the silica surface even under denaturing conditions, thereby facilitating affinity purification of recombinant proteins from inclusion bodies. A fusion protein of the Si-tag and a biotin acceptor peptide (AviTag), which was expressed as inclusion bodies in Escherichia coli, was used as a model protein. To simplify our purification method, we disrupted recombinant E. coli cells by sonication in the presence of 8M urea with concomitant solubilization of the inclusion bodies. The fusion protein was recovered with a purity of 90 ± 3% and yield of 92 ± 6% from the cleared cell lysate. We also discuss the binding mechanism of the Si-tag to a silica surface in the presence of high concentrations of denaturant. We propose that the intrinsic disorder of the polycationic Si-tag polypeptide plays an important role in its binding to the silica surface under denaturing conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Representations of affine Hecke algebras

    CERN Document Server

    Xi, Nanhua


    Kazhdan and Lusztig classified the simple modules of an affine Hecke algebra Hq (q E C*) provided that q is not a root of 1 (Invent. Math. 1987). Ginzburg had some very interesting work on affine Hecke algebras. Combining these results simple Hq-modules can be classified provided that the order of q is not too small. These Lecture Notes of N. Xi show that the classification of simple Hq-modules is essentially different from general cases when q is a root of 1 of certain orders. In addition the based rings of affine Weyl groups are shown to be of interest in understanding irreducible representations of affine Hecke algebras. Basic knowledge of abstract algebra is enough to read one third of the book. Some knowledge of K-theory, algebraic group, and Kazhdan-Lusztig cell of Cexeter group is useful for the rest

  11. Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography. (United States)

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


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

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

    DEFF Research Database (Denmark)

    Prentø, Jannick Cornelius; Bukh, Jens


    Hepatitis C virus (HCV) purification by ultracentrifugation is difficult because of the low and heterogeneous density of native and cultured viruses. It was recently shown that inserting flag tag into envelope protein 2 (E2) of HCV permitted virus purification by affinity chromatography. However...... to the original virus. Flag-tagged virus was susceptible to flag-specific antibody neutralization, and infected cells could be immuno-stained by anti-flag antibodies. Using affinity chromatography with anti-flag resin we repeatedly obtained ~30% recovery of infectious particles. The full viability and unaltered...

  13. Fragments of protein A eluted during protein A affinity chromatography. (United States)

    Carter-Franklin, Jayme N; Victa, Corazon; McDonald, Paul; Fahrner, Robert


    Protein A affinity chromatography is a common method for process scale purification of monoclonal antibodies. During protein A affinity chromatography, protein A ligand co-elutes with the antibody (commonly called leaching), which is a potential disadvantage since the leached protein A may need to be cleared for pharmaceutical antibodies. To determine the mechanism of protein A leaching and characterize the leached protein A, we fluorescently labeled the protein A ligand in situ on protein A affinity chromatography media. We found that intact protein A leaches when loading either purified antibody or unpurified antibody in harvested cell culture fluid (HCCF), and that additionally fragments of protein A leach when loading HCCF. The leaching of protein A fragments can be reduced by EDTA, suggesting that proteinases contribute to the generation of protein A fragments. We found that protein A fragments larger than about 6000 Da can be measured by enzyme linked immunosorbent assay, and that they can be more difficult to clear than whole protein A by cation-exchange chromatography.

  14. On nondegenerate umbilical affine hypersurfaces in recurrent affine manifolds

    Directory of Open Access Journals (Sweden)

    Zbigniew Olszak


    Full Text Available Let $widetilde{M}$ be a differentiable manifold of dimension $geqslant 5$, which is endowed with a (torsion-free affine connection $widetildeabla$ of recurrent curvature. Let $M$ be a nondegenerate umbilical affine hypersurface in $widetilde{M}$, whose shape operator does not vanish at every point of $M$. Denote by $abla$ and $h$, respectively, the affine connection and the affine metric induced on $M$ from the ambient manifold. Under the additional assumption that the induced connection $abla$ is related to the Levi-Civita connection $abla^{ast}$ of $h$ by the formula [ abla_XY = abla_X^{ast}Y + varphi(XY + varphi(YX + h(X,YE, ] $varphi$ being a $1$-form and $E$ a vector field on $M$, it is proved that the affine metric $h$ is conformally flat. Relations to totally umbilical pseudo-Riemannian hypersurfaces are also discussed. In this paper, certain ideas from my unpublished report [14] (cf. also [15] are generalized.

  15. Recovery and purification of ethylene (United States)

    Reyneke, Rian [Katy, TX; Foral, Michael J [Aurora, IL; Lee, Guang-Chung [Houston, TX; Eng, Wayne W. Y. [League City, TX; Sinclair, Iain [Warrington, GB; Lodgson, Jeffery S [Naperville, IL


    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. Nanomechanical Water Purification Device Project (United States)

    National Aeronautics and Space Administration — Seldon Laboratories, LLC, proposes a lightweight, low-pressure water purification device that harnesses the unique properties of carbon nanotubes and will operate...

  17. Automated small‐scale protein purification and analysis for accelerated development of protein therapeutics (United States)

    LeSaout, Xavier; Costioli, Matteo; Jordan, Lynn; Lambert, Jeremy; Beighley, Ross; Provencher, Laurel; McGuire, Kevin; Verlinden, Nico; Barry, Andrew


    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. PMID:27774045

  18. Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth. (United States)

    Yang, Chengli; Xing, Jianmin; Guan, Yueping; Liu, Huizhou


    An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.

  19. High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease. (United States)

    Bryan, Cassie M; Bhandari, Janhavi; Napuli, Alberto J; Leibly, David J; Choi, Ryan; Kelley, Angela; Van Voorhis, Wesley C; Edwards, Thomas E; Stewart, Lance J


    The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two ÄKTAexplorer 100 s and four ÄKTAprimes to perform immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography.

  20. Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells (United States)

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

  1. A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large.

    NARCIS (Netherlands)

    Waaijers, S.; Munoz, J.; Berends, C.; Ramalho, J.J.; Goerdayal, S.S.; Low, T.Y.; Zoumaro-Djayoon, A.D.; Hoffmann, M.; Koorman, T.; Tas, R.P.; Harterink, M.; Seelk, S.; Kerver, J.; Hoogenraad, C.C.; Bossinger, O.; Tursun, B.; Heuvel, S. van den; Heck, A.J.R. van; Boxem, M.


    BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in

  2. New downstream processing strategy for the purification of monoclonal antibodies from transgenic tobacco plants. (United States)

    Platis, D; Drossard, J; Fischer, R; Ma, J K-C; Labrou, N E


    Affinity chromatography on immobilized Protein A is the current method of choice for the purification of monoclonal antibodies (mAbs). Despite its widespread use it presents certain drawbacks, such as ligand instability, leaching, toxicity and high cost. In the present work, we report a new procedure for the purification of two human monoclonal anti-HIV (human immunodeficiency virus) antibodies (mAbs 2G12 and 4E10) from transgenic tobacco plants using stable and low cost chromatographic materials. The first step of the mAb 2G12 purification procedure is comprised of an aqueous two-phase partition system (ATPS) for the removal of polyphenols while providing an essential initial purification boost (2.01-fold purification). In the second step, mAb 2G12 was purified using cation-exchange chromatography (CEX) on S-Sepharose FF, by elution with 20mM sodium phosphate buffer pH 7.5, containing 0.1M NaCl. The eluted mAb was directly loaded onto an immobilized metal affinity chromatography column (IMAC, Zn(2+)-iminodiacetic acid-Sepharose 6B) and eluted by stepwise pH gradient. The proposed method offered 162-fold purification with 97.2% purity and 63% yield. Analysis of the antibody preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme immunosorbent assay (ELISA) and western blot showed that the mAb 2G12 was fully active and free of degraded variants, polyphenols and alkaloids. The effectiveness of the present purification protocol was evaluated by using a second transgenic human monoclonal anti-HIV mAb 4E10. The results showed that the same procedure can be successfully used for the purification of mAb 4E10. In the case of mAb 4E10, the proposed method offered 148-fold purification with 96.2% purity and 36% yield. Therefore, the proposed protocol may be of generic use for the purification of mAbs from transgenic tobacco plants.

  3. Isolation and Purification of Jacalin from Artocarpus Heterophyllus Lam

    Directory of Open Access Journals (Sweden)

    M.R. Othman


    Full Text Available This paper presents investigation results of saturation conditions needed for purification of jacalin lectin from the extract seeds of Artocarpus heterophyllus by ammonium precipitation and affinity chromatography on Galactose-Affi gel Hz. Three different aspects of parameters encompassing the percentage of saturation of ammonium sulfate precipitation, the presence of ammonium sulfate on Lowry method and the suitable galactose concentration for optimum elution of the protein from Galactose-Affi gel Hz were investigated. With three different sets of fractional saturation of jacalin purification using ammonium sulfate precipitation, the maximum yield of 0.463 g/g was achieved at 0-90% saturation range in the absence of dialysis. Maximum yield of 0.425 g/g was obtained at 30-60% and 0-90% saturation range in the presence of dialysis. The result from this work also indicates that excessive quantity of NH4SO4 interferes with Lowry method for protein determination substantially. The 0-90% saturation range was found to be more potentially appropriate for large scale application than 30-60% saturation, since the former involves only 1 step NH4SO4 addition. From the affinity chromatography, elution of 0.2 M galactose (in 0.15 M NaCl from Galactose-Affi gel Hz produced the maximum peak profile and jacalin concentration. A reduction or increase in galactose concentration of more than 0.2 M did not increase concentration of purified jacalin purified using this method.

  4. A comprehensive review on biodiesel purification and upgrading

    Directory of Open Access Journals (Sweden)

    Hamed Bateni


    Full Text Available Serious environmental concerns regarding the use of fossil-based fuels have raised awareness regarding the necessity of alternative clean fuels and energy carriers. Biodiesel is considered a clean, biodegradable, and non-toxic diesel substitute produced via the transesterification of triglycerides with an alcohol in the presence of a proper catalyst. After initial separation of the by-product (glycerol, the crude biodiesel needs to be purified to meet the standard specifications prior to marketing. The presence of impurities in the biodiesel not only significantly affects its engine performance but also complicates its handling and storage. Therefore, biodiesel purification is an essential step prior to marketing. Biodiesel purification methods can be classified based on the nature of the process into equilibrium-based, affinity-based, membrane-based, reaction-based, and solid-liquid separation processes. The main adverse properties of biodiesel – namely moisture absorption, corrosiveness, and high viscosity – primarily arise from the presence of oxygen. To address these issues, several upgrading techniques have been proposed, among which catalytic (hydrodeoxygenation using conventional hydrotreating catalysts, supported metallic materials, and most recently transition metals in various forms appear promising. Nevertheless, catalyst deactivation (via coking and/or inadequacy of product yields necessitate further research. This paper provides a comprehensive overview on the techniques and methods used for biodiesel purification and upgrading.

  5. Chitinase III in Euphorbia characias latex: Purification and characterization. (United States)

    Spanò, Delia; Pospiskova, Kristyna; Safarik, Ivo; Pisano, Maria Barbara; Pintus, Francesca; Floris, Giovanni; Medda, Rosaria


    This paper deals with the purification of a class III endochitinase from Euphorbia characias latex. Described purification method includes an effective novel separation step using magnetic chitin particles. Application of magnetic affinity adsorbent noticeably simplifies and shortens the purification procedure. This step and the subsequently DEAE-cellulose chromatography enable to obtain the chitinase in homogeneous form. One protein band is present on PAGE in non-denaturing conditions and SDS-PAGE profile reveals a unique protein band of 36.5 ± 2 kDa. The optimal chitinase activity is observed at 50 °C, pH 5.0. E. characias latex chitinase is able to hydrolyze colloidal chitin giving, as reaction products, N-acetyl-D-glucosamine, chitobiose and chitotriose. Moreover, we observed that calcium and magnesium ions enhance chitinase activity. Finally, we cloned the cDNA encoding the E. characias latex chitinase. The partial cDNA nucleotide sequence contains 762 bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Large-scale functional purification of recombinant HIV-1 capsid.

    Directory of Open Access Journals (Sweden)

    Magdeleine Hung

    Full Text Available During human immunodeficiency virus type-1 (HIV-1 virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.

  7. Purification and characterization of thermostable chitinase from a novel S. maltophilia strain

    Directory of Open Access Journals (Sweden)

    Javed, S.


    Full Text Available Aims: The presents study examines the purification and characterization of a chitinase from S. maltophilia SJ602 strainisolated from a soil sample collected from Jamia Hamdard, New Delhi.Methodology and Results: The purification steps included chitin affinity using colloidal chitin as the affinity matrix andcolumn chromatography using Sephadex G-100. The chitinase was purified to 66 fold having a yield of 17%. The molecular weight of the chitinase was found to be around 29 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. The pH and temperature optima of the purified chitinase were found to be at pH 5.5 and60 °C, respectively. Conclusion, Significance and Impact of the study: Besides showing a significant yield, the enzyme has a highthermal stability which has its applicability in the recycling of chitin waste.

  8. The potential role of self-cleaving purification tags in commercial-scale processes. (United States)

    Fong, Baley A; Wu, Wan-Yi; Wood, David W


    Purification tags are robust tools that can be used to purify a wide selection of target proteins, which makes them attractive candidates for implementation into platform processes. However, tag removal remains an expensive and significant issue that must be resolved before these tags can become widely used. One alternative is self-cleaving purification tags, which can provide the purity and versatility of conventional tags but eliminate the need for proteolytic tag removal. Many of these self-cleaving tags are based on inteins, but other emerging technologies, such as the FrpC and SrtAc proteins, have also been reported. In this review, we cover affinity and non-chromatographic self-cleaving purification tags and their potential industrial applications. Copyright 2010 Elsevier Ltd. All rights reserved.

  9. Water Purification Systems (United States)


    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.

  10. Affinity-Selected Filamentous Bacteriophage as a Probe for Acoustic Wave Biodetectors of Salmonella typhimurium

    National Research Council Canada - National Science Library

    Olsen, Eric V; Sorokulova, Iryna B; Petrenko, Valery A; Chen, I-Hsuan; Barbaree, James M; Vodyanoy, Vitaly J


    Proof-in-concept biosensors were prepared for the rapid detection of Salmonella typhimurium in solution, based on affinity-selected filamentous phage prepared as probes physically adsorbed to piezoelectric transducers...

  11. Protein separation using affinity-based reversed micelles (United States)

    Sun; Gu; Tong; Bai; Ichikawa; Furusaki


    Reversed micellar two-phase extraction is a developing technique for protein separation. Introduction of an affinity ligand is considered to be an effective approach to increase the selectivity and capacity of reversed micelles. In this article, Cibacron Blue F3G-A (CB) as an affinity ligand was immobilized to reversed micelles composed of soybean lecithin by a two-phase reaction. The affinity partitioning of lysozyme and bovine serum albumin (BSA) to the CB-lecithin micelles was studied. Formation of mixed micelles by additionally introducing a nonionic surfactant, Tween 85, to the CB-lecithin micelles was effective to increase the solubilization of lysozyme due to the increase of W0 (water/surfactant molar ratio)/micellar size. The partitioning isotherms of lysozyme to the CB-lecithin micelles with and without Tween 85 were expressed by the Langmuir equation. The dissociation constants in the Langmuir equation decreased on addition of Tween 85, indicating the increase of the effectiveness of lysozyme binding to the immobilized CB. On addition of 20 g/L Tween 85 to 50 g/L lecithin/hexane micellar phase containing 0.1 mmol/L CB, the extraction capacity for lysozyme could be increased by 42%. Moreover, the CB-lecithin micelles with or without Tween 85 showed significant size exclusion for BSA due to its high molecular weight. Thus, lysozyme and BSA were separated from artificial solutions containing the two proteins. In addition, the affinity-based reversed micellar phase containing Tween 85 was recycled three times for lysozyme purification from crude egg-white solutions. Lysozyme purity increased by 16-18-fold, reaching 60-70% in the recycled use.

  12. Purification of Recombinant Ebola Virus Glycoprotein and VP40 from a Human Cell Line (United States)


    secreted into the culture medium. Next, purification proceeded using immobilized metal affinity chromatography and anion exchange chromatography. The...typical yield for each of these proteins was approximately 1 mg/30 mL of cell culture with >90% purity. The procedures described herein may be useful...endorsement of any commercial products. This report may not be cited for purposes of advertisement . This report has been approved for public release

  13. Purification and characterisation of anti-pneumococcal capsular polysaccharide IgG immunoglobulins. (United States)

    Parker, Antony R; Lock, Emma; Iftikhar, Asma; Barber, Richard; Stubbs, Phil D; Harding, Stephen; Wallis, Gregg L F


    The production of reference materials for quantifying pneumococcal antibody concentrations relies upon large scale vaccination. An alternative simple, reproducible protocol has been developed for the affinity purification of 23 serotype anti-pneumococcal capsular polysaccharide (PCP) IgG immunoglobulins. The purification protocol utilised IgG fractionation, capsular polysaccharide (CPS) adsorption, and affinity chromatography using Pneumovax®-Sepharose. Purification efficiency and method reproducibility were assessed by comparison of 4 batches of anti-PCP IgG. Immunoglobulin composition was determined using nephelometry and functionality was evaluated using VaccZyme™ ELISAs. Anti-PCP IgG preparations were ≥95% pure by SDS-PAGE analysis with no contaminating IgA or IgM immunoglobulins or IgG antigen specific antibodies towards haemophilus influenzae b, diphtheria toxoid or tetanus toxoid. The predominant IgG subclass in the preparation was IgG2. This novel purification procedure produced highly specific anti-PCP IgG preparations that compared well to both Lot 89SF and 007sp international serum standards and could be used as an alternative method for the production of reference materials. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Affinity biosensors: techniques and protocols

    National Research Council Canada - National Science Library

    Rogers, Kim R; Mulchandani, Ashok


    ..., and government to begin or expand their biosensors research. This volume, Methods in Biotechnology vol. 7: Affinity Biosensors: Techniques and Protocols, describes a variety of classical and emerging transduction technologies that have been interfaced to bioaffinity elements (e.g., antibodies and receptors). Some of the reas...

  15. Gravity theory through affine spheres (United States)

    Minguzzi, E.


    In this work it is argued that in order to improve our understanding of gravity and spacetime our most successful theory, general relativity, must be destructured. That is, some geometrical assumptions must be dropped and recovered just under suitable limits. Along this line of thought we pursue the idea that the roundness of the light cone, and hence the isotropy of the speed of light, must be relaxed and that, in fact, the shape of light cones must be regarded as a dynamical variable. Mathematically, we apply some important results from affine differential geometry to this problem, the idea being that in the transition we should preserve the identification of the spacetime continuum with a manifold endowed with a cone structure and a spacetime volume form. To that end it is suggested that the cotangent indicatrix (dispersion relation) must be described by an equation of Monge-Ampère type determining a hyperbolic affine sphere, at least whenever the matter content is negligible. Non-relativistic spacetimes fall into this description as they are recovered whenever the center of the affine sphere is at infinity. In the more general context of Lorentz-Finsler theories it is shown that the lightlike unparametrized geodesic flow is completely determined by the distribution of light cones. Moreover, the transport of lightlike momenta is well defined though there could be no notion of affine parameter. Finally, we show how the perturbed indicatrix can be obtained from the perturbed light cone.

  16. Separation and purification of enzymes by continuous pH-parametric pumping

    Energy Technology Data Exchange (ETDEWEB)

    Huang, S.Y.; Lin, C.K.; Juang, L.Y.


    Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. e-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield, e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes. 19 references.


    Directory of Open Access Journals (Sweden)

    Azam Khodashenas Pelko


    Full Text Available The Jainism emphasizes three major teachings about the purification of the soul (jiva, Ahimsa, Aparigrapha and anekantwad. Jainism, The focus of this religion has been purification of the soul by means of right conduct, right faith and right knowledge. The ultimate goal of Hinduism is Moksha or liberation (total freedom. In Hinduism, purification of the soul is a goal that one must work to attain. The Buddhism is the science of pursuing the aim of making the human mind perfect, and of purifying the human soul. The knowledge of purifying of the soul and softening of the hearts is as essential for human. They having the correct motivations means purifying our souls from hypocrisy, caprice, and heedlessness. The primary goal of Taoism may be described as the mystical intuition of the Tao, which is the way, the undivided unity, and the ultimate Reality. According to the Christianity access to truth cannot be conceived without purity of the soul

  18. Purification of Water by Aquatic Plants


    Morimitsu, Katsuhito; Kawahigashi, Tatsuo


    [Abstract] Water quality purification of many water systems including those occurring in rivers depends to a great degree on water quality purification activities of aquatic plants and microbes. This paper presents a discussion of results, based on laboratory experiments, of purification by aquatic plants.

  19. A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications. (United States)

    Fowler, Daniel K; Stewart, Scott; Seredick, Steve; Eisen, Judith S; Stankunas, Kryn; Washbourne, Philip


    Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research.

  20. Boronate affinity materials for separation and molecular recognition: structure, properties and applications. (United States)

    Li, Daojin; Chen, Yang; Liu, Zhen


    Boronate affinity materials, as unique sorbents, have emerged as important media for the selective separation and molecular recognition of cis-diol-containing compounds. With the introduction of boronic acid functionality, boronate affinity materials exhibit several significant advantages, including broad-spectrum selectivity, reversible covalent binding, pH-controlled capture/release, fast association/desorption kinetics, and good compatibility with mass spectrometry. Because cis-diol-containing biomolecules, including nucleosides, saccharides, glycans, glycoproteins and so on, are the important targets in current research frontiers such as metabolomics, glycomics and proteomics, boronate affinity materials have gained rapid development and found increasing applications in the last decade. In this review, we critically survey recent advances in boronate affinity materials. We focus on fundamental considerations as well as important progress and new boronate affinity materials reported in the last decade. We particularly discuss on the effects of the structure of boronate ligands and supporting materials on the properties of boronate affinity materials, such as binding pH, affinity, selectivity, binding capacity, tolerance for interference and so on. A variety of promising applications, including affinity separation, proteomics, metabolomics, disease diagnostics and aptamer selection, are introduced with main emphasis on how boronate affinity materials can solve the issues in the applications and what merits boronate affinity materials can provide.

  1. New protein purification system using gold-magnetic beads and a novel peptide tag, "the methionine tag". (United States)

    Okada, Yoshiaki; Takano, Tomoko Y; Kobayashi, Nozomi; Hayashi, Arisa; Yonekura, Masaaki; Nishiyama, Yuji; Abe, Tomohiro; Yoshida, Takuya; Yamamoto, Takao A; Seino, Satoshi; Doi, Takefumi


    Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.

  2. The affine quantum gravity programme

    CERN Document Server

    Klauder, J R


    The central principle of affine quantum gravity is securing and maintaining the strict positivity of the matrix left brace g-hat sub a sub b (x)right brace composed of the spatial components of the local metric operator. On spectral grounds, canonical commutation relations are incompatible with this principle, and they must be replaced by noncanonical, affine commutation relations. Due to the partial second-class nature of the quantum gravitational constraints, it is advantageous to use the recently developed projection operator method, which treats all quantum constraints on an equal footing. Using this method, enforcement of regularized versions of the gravitational operator constraints is formulated quite naturally by means of a novel and relatively well-defined functional integral involving only the same set of variables that appears in the usual classical formulation. It is anticipated that skills and insight to study this formulation can be developed by studying special, reduced-variable models that sti...

  3. Profiling of drug binding proteins by monolithic affinity chromatography in combination with liquid chromatography-tandem mass spectrometry. (United States)

    Zhang, Xuepei; Wang, Tongdan; Zhang, Hanzhi; Han, Bing; Wang, Lishun; Kang, Jingwu


    A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC-MS/MS is reported. Two immunosuppresive drugs, namely FK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate. By combining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported target proteins of these two drugs were identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins. The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Electron affinity of liquid water

    Energy Technology Data Exchange (ETDEWEB)

    Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco; Paesani, Francesco; Galli, Giulia


    Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1–0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential of the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.

  5. Purification and properties of dialkylfluorophosphatase

    NARCIS (Netherlands)

    Cohen, J.A.; Warringa, M.G.P.J.


    1. 1. Zone electrophoresis on starch columns of purified preparations of fluorophosphatase resulted in a further purification. The preparations thus obtained differed in various respects from the cruder ones so far described. 2. 2. In the course of this electrophoresis fractions were obtained,

  6. Bioinspired Materials for Water Purification

    Directory of Open Access Journals (Sweden)

    Alfredo Gonzalez-Perez


    Full Text Available Water scarcity issues associated with inadequate access to clean water and sanitation is a ubiquitous problem occurring globally. Addressing future challenges will require a combination of new technological development in water purification and environmental remediation technology with suitable conservation policies. In this scenario, new bioinspired materials will play a pivotal role in the development of more efficient and environmentally friendly solutions. The role of amphiphilic self-assembly on the fabrication of new biomimetic membranes for membrane separation like reverse osmosis is emphasized. Mesoporous support materials for semiconductor growth in the photocatalytic degradation of pollutants and new carriers for immobilization of bacteria in bioreactors are used in the removal and processing of different kind of water pollutants like heavy metals. Obstacles to improve and optimize the fabrication as well as a better understanding of their performance in small-scale and pilot purification systems need to be addressed. However, it is expected that these new biomimetic materials will find their way into the current water purification technologies to improve their purification/removal performance in a cost-effective and environmentally friendly way.

  7. Molecular modification of Protein A to improve the elution pH and alkali resistance in affinity chromatography. (United States)

    Xia, Hai-Feng; Liang, Zhen-Dong; Wang, Sha-Li; Wu, Pu-Qiang; Jin, Xiong-Hua


    Protein A of Staphylococcus aureus has been widely used as an affinity ligand for the purification of immunoglobulin. However, the low elution pH and the sensitivity to alkaline condition restricted the large-scale application of antibody purification. To overcome these disadvantages, the B domain was selected and mutated to Z domain and the recombinant Protein A was reconstructed by linking five Z domains. First, a section of six glycines was inserted into the second loop of Z domain, Z (6G). This increased the elution pH to 4.0-5.0. Then, the site-specific mutagenesis was conducted by replacing the 23rd asparagines to threonine and 30th phenylalanine to alanine, Z (N23T, F30A). These mutations made the recombinant Protein A shown a higher alkaline resistance than the nature Protein A. The work confirmed the modification of Protein A and exhibited the characteristics of recombinant Staphylococcal Protein A for antibody purification.

  8. Statistical and Judgmental Criteria for Scale Purification

    DEFF Research Database (Denmark)

    Wieland, Andreas; Durach, Christian F.; Kembro, Joakim


    of scale purification, to critically analyze the current state of scale purification in supply chain management (SCM) research and to provide suggestions for advancing the scale-purification process. Design/methodology/approach A framework for making scale-purification decisions is developed and used...... of methodological rigor and coherence is identified when it comes to current purification practices in empirical SCM research. Suggestions for methodological improvements are provided. Research limitations/implications The framework and additional suggestions will help to advance the knowledge about scale...... to analyze and critically reflect on the application of scale purification in leading SCM journals. Findings This research highlights the need for rigorous scale-purification decisions based on both statistical and judgmental criteria. By applying the proposed framework to the SCM discipline, a lack...

  9. Spectral affinity in protein networks

    Directory of Open Access Journals (Sweden)

    Teng Shang-Hua


    Full Text Available Abstract Background Protein-protein interaction (PPI networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. Results We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. Conclusion We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at, that allows the user to

  10. Spectral affinity in protein networks. (United States)

    Voevodski, Konstantin; Teng, Shang-Hua; Xia, Yu


    Protein-protein interaction (PPI) networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at, that allows the user to quickly find nodes closest to a queried vertex in any protein

  11. Using Affinity Diagrams to Evaluate Interactive Prototypes


    Lucero, Andrés


    International audience; Affinity diagramming is a technique used to externalize, make sense of, and organize large amounts of unstructured, far-ranging, and seemingly dissimilar qualitative data. HCI and interaction design practitioners have adopted and used affinity diagrams for different purposes. This paper discusses our particular use of affinity diagramming in prototype evaluations. We reflect on a decade’s experience using affinity diagramming across a number of projects, both in indust...

  12. [Role of hemoglobin affinity to oxygen in adaptation to hypoxemia]. (United States)

    Kwasiborski, Przemysław Jerzy; Kowalczyk, Paweł; Zieliński, Jakub; Przybylski, Jacek; Cwetsch, Andrzej


    states showed that acidosis and increased tissue oxygen demand lead to a broadened arterial blood pO2 range, in which the high-affinity hemoglobin is more efficient. Contrary to the widely held view that the only response to hypoxemia is a decrease in haemoglobin oxygen affinity, it was shown that under extreme hypoxemic conditions, an increased haemoglobin oxygen affinity improves the oxygenation of tissues. It was also shown that the dominance of hemoglobin with a high oxygen affinity rapidly exceeds hemoglobin with low oxygen affinity in the case of acidosis with its accompanying high tissue oxygen extraction. In cases of extreme disruptions of the acid-base equilibrium, the dominance of high-oxygen-affinity hemoglobin spans over the entire possible range of pO2 in arterial blood.

  13. Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp paracasei CHCC 2115

    DEFF Research Database (Denmark)

    Thage, B.V.; Rattray, F.P.; Laustsen, M.W.


    Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp. paracasei CHCC 2115. Methods and Results: The purification protocol consisted of anion exchange chromatography, affinity chromatography...... and hydrophobic interaction chromatography. The enzyme was found to exist as a monomer with a molecular mass of 40-50 kDa. The AT converted isoleucine, leucine and valine at a similar rate with alpha-ketoglutarate as the amino group acceptor; minor activity was shown for methionine. The enzyme had p...

  14. Liquid-liquid extraction of enzymes by affinity aqueous two-phase systems

    Directory of Open Access Journals (Sweden)

    Xu Yan


    Full Text Available From analytical to commercial scale, aqueous two-phase systems have their application in the purification, characterization and study of biomaterials. In order to improve the selectivity of the systems, the biospecific affinity ligands were introduced. In the affinity partitioning aqueous two-phase system, have many enzymes been purified. This review discusses the partitioning of some enzymes in the affinity aqueous two-phase systems in regard to the different ligands, including reactive dyes, metal ions and other ligands. Some integration of aqueous two-phase system with other techniques for more effective purification of enzymes are also presented.Tanto em escala de laboratório como industrial, os sistemas de duas fases aquosas podem ser utilizados para a purificação, caracterização e estudos de biomateriais. Para aumentar a seletividade desse sistema, ligantes de afinidade bioespecíficos podem ser utilizados. No sistema de duas fases aquosas por afinidade, muitas enzimas podem ser purificadas. Neste artigo de revisão, a partição de algumas enzimas por esse tipo de afinidade, utilizando diferentes ligantes como corantes e íons metálicos, são discutidas. Além disso, a integração desse sistema de duas fases aquosas com outras técnicas de purificação estão sendo apresentados, com o objetivo mostrar a melhoria da eficiência do processo.

  15. Affinity Spaces and 21st Century Learning (United States)

    Gee, James Paul


    This article discusses video games as "attractors" to "affinity spaces." It argues that affinity spaces are key sites today where people teach and learn 21st Century skills. While affinity spaces are proliferating on the Internet as interest-and-passion-driven sites devoted to a common set of endeavors, they are not new, just…

  16. Manifolds with integrable affine shape operator

    Directory of Open Access Journals (Sweden)

    Daniel A. Joaquín


    Full Text Available This work establishes the conditions for the existence of vector fields with the property that theirs covariant derivative, with respect to the affine normal connection, be the affine shape operatorS in hypersurfaces. Some results are obtained from this property and, in particular, for some kind of affine decomposable hypersurfaces we explicitely get the actual vector fields.

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

    Directory of Open Access Journals (Sweden)

    Mayank Saraswat


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

  18. Preparation of immunoaffinity column for rapid purification of human ...

    African Journals Online (AJOL)

    Mammalian deoxyribonucleic acid (DNA) polymerase delta (pol δ) is well characterized as a tightly associated heterotetrameric complex. It is thought to play a central role in chromosomal DNA replication and various DNA repair processes. However, the availability of highly purified active pol δ becomes one of the major ...

  19. Using Affinity Diagrams to Evaluate Interactive Prototypes

    DEFF Research Database (Denmark)

    Lucero, Andrés


    our particular use of affinity diagramming in prototype evaluations. We reflect on a decade’s experience using affinity diagramming across a number of projects, both in industry and academia. Our affinity diagramming process in interaction design has been tailored and consists of four stages: creating......Affinity diagramming is a technique used to externalize, make sense of, and organize large amounts of unstructured, far-ranging, and seemingly dissimilar qualitative data. HCI and interaction design practitioners have adopted and used affinity diagrams for different purposes. This paper discusses...

  20. Applications of silica supports in affinity chromatography. (United States)

    Schiel, John E; Mallik, Rangan; Soman, Sony; Joseph, Krina S; Hage, David S


    The combined use of silica-based chromatographic supports with immobilized affinity ligands can be used in many preparative and analytical applications. One example is the use of silica-based affinity columns in HPLC, giving rise to a method known as high-performance affinity chromatography (HPAC). This review discusses the role that silica has played in the development of affinity chromatography and HPAC and the applications of silica in these methods. This includes a discussion of the types of ligands that have been employed with silica and the methods by which these ligands have been immobilized. Various formats have also been presented for the use of silica in affinity chromatographic methods, including assays involving direct or indirect analyte detection, on-line or off-line affinity extraction, and chiral separations. The use of silica-based affinity columns in studies of biological systems based on zonal elution and frontal analysis methods will also be considered.

  1. Affinity partitioning of proteins tagged with choline-binding modules in aqueous two-phase systems. (United States)

    Maestro, Beatriz; Velasco, Isabel; Castillejo, Isabel; Arévalo-Rodríguez, Miguel; Cebolla, Angel; Sanz, Jesús M


    We present a novel procedure for affinity partitioning of recombinant proteins fused to the choline-binding module C-LytA in aqueous two-phase systems containing poly(ethylene glycol) (PEG). Proteins tagged with the C-LytA module and exposed to the two-phase systems are quantitatively localized in the PEG-rich phase, whereas subsequent addition of the natural ligand choline specifically shifts their localization to the PEG-poor phase by displacement of the polymer from the binding sites. The described procedure is simple, scalable and reproducible, and has been successfully applied to the purification of four diverse proteins, resulting in high yields and purity.

  2. Water purification using organic salts (United States)

    Currier, Robert P.


    Water purification using organic salts. Feed water is mixed with at least one organic salt at a temperature sufficiently low to form organic salt hydrate crystals and brine. The crystals are separated from the brine, rinsed, and melted to form an aqueous solution of organic salt. Some of the water is removed from the aqueous organic salt solution. The purified water is collected, and the remaining more concentrated aqueous organic salt solution is reused.

  3. Development of tantalum–zirconium alloy for hydrogen purification

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Sanjay, E-mail: [Fusion Reactor Materials Section, MG, BARC, Mumbai 85 (India); IAMR, Hiroshima University, Higashihiroshima 739-8530 (Japan); Singh, Anamika [GSASM Hiroshima University, Higashihiroshima 739-8530 (Japan); Jain, Uttam; Dey, Gautam Kumar [Fusion Reactor Materials Section, MG, BARC, Mumbai 85 (India)


    Highlights: • Terminal solid solubility of Ta increases with Zr addition. • Increase in lattice parameters of Ta due to Zr addition may be the possible reason. • Enhance H solubility could also be explained on the change in e-DOS of Ta–Zr alloys. • Ta–Zr alloys could be possible combination for hydrogen purification membrane. - Abstract: Terminal solid solubility of hydrogen in Ta–Zr alloys has been studied in connection with the development of tantalum based metallic membrane for hydrogen/tritium purification. The alloys were prepared by vacuum arc melting technique and subsequently cold rolled to 0.2 mm thickness. The terminal solid solubility of hydrogen in these cold rolled samples was investigated in a modified Sieverts apparatus. The terminal solid solubility of hydrogen was marginally increased with zirconium content. The change in the lattices parameter of tantalum upon zirconium addition and the higher affinity of zirconium for hydrogen as compared to tantalum could be the possible reasons.

  4. Multimodal charge-induction chromatography for antibody purification. (United States)

    Tong, Hong-Fei; Lin, Dong-Qiang; Chu, Wen-Ning; Zhang, Qi-Lei; Gao, Dong; Wang, Rong-Zhu; Yao, Shan-Jing


    Hydrophobic charge-induction chromatography (HCIC) has advantages of high capacity, salt-tolerance and convenient pH-controlled elution. However, the binding specificity might be improved with multimodal molecular interactions. New ligand W-ABI that combining tryptophan and 5-amino-benzimidazole was designed with the concept of mutimodal charge-induction chromatography (MCIC). The indole and benzimidazole groups of the ligand could provide orientated mutimodal binding to target IgG under neutral pH, while the imidazole groups could induce the electrostatic repulsion forces for efficient elution under acidic pH. W-ABI ligand was coupled successfully onto agarose gel, and IgG adsorption behaviors were investigated. High affinity to IgG was found with the saturated adsorption capacity of 70.4 mg/ml at pH 7, and the flow rate of mobile phase showed little impact on the dynamic binding capacity. In addition, efficient elution could be achieved at mild acidic pH with high recovery. Two separation cases (IgG separation from albumin containing feedstock and monoclonal antibody purification from cell culture supernatant) were verified with high purity and recovery. In general, MCIC with the specially-designed ligand is an expanding of HCIC with improved adsorption selectivity, which would be a potential alternative to Protein A-based capture for the cost-effective purification of antibodies. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. SNO+ Scintillator Purification and Assay (United States)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.


    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  6. Technological assumptions for biogas purification. (United States)

    Makareviciene, Violeta; Sendzikiene, Egle


    Biogas can be used in the engines of transport vehicles and blended into natural gas networks, but it also requires the removal of carbon dioxide, hydrogen sulphide, and moisture. Biogas purification process flow diagrams have been developed for a process enabling the use of a dolomite suspension, as well as for solutions obtained by the filtration of the suspension, to obtain biogas free of hydrogen sulphide and with a carbon dioxide content that does not exceed 2%. The cost of biogas purification was evaluated on the basis of data on biogas production capacity and biogas production cost obtained from local water treatment facilities. It has been found that, with the use of dolomite suspension, the cost of biogas purification is approximately six times lower than that in the case of using a chemical sorbent such as monoethanolamine. The results showed travelling costs using biogas purified by dolomite suspension are nearly 1.5 time lower than travelling costs using gasoline and slightly lower than travelling costs using mineral diesel fuel.

  7. Heparin-binding proteins of human seminal plasma: purification and characterization. (United States)

    Kumar, Vijay; Hassan, Md Imtaiyaz; Kashav, Tara; Singh, Tej P; Yadav, Savita


    Human seminal plasma (HuSP) contains several proteins that bind heparin and related glycosaminoglycans. Heparin binding proteins (HBPs) from seminal plasma have been shown to participate in modulation of capacitation or acrosome reaction and thus have been correlated with fertility in some species. However, these have not been studied in detail in human. The objective of this study was to purify major HBPs from HuSP in order to characterize these proteins. HBPs were isolated by affinity-chromatography on Heparin-Sepharose column, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and Size-exclusion chromatography and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Identification of HBPs was done by matrix-assisted laser desorption-ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Here we report the purification and identification of seven HBPs in seminal fluid. The major HBPs are lactoferrin and its fragments, semenogelin I fragments, semenogelin II, prostate specific antigen, homolog of bovine seminal plasma-proteins (BSP), zinc finger protein (Znf 169) and fibronectin fragments. In this study we are reporting for the first time the purification and identification of BSP-homolog and Znf 169 from HuSP and classified them as HBPs. Here we report the purification of seven clinically important proteins from human seminal fluid through heparin affinity chromatography and RP-HPLC, in limited steps with higher yield. (c) 2008 Wiley-Liss, Inc.

  8. Novel purification method of human immunoglobulin by using a thermo-responsive protein A. (United States)

    Koguma, Ichiro; Yamashita, Shuntaro; Sato, Satoshi; Okuyama, Kazuo; Katakura, Yoshinori


    We attempted to evaluate a novel purification method of immunoglobulins (IgGs) by using a mutant type of protein A. Although this mutant protein A binds to IgGs at 5°C, the IgGs are released at 40°C; hence, it was designated as thermo-responsive protein A (TRPA). We aimed to purify IgG1 from the culture supernatant of CHO cells producing AE6F4 human monoclonal IgG1. AE6F4 IgG1 was purified using only a TRPA-filled column and by modifying the temperature, without any exposure to acidic conditions. Furthermore, the purified AE6F4 IgG1 maintained the inherent binding affinity to antigen, while this property was lost in AE6F4 IgG1 purified using a conventional protein A (CPA) column possibly because of product aggregation and fragmentation. These data suggest that IgG is sensitive to acid treatment; however, it can be highly purified with retention of high affinity by using a TRPA column. Further, this purification method can be used on an industrial scale for the purification of antibody drugs. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Quantitative affinity purification mass spectrometry: a versatile technology to study protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Katrina eMeyer


    Full Text Available While the genomic revolution has dramatically accelerated the discovery of disease-associated genes, the functional characterization of the corresponding proteins lags behind. Most proteins fulfill their tasks in complexes with other proteins, and analysis of Protein-Protein Interactions (PPIs can therefore provide insights into protein function. Several methods can be used to generate large-scale protein interaction networks. However, most of these approaches are not quantitative and therefore cannot reveal how perturbations affect the network. Here, we illustrate how a clever combination of quantitative mass spectrometry with different biochemical methods provides a rich toolkit to study different aspects of PPIs including topology, subunit stoichiometry, and dynamic behavior.

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

    Directory of Open Access Journals (Sweden)

    Giordanni C. Dantas


    Full Text Available Abstract 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.

  11. Monoclonal antibody affinity purification of a 78 kDa membrane ...

    Indian Academy of Sciences (India)


    pension was incubated at 4°C for 20–25 min with occa- sional stirring and centrifuged at 10,000 g for 10 min. The supernatant (5 ml, diluted to 1⋅0 mg/ml protein with the same buffer) was applied to IgG-sepharose 4B col- umn (1⋅0 ml bed volume) pre-equilibriated with the same buffer. After thorough washing with 20⋅0 ml ...

  12. Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo

    Czech Academy of Sciences Publication Activity Database

    Majerská, J.; Schrumpfová, P.; Dokládal, Ladislav; Schorová, Š.; Stejskal, K.; Obořil, M.; Honys, D.; Kozáková, L.; Polanská, P.; Sýkorová, Eva


    Roč. 254, č. 4 (2017), s. 1547-1562 ISSN 0033-183X R&D Projects: GA ČR GA13-06943S Institutional support: RVO:68081707 Keywords : single-stranded-dna * genome-wide screen * arabidopsis-thaliana * reverse-transcriptase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.870, year: 2016

  13. Identification of Drosophila centromere associated proteins by quantitative affinity purification-mass spectrometry

    Directory of Open Access Journals (Sweden)

    Teresa K. Barth


    The data accompanying the manuscript on this approach (Barth et al., 2015, Proteomics 14:2167-78, DOI: 10.1002/pmic.201400052 has been deposited to the ProteomeXchange Consortium ( via the PRIDE partner repository with the dataset identifier PXD000758.

  14. A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri. (United States)

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


    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. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  15. Nanostructured Catalytic Reactors for Air Purification Project (United States)

    National Aeronautics and Space Administration — This SBIR Phase I project proposes the development of lightweight compact nanostructured catalytic reactors for air purification from toxic gaseous organic...

  16. Nanostructured Catalytic Reactors for Air Purification Project (United States)

    National Aeronautics and Space Administration — This SBIR Phase II project proposes the development of lightweight compact nanostructured catalytic reactors for air purification from toxic gaseous organic...

  17. Quantitative analysis of fibrin-binding affinity of fibrinolytic components by frontal affinity chromatography. (United States)

    Kazama, M; Tahara, C; Abe, T; Kasai, K


    Binding affinity of fibrinolytic factors to insolubilized lysine and fibrin was quantitatively measured by frontal affinity chromatography using lysine-Toyopearl and fibrin-Sepharose column. The highest binding affinity was found with recombinant tissue-type plasminogen activator (t-PA), followed by lysyl-plasminogen and glutamyl-plasminogen (Glu-PLg) with intermediate affinity, but very low affinity by single chain UK-type plasminogen activator, high molecular weight UK and low molecular weight UK. At the coexistence of EACA, fibrin-binding affinity of Glu-PLg was greatly reduced, but those of UK's were substantially unchanged. It was concluded that high fibrin-binding affinity of t-PA and plasminogens were largely related to the lysine-binding affinity of these enzymes, but that of UK's would be related to the other binding affinity.

  18. Introduction of structural affinity handles as a tool in selective nucleic acid separations (United States)

    Willson, III, Richard Coale (Inventor); Cano, Luis Antonio (Inventor)


    The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

  19. Comparing Russian and Finnish standards of water purification


    Maria, Pupkova


    The subject of this thesis is water purification. The first aim of this thesis is to consider different ways of water purification. The second aim is to compare Finnish and Russian standards of water purification. The third one is to show water purification methods on the pattern of Mikkeli water purification plan. Water purification methods of water intended for human consumption will be described.Combined tables will be done according to the quality requirement of drinking water of both,...

  20. Proton affinities of hydrated molecules (United States)

    Valadbeigi, Younes


    Proton affinities (PA) of non-hydrated, M, and hydrated forms, M(H2O)1,2,3, of 20 organic molecules including alcohols, ethers, aldehydes, ketones and amines were calculated by the B3LYP/6-311++G(d,p) method. For homogeneous families, linear correlations were observed between PAs of the M(H2O)1,2,3 and the PAs of the non-hydrated molecules. Also, the absolute values of the hydration enthalpies of the protonated molecules decreased linearly with the PAs. The correlation functions predicted that for an amine with PA amine with PA > 1100 kJ/mol the PA(M(H2O)) is smaller than the PA.

  1. Assouad type dimensions for partially affine sponges


    Howroyd, Douglas


    Recently self-affine sponges have been shown to be interesting examples and counter-examples to several previously open problems. One class of recently discovered sponges are partially affine Bedford-McMullen sponges whose Assouad type dimensions cannot be calculated like the dimensions of regular Bedford-McMullen sponges are. We calculate the Assouad type dimensions for such partially affine sponges and discuss some of their more subtle details.

  2. Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein

    DEFF Research Database (Denmark)

    Balogh, Ria K.; Gyurcsik, Béla; Hunyadi-Gulyás, Éva


    . A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes...... any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure....

  3. Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization.


    Subramanian, N; Adiga, P R


    Chicken egg yolk biotin-binding protein-I (BBP-I) has been purified to homogeneity along with the tetrameric BBP-II by a common protocol. The purification includes delipidation of egg yolk by butanol extraction, DEAE-Sephacel chromatography, treatment with guanidinium chloride and biotin-aminohexyl-Sepharose affinity chromatography. The identity of purified BBP-I was ascertained by its physicochemical properties as well as by its immunological cross-reactivity and precursor-product relationsh...

  4. A purification method for a molecular complex in which a scaffold molecule is fully loaded with heterogeneous molecules.

    Directory of Open Access Journals (Sweden)

    Shoji J Ohuchi

    Full Text Available An affinity resin-based pull-down method is convenient for the purification of biochemical materials. However, its use is difficult for the isolation of a molecular complex fully loaded with multiple components from a reaction mixture containing the starting materials and intermediate products. To overcome this problem, we have developed a new purification procedure that depends on sequential elimination of the residues. In practice, two affinity resins were used for purifying a triangular-shaped RNP (RNA-protein complex consisting of three ribosomal proteins (L7Ae bound to an RNA scaffold. First, a resin with immobilized L7Ae protein captured the incomplete RNP complexes and the free RNA scaffold. Next, another resin with an immobilized chemically modified RNA of a derivative of Box C/D motif, the binding partner of L7Ae, was used to capture free protein. The complete triangular RNP was successfully purified from the mixture by these two steps. Obviously, the purified triangular RNP displaying three protein-binding peptides exhibited an improved performance when compared with the unrefined product. Conceptually, this purification procedure should be applicable for the purification of a variety of complexes consisting of multiple components other than RNP.

  5. Affine Flag Manifolds and Principal Bundles

    CERN Document Server

    Schmitt, Alexander HW


    Affine flag manifolds are infinite dimensional versions of familiar objects such as Gramann varieties. The book features lecture notes, survey articles, and research notes - based on workshops held in Berlin, Essen, and Madrid - explaining the significance of these and related objects (such as double affine Hecke algebras and affine Springer fibers) in representation theory (e.g., the theory of symmetric polynomials), arithmetic geometry (e.g., the fundamental lemma in the Langlands program), and algebraic geometry (e.g., affine flag manifolds as parameter spaces for principal bundles). Novel

  6. A Novel Vertex Affinity for Community Detection

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Andy [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Sanders, Geoffrey [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Henson, Van [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vassilevski, Panayot [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)


    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  7. Novel trends in affinity biosensors: current challenges and perspectives (United States)

    Arugula, Mary A.; Simonian, Aleksandr


    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives.

  8. [Purification and properties of NAG A from human kidney]. (United States)

    Yoshida, K


    In the present paper, we have reported the purification procedures of N-acetyl-beta, D-glucosaminidase (NAG) A from human renal tissue as well as the enzymatic properties of NAG A. NAG A was purified to homogeneity by gel filtration methods using Sephacry S-400 and S-200, followed by affinity chromatography with TSK DEAE 5-PW. The final activity of the enzyme was 1001 U/ml protein which was 506.6-fold that of the crude extract (supernatant of 20,000 x G of the homogenate). The molecular weight of NAG A was 140 kDa, consisting of two subunits of 30 kDa and 57 kDa. The isoelectric point of the enzyme was 5.60. The optimal pH of the enzyme was between 4.7 and 4.9. The Km value of the enzyme for sodio-m-cresol sulfophtaleinyl-N-acetyl-beta, D-glucosaminide was found 0.177 x 10(-3) mol/l. Lectin affinity chromatographies using concanavalin A and wheat germagglutinin have demonstrated that major sugar-chains of the enzyme were the high mannose type and hybrid type with a fucose residue, and that a small amount of the complex type was contained.

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

    Directory of Open Access Journals (Sweden)

    Aimee Shen


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

  10. Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin. (United States)

    Gabe, Claire M; Brookes, Steven J; Kirkham, Jennifer


    Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli ) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic

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

    Directory of Open Access Journals (Sweden)

    Claire M. Gabe


    Full Text Available Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We

  12. Synthesis and Charaterization of Silica-Based Aldehyde Chitosan Hybrid Material for Biodiesel Purification

    Directory of Open Access Journals (Sweden)

    Sandra Rodrigues da Silva


    Full Text Available This study concerns the development and charaterization of Silica-based aldehyde Chitosan hybrid material as an adsorbent for biodiesel purification. This biocomposite was prepared by sol-gel route and oxidation with periodate, and then characterized. FTIR experiments showed that the hybrid formed presents absorption bands similar to those of Chitosan-Silica, with the exception of the vibrations at 1480 cm−1 and 1570 cm−1 attributed to the symmetrical angular deformation in the N-H plane, and possess large N2 Brunauer–Emmett–Teller (BET surface areas. Thermogravimetric analysis (TG and scanning electron microscopy (SEM was also carried out. Adsorption studies of bioadsorbents involving the analysis of free glycerol, soap, acidity, diglycerides, triglycerides, and fluorescence spectroscopy showed that silica-based aldehyde chitosan has a good affinity for glycerol and a good purification process.

  13. Synthesis and Charaterization of Silica-Based Aldehyde Chitosan Hybrid Material for Biodiesel Purification. (United States)

    da Silva, Sandra Rodrigues; de Albuquerque, Nilson J A; de Almeida, Rusiene M; de Abreu, Fabiane C


    This study concerns the development and charaterization of Silica-based aldehyde Chitosan hybrid material as an adsorbent for biodiesel purification. This biocomposite was prepared by sol-gel route and oxidation with periodate, and then characterized. FTIR experiments showed that the hybrid formed presents absorption bands similar to those of Chitosan-Silica, with the exception of the vibrations at 1480 cm-1 and 1570 cm-1 attributed to the symmetrical angular deformation in the N-H plane, and possess large N₂ Brunauer-Emmett-Teller (BET) surface areas. Thermogravimetric analysis (TG) and scanning electron microscopy (SEM) was also carried out. Adsorption studies of bioadsorbents involving the analysis of free glycerol, soap, acidity, diglycerides, triglycerides, and fluorescence spectroscopy showed that silica-based aldehyde chitosan has a good affinity for glycerol and a good purification process.

  14. Purification of rhamnolipid using colloidal magnetic nanoparticles ...

    African Journals Online (AJOL)

    Phospholipid-coated colloidal magnetic nanoparticles with mean magnetite core size of 9 nm are shown to be effective ion exchange media for the recovery and purification of Rhaminolipid from culture mixtures. These particles have high adsorption capacity for purification (an order of magnitude larger than the best ...

  15. Water purification in low background experiments (United States)

    Giammarchi, Marco


    Water purification is an important technique in high-mass low radioactivity experiments in modern physics. Water is frequently used both as a shielding and as the sensitive part of a particle detector in underground arrangements, especially in the frame of Astroparticle Physics studies. In this paper, I will describe the main purification techniques and discuss some of its performances.


    DEFF Research Database (Denmark)

    Nielsen, Joan Maj; Dahi, Elian


    Purification of fluoride contaminated magadi is studied using bone char sorption and calcium precipitation. The bone char treatment is found to be workable both in columns and in batches where the magadi is dissolved in water prior to treatment. The concentrations in the solutions were 89 g magadi...... treatment method. A procedure for purification of fluoride contaminated magadi at household level is described....

  17. Purification and characterization of amidase from acrylamide ...

    African Journals Online (AJOL)

    An amidase from a newly isolated acrylamide-degrading bacterium Burkholderia sp. strain DR.Y27 was purified to homogeneity by a combination of anion exchange and gel filtration chromatography. The purification strategy achieved 11.15 of purification fold and a yield of 1.55%. The purified amidase consisted of four ...

  18. pMHC affinity controls duration of CD8+ T cell-DC interactions and imprints timing of effector differentiation versus expansion. (United States)

    Ozga, Aleksandra J; Moalli, Federica; Abe, Jun; Swoger, Jim; Sharpe, James; Zehn, Dietmar; Kreutzfeldt, Mario; Merkler, Doron; Ripoll, Jorge; Stein, Jens V


    During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity-primed T cells acquired cytotoxic activity earlier than high affinity-primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity-stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. © 2016 Ozga et al.


    Skeggs, Leonard T.; Marsh, Walton H.; Kahn, Joseph R.; Shumway, Norman P.


    The purification of hypertensin I has been described. The final product which is four times as powerful a pressor agent as l-arterenol, is obtained with an over-all recovery of 40 per cent. The product consists of a single component in countercurrent distribution, having a nitrogen content of 15.97 per cent and a specific activity of 7050 Goldblatt units per mg. of N or 1125 units per mg. of solid. Acid hydrolysis and paper chromatography indicate in a preliminary fashion that there are about nine amino acids present in the intact polypeptide. PMID:13201713

  20. The fundamentals of RNA purification. (United States)

    Nilsen, Timothy W


    The ability to purify, analyze, and manipulate RNA is now essential for many laboratories working in the life sciences; however, the skills and practices required to work with RNA are not present in every laboratory, and initiating RNA research can be intimidating. In this article, we provide an overview of RNA purification procedures and discuss strategies to prevent RNA degradation, so that any competent researcher can confidently purify RNA and use it to perform meaningful experiments from the most basic to the highly sophisticated.

  1. Electrophoretic affinity chromatography: method validation. (United States)

    Liu, Z; Feng, S; Guo, S; Shen, Z; Ding, F; Yuan, N


    A new method for preparative-scale separation of biomolecules, electrophoretic affinity chromatography (EAC), is proposed in this paper. Separation by EAC is carried out in a long and ribbon-like multicompartment electrolyser separated by membranes, in which the two central compartments are used for packing the gel matrix and for sample loading respectively. Next to the central compartments are the elution compartments and electrode compartments. The electric field is applied perpendicular to the fluid flow in the compartments. Adsorption and desorption steps may both be carried out in the presence of an electric field, which transports the target components into the gel compartment for adsorption and the impurities into the elution compartments for washing. After the adsorption step an elution solution is introduced and the product is released from the gel matrix and washed out. Separation of human serum albumin (HSA) from human serum gives HSA product of high purity, as demonstrated by isoelectric focusing analysis. The characteristics of electrophoretic binding of HSA on Blue Sepharose Fast Flow are examined. The preliminary results show that this new method has advantages in terms of high rate of mass transfer and ease of scaling up, which are of particular interest when large-scale separation of biomolecules is considered.

  2. On affine non-negative matrix factorization

    DEFF Research Database (Denmark)

    Laurberg, Hans; Hansen, Lars Kai


    We generalize the non-negative matrix factorization (NMF) generative model to incorporate an explicit offset. Multiplicative estimation algorithms are provided for the resulting sparse affine NMF model. We show that the affine model has improved uniqueness properties and leads to more accurate id...

  3. Phosphopeptide enrichment by immobilized metal affinity chromatography

    DEFF Research Database (Denmark)

    Thingholm, Tine E.; Larsen, Martin R.


    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively...

  4. Global affine differential geometry of hypersurfaces

    CERN Document Server

    Li, An-Min; Zhao, Guosong; Hu, Zejun


    This book draws a colorful and widespread picture of global affine hypersurface theory up to the most recent state. Moreover, the recent development revealed that affine differential geometry- as differential geometry in general- has an exciting intersection area with other fields of interest, like partial differential equations, global analysis, convex geometry and Riemann surfaces.

  5. Development of monoclonal antibodies and immunochromatographic lateral flow device for rapid test of alanine aminotransferase isoenzyme 1. (United States)

    Hu, Xiaomei; Cheng, Shiliang; Liu, Xinfeng; Li, Jie; Zheng, Wen; Lu, Gang; Zhang, Jun; Zheng, Jian; Zhang, Juan


    Alanine aminotransferase (ALT) has been used as a sensitive marker for liver injury in people and in preclinical toxicity studies. But measurement of ALT isoenzymes, ALT1 and ALT2, was reported to be of more diagnostic value. The aim of this study is to develop an ideal pair of anti-ALT1 monoclonal antibodies (MAbs) of high specificity and affinity, and subsequently prepare a Immunochromatographic lateral flow device (LFD) for rapid test of ALT1 in human serums. The complete coding sequence of ALT1 gene (1500 bp) was cloned from human hepatoma G2 cells (HepG2) and inserted into the expression vector pET-32a(+). ALT1 recombinant protein was routinely prepared by E. coli BL21 (DE3) expression and Ni(2+) affinity purification. Balb/c mice were immunized with purified ALT1 and the splenocytes were fused with Sp2/0 myeloma cells. The positive clones, verified by indirect enzyme-linked immunosorbent assay (ELISA) using purified ALT1, were subcloned to single clones by limiting dilution process. A MAb pair was selected from the obtained MAbs according the sandwich ELISA pairing results and then used for lateral flow device (LFD) production. After evaluation of the sensitivity and specificity, the LFD strips were employed to test human serum samples with known ALT activity levels. ALT1 recombinant protein was expectedly prepared by expression and purification. A total of 8 stable clones that produced antibodies specifically recognizing ALT1 protein were developed. After sandwich ELISA pairing, an ideal pair of anti-ALT1 MAbs, designated as BD7 and DG3, were selected and proved to be of high specificity, titer and affinity. Based on the MAb pair, LFD strips specifically for ALT1 rapid test were subsequently prepared. The detection threshold of the LFD strips was 12 U/L. No cross reaction was found. The ALT1 LFD with high sensitivity and specificity was successfully developed. It is valuable for testing ALT1 protein in human sera and can be a beneficial complement for

  6. Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography. (United States)

    Clark, Kathleen M; Fedoriw, Nadia; Robinson, Katrina; Connelly, Sara M; Randles, Joan; Malkowski, Michael G; DeTitta, George T; Dumont, Mark E


    To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by X-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow expression of reading frames fused to C-terminal His10 and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Initial purifications were based on expression of the genes under control of a galactose-inducible promoter, but higher cell densities and improved expression have been obtained through use of the yeast ADH2 promoter. Wide variations have been observed in the behavior of different TMP targets during purification; some can be readily purified, while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or remain tightly associated with the matrices even after cleavage of the affinity tags. The size, oligomeric state, and composition of purified protein-detergent complexes purified under different conditions were analyzed using a colorimetric assay of detergent concentrations and by analytical size-exclusion chromatography using static light scattering, refractive index, and UV absorption detection to monitor the elution profiles. Effective procedures were developed for obtaining high concentrations of purified TMPs without excessively concentrating detergents.

  7. Improving image segmentation by learning region affinities

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Lakshman [Los Alamos National Laboratory; Yang, Xingwei [TEMPLE UNIV.; Latecki, Longin J [TEMPLE UNIV.


    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  8. Purification of recombinant Aβ(1-42) and pGlu-Aβ(3-42) using preparative SDS-PAGE. (United States)

    Spahn, Claudia; Wermann, Michael; Eichentopf, Rico; Hause, Gerd; Schlenzig, Dagmar; Schilling, Stephan


    Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aβ, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was  >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aβ peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein. (United States)

    Hafrén, Anders; Mäkinen, Kristiina


    In order to be able to analyse post-translational modifications and protein interactions of viral genome-linked protein VPg taking place during potato virus A (PVA) infection, an affinity tag-based purification system was developed by inserting a sequence encoding a six-histidine and haemagglutinin (HisHA) tag to the 3' end of the VPg coding sequence within the infectious cDNA clone of PVA. The engineered virus was fully functional and the HisHA tag-encoding sequence remained stable in the PVA genome throughout the infection process. Purification under denaturing conditions resulted in a protein sample that contained multiple VPg and NIa forms carrying post-translational modifications that altered their isoelectric points. Non-modified tagged VPg (pI 8) was a minor product in the protein sample derived from total leaf proteins, but when the replication-associated membranes were used as starting material, its relative amount increased. Further characterization demonstrated that some of the PVA VPg isoforms were modified by multiple phosphorylation events. Purity of the proteins derived from the native purifications with either of the tags was evaluated. A clearly purer VPg sample was obtained by performing tandem affinity purification utilizing both tags sequentially. NIb, CI and HC-Pro co-purified in an affinity-tagged VPg-dependent manner, indicating that the system was able to isolate protein complexes operating during PVA infection.

  10. Experimental Immunization Based on Plasmodium Antigens Isolated by Antibody Affinity

    Directory of Open Access Journals (Sweden)

    Ali N. Kamali


    Full Text Available Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethal Plasmodium yoelii yoelii 17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses of P. yoelii yoelii 17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites.

  11. Purification and properties of an acid phosphatase from Entamoeba histolytica HM-1:IMSS. (United States)

    Aguirre-García, M M; Cerbón, J; Talamás-Rohana, P


    Entamoeba histolytica contains and secretes acid phosphatase, which has been proposed as a virulence factor in some pathogenic microorganisms. In this work, we purified and characterised a membrane-bound acid phosphatase (MAP) from E. histolytica HM-1:IMSS and studied the effect of different chemical compounds on the secreted acid phosphatase and MAP activities. MAP purification was accomplished by detergent solubilisation, and affinity and ion exchange chromatographies. The enzyme showed a pI of 5.5-6.2, an optimum pH of 5.5, and a Km value of 1.14 mM with p-nitrophenyl phosphate.

  12. Semiconductor grade, solar silicon purification project (United States)

    Ingle, W. M.; Rosler, R. R.; Thompson, S. W.; Chaney, R. E.


    Experimental apparatus and procedures used in the development of a 3-step SiF2(x) polymer transport purification process are described. Both S.S.M.S. and E.S. analysis demonstrated that major purification had occured and some samples were indistinguishable from semiconductor grade silicon (except possibly for phosphorus). Recent electrical analysis via crystal growth reveals that the product contains compensated phosphorus and boron. The low projected product cost and short energy payback time suggest that the economics of this process will result in a cost less than the goal of $10/Kg(1975 dollars). The process appears to be readily scalable to a major silicon purification facility.

  13. Identification, purification, and expression patterns of chitinase from psychrotolerant Pedobacter sp. PR-M6 and antifungal activity in vitro. (United States)

    Song, Yong-Su; Seo, Dong-Jun; Jung, Woo-Jin


    In this study, a novel psychrotolerant chitinolytic bacterium Pedobacter sp. PR-M6 that displayed strong chitinolytic activity on 0.5% colloidal chitin was isolated from the soil of a decayed mushroom. Chitinase activity of PR-M6 at 25 °C (C25) after 6 days of incubation with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins). Three chitinase isozymes (chiII, chiIII, and chiIV) from the crude enzyme at 25 °C (C25) incubation were expressed on SDS-PAGE gels at 25 °C. After purification by chitin-affinity chromatography, six chitinase isozymes (chiI, chiII, chiIII, chiIV, chiV, and chiVI) from C25-fractions were expressed on SDS-PAGE gels at 25 °C. Major bands of chitinase isozymes (chiI, chiII, and chiIII) from C4-fractions were strongly expressed on SDS-PAGE gels at 25 °C. Pedobacter sp. PR-M6 showed high inhibition rate of 60.9% and 57.5% against Rhizoctonia solani and Botrytis cinerea, respectively. These results indicated that psychrotolerant Pedobacter sp. PR-M6 could be applied widely as a microorganism agent for the biocontrol of agricultural phytopathogens at low temperatures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Reverse osmosis water purification system (United States)

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


    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.

  15. One-step non-chromatography purification of a low abundant fucosylated protein from complex plant crude extract

    Directory of Open Access Journals (Sweden)

    Lindsay Arnold


    Full Text Available Effective methods for isolation and purification of glycoproteins and other glycoconjugates are important to biopharmaceutical industry and diagnostic industry. They are also critical to an emerging field of glycoproteomics. In this work, we applied the newly-developed affinity ligand, a fusion protein of elastic like polymer (ELP and a bacterial lectin, in an affinity precipitation process to purify soybean peroxidase (SBP based on the presence of fucoseon the protein surface. We addressed, in particular, the challenge of purifying a low abundant protein from a complex dilute crude plant extract. The novel affinity precipitation developed in this work was very promising. One step binding and precipitation resulted in >95% recovery yield directly from crude extract and a 22.7 fold purification, giving a specific activity of 420 U/mg. The SBP isolated using this affinity precipitation meets or exceeds the quality specifications of reagent grade products by Sigma. We showed that the recovery yield had a strong dependence on the molar ratio of ligand to target fucosylated protein, with a ratio of three giving nearly full recovery, which could be predicted based on the total fucose content per protein molecule and the number of binding site per ligand molecule. We additionally developed a method of ligand regeneration and investigated its reuse. A simple wash with pH buffer was shown to be effective to regenerate the binding capacity for the ligand, and the ligand could be used for 10 times, giving an averaged 80% isolation yield based on initial input of soybean peroxidase. Taken together, an effective method of affinity precipitation was developed, which could be used to enrich a low abundant target glycoprotein from a complex mixture with a high recovery yield. The high selectivity for fucosylated protein and its ease of operation make this method particularly useful for purification of low abundant glycoprotein from natural sources. This work

  16. Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography

    DEFF Research Database (Denmark)

    Vorum, H; Pedersen, A O; Honoré, B


    of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid...

  17. Automorphisms in Birational and Affine Geometry

    CERN Document Server

    Ciliberto, Ciro; Flenner, Hubert; McKernan, James; Prokhorov, Yuri; Zaidenberg, Mikhail


    The main focus of this volume is on the problem of describing the automorphism groups of affine and projective varieties, a classical subject in algebraic geometry where, in both cases, the automorphism group is often infinite dimensional. The collection covers a wide range of topics and is intended for researchers in the fields of classical algebraic geometry and birational geometry (Cremona groups) as well as affine geometry with an emphasis on algebraic group actions and automorphism groups. It presents original research and surveys and provides a valuable overview of the current state of the art in these topics. Bringing together specialists from projective, birational algebraic geometry and affine and complex algebraic geometry, including Mori theory and algebraic group actions, this book is the result of ensuing talks and discussions from the conference “Groups of Automorphisms in Birational and Affine Geometry” held in October 2012, at the CIRM, Levico Terme, Italy. The talks at the conference high...

  18. On the Lp affine isoperimetric inequalities

    Indian Academy of Sciences (India)

    surface area measure on convex bodies. We also establish the reverse version of -Petty projection inequality and an affine isoperimetric inequality of − p K . Author Affiliations. Wuyang Yu1 Gangsong Leng2. Institute of Management Decision ...

  19. Solid State Air Purification System Project (United States)

    National Aeronautics and Space Administration — The purpose of this proposed research is to develop a new air purification system based on a liquid membrane, capable of purifying carbon dioxide from air in a far...

  20. Purification of Gaussian maximally mixed states

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Kabgyun [Center for Macroscopic Quantum Control, Department of Physics and Astronomy, Seoul National University, Seoul 08826 (Korea, Republic of); School of Computational Sciences, Korea Institute for Advanced Study, Seoul 02455 (Korea, Republic of); Lim, Youngrong, E-mail: [Center for Macroscopic Quantum Control, Department of Physics and Astronomy, Seoul National University, Seoul 08826 (Korea, Republic of)


    We find that the purifications of several Gaussian maximally mixed states (GMMSs) correspond to some Gaussian maximally entangled states (GMESs) in the continuous-variable regime. Here, we consider a two-mode squeezed vacuum (TMSV) state as a purification of the thermal state and construct a general formalism of the Gaussian purification process. Moreover, we introduce other kind of GMESs via the process. All of our purified states of the GMMSs exhibit Gaussian profiles; thus, the states show maximal quantum entanglement in the Gaussian regime. - Highlights: • Candidates of Gaussian maximally mixed state are proposed. • Obtaining Gaussian maximally entangled states using the purification process. • The suggested states can be applicable for the test of capacity problem in Gaussian regime.

  1. A simple and efficient purification platform for monoclonal antibody production based on chromatin-directed cell culture clarification integrated with precipitation and void-exclusion anion exchange chromatography. (United States)

    Chen, Quan; Abdul Latiff, Sarah Maria; Toh, Phyllicia; Peng, Xinying; Hoi, Aina; Xian, Mo; Zhang, Haibo; Nian, Rui; Zhang, Wei; Gagnon, Pete


    Protein A affinity chromatography, featured by its robustness and high-specificity, is still dominant as a first capture step for the purification of immunoglobulin G monoclonal antibodies (IgG mAbs). However, the material and operational costs of protein A are universally recognized as high, and its productivity is also limited as column mode. In order to overcome these limitations, industry is increasingly considering the use of non-protein A-based processes for IgG purification. In this study, sodium citrate precipitation (SCP) was developed as the primary purification step, and chromatin-directed cell culture clarification was demonstrated to significantly elevate the purification capability. Additional 0.05% (w/v) of Tween 20 was shown to effectively reduce the residual free antibody light chain (LC) during precipitation. The resuspended IgG was further polished by void-exclusion anion exchange chromatography (VEAX), which supported protein loading without buffer adjustment. The non-histone host cell protein (nh-HCP) content in the final product was about 5ppm and histone HCP below limit of detection (LOD). DNA was reduced to less than 1ppb, and aggregates/free LC less than 0.1%. The overall IgG recovery was 87.2%. A simple and efficient purification platform with only one-column step was therefore established, providing a more promising alternative to the current prevailing protein A-based purification platforms. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Learning Affinity via Spatial Propagation Networks


    Liu, Sifei; De Mello, Shalini; Gu, Jinwei; Zhong, Guangyu; Yang, Ming-Hsuan; Kautz, Jan


    In this paper, we propose spatial propagation networks for learning the affinity matrix for vision tasks. We show that by constructing a row/column linear propagation model, the spatially varying transformation matrix exactly constitutes an affinity matrix that models dense, global pairwise relationships of an image. Specifically, we develop a three-way connection for the linear propagation model, which (a) formulates a sparse transformation matrix, where all elements can be the output from a...

  3. Affinity Propagation Clustering Using Path Based Similarity


    Yuan Jiang; Yuliang Liao; Guoxian Yu


    Clustering is a fundamental task in data mining. Affinity propagation clustering (APC) is an effective and efficient clustering technique that has been applied in various domains. APC iteratively propagates information between affinity samples, updates the responsibility matrix and availability matrix, and employs these matrices to choose cluster centers (or exemplars) of respective clusters. However, since it mainly uses negative Euclidean distance between exemplars and samples as the simila...

  4. Collaborating on Affinity Diagrams Using Large Displays


    Judge, Tejinder K.; McCrickard, D. Scott


    Gathering and understanding user requirements is an essential part of design. Techniques like affinity diagramming are useful for gathering and understanding user data but have shortcomings such as the difficulty to preserve the diagram after its creation, problems during the process such as searching for notes, and loss of shared awareness. We propose an early prototype that solves problems in the process of creating an affinity diagram and enhances it using a...

  5. Purification and characterization of thermostable glucoamylase from ...

    African Journals Online (AJOL)

    Thermostable glucoamylase from Rhizopus oligosporus SK5 mutant was purified in a 3-step purification using Imarsil, activated charcoal and Sephadex-G-100 to achieve a 40-fold purification. The enzyme was optimally active at pH 5.0 and temperature of 80 °C. It exhibited a half-life of 60 minutes at 70 °C. Its stability was ...

  6. Glycoproteins of axonal transport: affinity chromatography on fucose-specific lectins

    Energy Technology Data Exchange (ETDEWEB)

    Gustavsson, S.; Ohlson, C.; Karlsson, J.O.


    Rapidly transported fucose-labeled glycoproteins from axons of rabbit retinal ganglion cells were solubilized with nonionic detergents. The solubilized components were subjected to affinity chromatography on three different fucose-specific lectins. A recently characterized fucose-specific lectin from Aleuria aurantia bound reversibly approximately 60% of the applied protein-bound radioactivity. The lectins from Lotus tetragonolobus and Ulex europaeus bound are very small proportions of the labeled rapidly transported glycoproteins.

  7. Cleavage of fusion proteins on the affinity resins using the TEV protease variant. (United States)

    Zhu, Kejun; Zhou, Xuan; Yan, Yanping; Mo, Hongmei; Xie, Yifan; Cheng, Beijiu; Fan, Jun


    It is documented that the tobacco etch virus protease (TEVp) variant TEVp3M is less efficient in cleaving the fusion protein bound to Ni-NTA resin at relatively low temperature. Here, we determined that, using the GFP fusion substrate bound to Ni-NTA or Strep-tactin agarose, activity of the TEVp5M variant was higher than that of the other TEVp construct, and about 15% higher than that of the TEVp3M. The GST fusion proteins immobilized on Strep-tactin agarose or Glutathione Sepharose were efficiently cleaved by purified TEVp5M at specified conditions using GFP reporter for visual track and detection. After on-column cleavage of three fusion constructs using the cognate TEVp5M constructs, two target proteins with relatively high purity were separated from Ni-NTA or Amylose agarose. With elution of the buffer containing 1 M NaCl, maize sulfiredoxin was released from Ni-NTA resin via on-column cleavage. Our results confirmed that TEVp5M efficiently cleaved the fusion proteins bound to the four affinity matrices. By combination with appropriate affinity handles, the cognate TEVp5M mediating tag removal enabled purification and cleavage of the fusion proteins, removal of the protease, and separation of the target proteins from the affinity resin to be accomplished in one step. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography. (United States)

    Müller, Egbert; Vajda, Judith


    Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    Energy Technology Data Exchange (ETDEWEB)

    Orcutt, Kelly Davis; Slusarczyk, Adrian L. [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Cieslewicz, Maryelise [Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Ruiz-Yi, Benjamin [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Bhushan, Kumar R. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Frangioni, John V. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Wittrup, K. Dane, E-mail: wittrup@mit.ed [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)


    Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2{+-}1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a {sup 111}In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

  10. Purification and functional characterization of a protein: Bombyx mori human growth hormone like protein in silkworm pupa. (United States)

    Chen, Jianqing; Shu, Tejun; Lv, Zhengbing; Nie, Zuoming; Chen, Jian; Chen, Hao; Yu, Wei; Gai, Qijing; Zhang, Yaozhou


    Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.

  11. Purification and functional characterization of a protein: Bombyx mori human growth hormone like protein in silkworm pupa.

    Directory of Open Access Journals (Sweden)

    Jianqing Chen

    Full Text Available Human growth hormone (hGH is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.

  12. Automated purification of Borrelia burgdorferi s.l. PCR products with KingFisher{sup TM} magnetic particle processor prior to genome sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Maekinen, Johanna E-mail:; Marttila, Harri; Viljanen, Matti K


    Borrelia burgdorferi sensu lato genospecies were differentiated by PCR-based sequencing of the borrelial flagellin gene. To evaluate the usefulness of KingFisher{sup TM} magnetic particle processor in PCR product purification, borrelia PCR products were purified with KingFisher{sup TM} magnetic particle processor prior to cycle sequencing and the quality of the sequence data received was analyzed. KingFisher was found to offer a rapid and reliable alternative for borrelial PCR product purification.

  13. Automated purification of Borrelia burgdorferi s.l. PCR products with KingFisher™ magnetic particle processor prior to genome sequencing (United States)

    Mäkinen, Johanna; Marttila, Harri; Viljanen, Matti K.


    Borrelia burgdorferi sensu lato genospecies were differentiated by PCR-based sequencing of the borrelial flagellin gene. To evaluate the usefulness of KingFisher™ magnetic particle processor in PCR product purification, borrelia PCR products were purified with KingFisher™ magnetic particle processor prior to cycle sequencing and the quality of the sequence data received was analyzed. KingFisher was found to offer a rapid and reliable alternative for borrelial PCR product purification.

  14. Optimization of purification method and characterization of recombinant human Centrin-1. (United States)

    Phanindranath, Regur; Sudhakar, Digumarthi V S; Sharma, Anand Kumar; Thangaraj, Kumarasamy; Sharma, Yogendra


    Centrins are acidic proteins, present in all eukaryotes to perform imperative roles in centrosome positioning and segregation. Existing methods for the purification of centrins for biophysical studies involves either multiple steps or yields protein with an affinity tag, which pins additional tag-cleavage step. Therefore, we have made an attempt to develop a simple and single step method for protein purification. We have performed categorical evaluation of existing methods, and describe a one-step procedure based on cleavable Intein-tag, which can be utilized for routine preparation of any isoform of centrins. Since human Centrin-1 and Centrin-2 are devoid of Trp, we exploit this feature to assess the purity of the protein using Tyr fluorescence; an essential point ignored generally. In addition, we report important spectral and hydrodynamic characteristics of human Centrin-1, accounting that HsCentrin-1 has moderate affinity for Ca(2+). Centrin-1 does not gain structure as seen by far- and near-UV circular dichroism, rather there is a loss of ellipticity, though inconsiderable upon binding Ca(2+). Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Purification of aflatoxin B1 antibody for the development of aflatoxin biosensor (United States)

    Prihantoro, E. A. B.; Saepudin, E.; Ivandini, T. A.


    Aflatoxin B1 (AFB1) is produced from agricultural products especially peanuts overgrown with aspergillus flavus during the post-harvest process. Aflatoxin is classified as a highly toxic and carcinogenic substance to humans by the International Agency for Research on Cancer (IARC), WHO. This research was conducted to develop the AFB1 biosensor using antibody that specifically binds to aflatoxin B1. This antibody was produced by injecting an AFB1 hapten-protein (immunogen) to a rabbit. Antibody was obtained from rabbit's blood serum and purified using Protein A affinity chromatography and precipitation at the isoelectric point. The result showed that purification using protein A contains antibody of 4.0 mg/mL, whereas purification using precipitation at isoelectric pH contains antibody of 0.3 mg/mL. The pure antibody was tested for its specificity against AFB1, tetrahydrofuran (THF), dimethyl formamide (DMF), bovine serum albumin (BSA), and ethanol. The result revealed that THF, BSA, and ethanol were bound to antibody, while DMF showed no interaction. It was concluded that the polyclonal antibody which have been successfully purified from rabbit's blood serum using protein A affinity chromatography and precipitation methods showed an unspecific identification.


    Directory of Open Access Journals (Sweden)

    Ana Carla Penteado Feltrin

    Full Text Available This study established the conditions for the extraction of the enzyme peroxidase (PO from soybean meal (SBM. An experimental design methodology was carried out in order to evaluate the effects of stirring rate time, pH and extracting solvent volume on the enzyme extraction. By using 5 g SBM and 50 mL phosphate buffer 10 mmol L-1 pH 4.7, 60 min stirring rate at 100 rpm, an enzyme with specific activity of 100 U mg-1 for SBM was obtained. Two techniques of purification were tested and compared for purification of peroxidase from soybean meal: triphasic partition (TPP and molecular exclusion chromatography. TPP showcased a greater efficiency with 50% recuperation and a purification factor of 13.6. Peroxidase in crude and pure forms was characterized for kinetics, thermodynamics and biochemistry. The parameter of thermal inactivation indicates high stability to exposure time and temperature increase, showing that enzyme activity is not altered by the presence of constituents of the reaction medium. Peroxidase in crude form represented a greater upkeep in activity, keeping 50% activity for 114 days at 0 °C. Peroxidase in pure form had greater affinity for substrate and reduced Deoxynivalenol levels by 80%, 20% more than the crude form.

  17. Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase). (United States)

    Mahn, Andrea; Angulo, Alejandro; Cabañas, Fernanda


    Myrosinase (β-thioglucosidase glucohydrolase, EC from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

  18. Biotinylated-sortase self-cleavage purification (BISOP method for cell-free produced proteins

    Directory of Open Access Journals (Sweden)

    Endo Yaeta


    Full Text Available Abstract Background Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in Escherichia coli. This method, however, is yet to be applied to the cell-free based protein production. Results The histidine tag-based self-cleavage method for purifying proteins produced by the wheat cell-free protein synthesis system showed high background, low recovery, and unexpected cleavage between the N-terminally fused sortase and target protein during the protein synthesis. Addition of calcium chelator BAPTA to the cell-free reaction inhibited the cleavage. In order to adapt the sortase-based purification method to the cell-free system, we next used biotin as the affinity tag. The biotinylated sortase self-cleavage purification (BISOP method provided tag-free, highly purified proteins due to improved recovery of proteins from the resin. The N-terminal sequence analysis of the GFP produced by the BISOP method revealed that the cleavage indeed occurred at the right cleavage site. Using this method, we also successfully purified the E2 heterocomplex of USE2N and USE2v1. The c-terminal src kinase (CSK obtained by the BISOP method showed high activity in phosphorylating the Src protein. Furthermore, we demonstrated that this method is suitable for automatically synthesizing and purifying proteins using robots. Conclusion We demonstrated that the newly developed BISOP method is very useful for obtaining

  19. Purification processes for coal gasification

    Energy Technology Data Exchange (ETDEWEB)

    Fleming, D.K.; Primack, H.S.


    It is apparent from the discussion that many routes can be taken to achieve acid-gas removal and sulfur recovery from coal gas. The selection of the optimum purification system is a major task. The type of coal, type of gasifier and the upstream processing all strongly influence the selection. Several generalizations can be made: (1) The cost of the purification sections of a high-Btu gas plant is significant--perhaps 10 to 30% of the capital cost of the coal conversion facility. (2) The cost of purifying gas produced from high-sulfur coal feed is more expensive than the cost for purifying gas produced from low-sulfur coal. (3) The choice of an acid-gas removal system will often be a function of system pressure. The economical choice will usually be: (a) amine-based systems at atmospheric pressure; (b) hot-carbonate systems at moderate pressure or (c) physical-solvent systems at higher pressure. (4) For a high-Btu, high-sulfur case: (a) A selective acid-gas removal system with a Claus plant is probably more economical than a non-selective acid-gas system with liquid oxidation of the H/sub 2/S in the regenerator off-gas. (b) Even moderately selective systems can produce an H/sub 2/S-rich gas suitable for a Claus plant. The CO/sub 2/-rich gas may or may not require further sulfur removal, depending on the selectivity. (5) For a high-Btu, low-sulfur case: (a) The hot carbonate and tertiary amine systems may not be sufficiently selective to produce a gas suitable for feed to a Claus process while a physical solvent system may be. Therefore, the physical solvent system may be expected to be more economical. (b) The regenerated gas from the bulk CO/sub 2/ removal system following a selective physical solvent system may require further sulfur removal, depending upon the sulfur level in the initial feedstock and the selectivity of the system selected.

  20. Lis1 Regulates Germinal Center B Cell Antigen Acquisition and Affinity Maturation. (United States)

    Chen, Jingjing; Cai, Zhenming; Zhang, Le; Yin, Yuye; Chen, Xufeng; Chen, Chao; Zhang, Yang; Zhai, Sulan; Long, Xuehui; Liu, Xiaolong; Wang, Xiaoming


    The germinal center (GC) is the site where activated B cells undergo rapid expansions, somatic hypermutation, and affinity maturation. Affinity maturation is a process of Ag-driven selection. The amount of Ag acquired and displayed by GC B cells determines whether it can be positively selected, and therefore Ag acquisition has to be tightly regulated to ensure the efficient affinity maturation. Cell expansion provides sufficient quantity of GC B cells and Abs, whereas affinity maturation improves the quality of Abs. In this study, we found that Lis1 is a cell-intrinsic regulator of Ag acquisition capability of GC B cells. Lack of Lis1 resulted in redistribution of polymerized actin and accumulation of F-actin at uropod; larger amounts of Ags were acquired and displayed by GC B cells, which presumably reduced the selection stringency. Affinity maturation was thus compromised in Lis1-deficient mice. Consistently, overexpression of Lis1 in GC B cells led to less Ag acquisition and display. Additionally, Lis1 is required for GC B cell expansion, and Lis1 deficiency blocked the cell cycle at the mitotic phase and GC B cells were prone to apoptosis. Overall, we suggest that Lis1 is required for GC B cell expansion, affinity maturation, and maintaining functional intact GC response, thus ensuring both the quantity and quality of Ab response. Copyright © 2017 by The American Association of Immunologists, Inc.

  1. High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.

    Directory of Open Access Journals (Sweden)

    Audrey D McConnell

    Full Text Available A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID. Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.

  2. Classification of neocortical interneurons using affinity propagation (United States)

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael


    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  3. Interactions between microbial activity and distribution and mineral coatings on sand grains from rapid sand filters treating groundwater

    DEFF Research Database (Denmark)

    Gülay, Arda; Tatari, Karolina; Musovic, Sanin

    Rapid sand filtration is a traditional and widespread technology for drinking water purification which combines biological, chemical and physical processes together. Granular media, especially sand, is a common filter material that allows several oxidized compounds to accumulate on its surface...

  4. Congophilicity (Congo red affinity) of different beta2-microglobulin conformations characterized by dye affinity capillary electrophoresis

    DEFF Research Database (Denmark)

    Heegaard, N H; Sen, J W; Nissen, Mogens Holst


    -affinities of native and abnormally folded beta2-microglobulin. We find that native beta2-microglobulin has an intermediate affinity for Congo red at pH 7.3 and that binding involves electrostatic interactions. The conformational variant of beta2-microglobulin that appears in acetonitrile solutions binds Congo red...

  5. Relationship between intracellular Na+ concentration and reduced Na+ affinity in Na+,K+-ATPase mutants causing neurological disease

    DEFF Research Database (Denmark)

    Toustrup-Jensen, Mads Schak; Einholm, Anja P.; Schack, Vivien


    The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na+,K+-ATPase α2- and α3-isoforms, expressed in glial and neuronal cells, respectively. Although these disorders...... mutations that increase Na+ affinity were found to reduce [Na+]i. It is concluded that the Na+ affinity of the Na+,K+-ATPase is an important determinant of [Na+]i....

  6. Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence. (United States)

    Gromek, Katarzyna A; Meddaugh, Hannah R; Wrobel, Russell L; Suchy, Fabian P; Bingman, Craig A; Primm, John G; Fox, Brian G


    Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis. (United States)

    Sugimoto, S; Iwase, T; Sato, F; Tajima, A; Shinji, H; Mizunoe, Y


    Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression-secretion system. The esp gene was fused with the N-terminal Sec-dependent signal sequence of the B. choshinensis cell wall protein and a C-terminal hexa-histidine-tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity. The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20-ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1-l culture). Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  8. Expression Optimizing and Purification of Recombinant Human Leukemia Inhibitory Factor Produced in E. coli Strain BL21

    Directory of Open Access Journals (Sweden)

    Houman Kahroba


    Full Text Available Background: Leukemia inhibitory factor (LIF is a glycoprotein, categorized as a subfamily of interleukin 6 cytokines which is known in many mammolals. A pluripotent cytokine with a wide biological function range has numerous effects on target cells. The LIF regulates neuron survival, hematopoiesis and seen in LIF-/- knockout mice affects blastocyst implantation, also acts as pre-inflammolatory cytokine, and regulates immolune response. Further, it is able to maintain stem cells poly potency. The main object of present work was expression, optimizing, and purification of recombinant human leukemia inhibitory factor (rhLIF. Materials and Methods: In this experimental study, Pet28 (+ carrying the LIF gene and kanamycin resistance marker was cloned in E. coli strain BL21. The induction was optimized by altering 3 factors including the temperature, the induction time, and the concentration of the Isopropyl β-D-1-thiogalactopyranoside (IPTG as inducer. The purification of the recombinant human LIF (rhLIF was done by single step affinity chromatography. After the purification, method accuracy was proved by Sodium dodecyl sulfate (SDS -PAGE electrophoresis and Western blotting. Results: Optimizing of the expression was reached by changing various parameters, and purification has been done successful. Conclusion: rhLIF undergoes modification by glycosylation to get its full functionality. The produced rhLIF in prokaryotic host in this work is lacking of glycosylation. However, its proper function should be evaluated in further studies.

  9. Affine coherent states and Toeplitz operators (United States)

    Hutníková, Mária; Hutník, Ondrej


    We study a parameterized family of Toeplitz operators in the context of affine coherent states based on the Calderón reproducing formula (= resolution of unity on L_2( {R})) and the specific admissible wavelets (= affine coherent states in L_2( {R})) related to Laguerre functions. Symbols of such Calderón-Toeplitz operators as individual coordinates of the affine group (= upper half-plane with the hyperbolic geometry) are considered. In this case, a certain class of pseudo-differential operators, their properties and their operator algebras are investigated. As a result of this study, the Fredholm symbol algebras of the Calderón-Toeplitz operator algebras for these particular cases of symbols are described. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’.

  10. On Affine Fusion and the Phase Model

    Directory of Open Access Journals (Sweden)

    Mark A. Walton


    Full Text Available A brief review is given of the integrable realization of affine fusion discovered recently by Korff and Stroppel. They showed that the affine fusion of the su(n Wess-Zumino-Novikov-Witten (WZNW conformal field theories appears in a simple integrable system known as the phase model. The Yang-Baxter equation leads to the construction of commuting operators as Schur polynomials, with noncommuting hopping operators as arguments. The algebraic Bethe ansatz diagonalizes them, revealing a connection to the modular S matrix and fusion of the su(n WZNW model. The noncommutative Schur polynomials play roles similar to those of the primary field operators in the corresponding WZNW model. In particular, their 3-point functions are the su(n fusion multiplicities. We show here how the new phase model realization of affine fusion makes obvious the existence of threshold levels, and how it accommodates higher-genus fusion.

  11. Photocatalytic materials and technologies for air purification. (United States)

    Ren, Hangjuan; Koshy, Pramod; Chen, Wen-Fan; Qi, Shaohua; Sorrell, Charles Christopher


    Since there is increasing concern for the impact of air quality on human health, the present work surveys the materials and technologies for air purification using photocatalytic materials. The coverage includes (1) current photocatalytic materials for the decomposition of chemical contaminants and disinfection of pathogens present in air and (2) photocatalytic air purification systems that are used currently and under development. The present work focuses on five main themes. First, the mechanisms of photodegradation and photodisinfection are explained. Second, system designs for photocatalytic air purification are surveyed. Third, the photocatalytic materials used for air purification and their characteristics are considered, including both conventional and more recently developed photocatalysts. Fourth, the methods used to fabricate these materials are discussed. Fifth, the most significant coverage is devoted to materials design strategies aimed at improving the performance of photocatalysts for air purification. The review concludes with a brief consideration of promising future directions for materials research in photocatalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Purification and Some Biological Properties of Asparaginase from Azotobacter vinelandii (United States)

    Gaffar, S. A.; Shethna, Y. I.


    Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37°C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48°C. The approximate molecular weight was 84,000. The apparent Km value for the substrate was 1.1 × 10-4 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu2+, Zn2+, and Hg2+ showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells. Images PMID:16345199

  13. Extraction purification and characterization of trypsin inhibitors from Andean seeds

    Directory of Open Access Journals (Sweden)

    Patricio Castillo


    Full Text Available This work established the conditions of covalent immobilization of trypsin on a Sepharose matrix, which could be applied for the purification of trypsin inhibitors. The higher values of retention of enzymatic activity and immobilized enzymatic activity were obtained with a Sepharose 6B-CL matrix, at room temperature, a pH value of 10.5, an enzymatic load of 25 mg/mL, and a minimum immobilization time of 12 hours, in order to obtain a stable immobilization. The most active trypsin inhibitors were selected through the comparison of, extracts obtained from the seeds of amaranth (Amaranthus caudatus L., pea (Pisum sativum, lupine or “chocho” (Lupinus mutabilis, bean (Phaseolus vulgaris and “sangorache” (Amaranthus hybridus L.. The inhibitors were partially purified using centrifugal ultrafiltration, heat treatment, and TCA precipitation. The permeated and retained fractions of “sangorache” were selected as the most active trypsin inhibitors, and they were selectively purified using affinity chromatography in a Trypsin - Glyoxyl - Sepharose 6B-CL matrix. The kinetic characterization showed the presence of two inhibitors; the first one corresponded to a competitive inhibitor, while the second one behaved as a mixed inhibitor.

  14. Simplified riboprobe purification using translucent straws as gel tubes. (United States)

    Kol, S; Ben-Shlomo, I; Adashi, E Y; Rohan, R M


    Gel purification of radioactive riboprobes enhances the quality of the ribonuclease protection assay. A simple and effective method for riboprobe purification is described. The method uses acrylamide gels in plastic tubes to achieve electrophoretic separation of the RNA polymerase products.

  15. Ionic behavior of treated water at a water purification plant


    Yanagida, Kazumi; Kawahigashi, Tatsuo


    [Abstract] Water at each processing stage in a water purification plant was extracted and analyzed to investigate changes of water quality. Investigations of water at each processing stage at the water purification plant are discussed herein.

  16. Cation affinity numbers of Lewis bases

    Directory of Open Access Journals (Sweden)

    Christoph Lindner


    Full Text Available Using selected theoretical methods the affinity of a large range of Lewis bases towards model cations has been quantified. The range of model cations includes the methyl cation as the smallest carbon-centered electrophile, the benzhydryl and trityl cations as models for electrophilic substrates encountered in Lewis base-catalyzed synthetic procedures, and the acetyl cation as a substrate model for acyl-transfer reactions. Affinities towards these cationic electrophiles are complemented by data for Lewis-base addition to Michael acceptors as prototypical neutral electrophiles.

  17. Control and estimation of piecewise affine systems

    CERN Document Server

    Xu, Jun


    As a powerful tool to study nonlinear systems and hybrid systems, piecewise affine (PWA) systems have been widely applied to mechanical systems. Control and Estimation of Piecewise Affine Systems presents several research findings relating to the control and estimation of PWA systems in one unified view. Chapters in this title discuss stability results of PWA systems, using piecewise quadratic Lyapunov functions and piecewise homogeneous polynomial Lyapunov functions. Explicit necessary and sufficient conditions for the controllability and reachability of a class of PWA systems are

  18. Applications of Affine and Weyl geometry

    CERN Document Server

    García-Río, Eduardo; Nikcevic, Stana


    Pseudo-Riemannian geometry is, to a large extent, the study of the Levi-Civita connection, which is the unique torsion-free connection compatible with the metric structure. There are, however, other affine connections which arise in different contexts, such as conformal geometry, contact structures, Weyl structures, and almost Hermitian geometry. In this book, we reverse this point of view and instead associate an auxiliary pseudo-Riemannian structure of neutral signature to certain affine connections and use this correspondence to study both geometries. We examine Walker structures, Riemannia

  19. Asymptotic Representations of Quantum Affine Superalgebras (United States)

    Zhang, Huafeng


    We study representations of the quantum affine superalgebra associated with a general linear Lie superalgebra. In the spirit of Hernandez-Jimbo, we construct inductive systems of Kirillov-Reshetikhin modules based on a cyclicity result that we established previously on tensor products of these modules, and realize their inductive limits as modules over its Borel subalgebra, the so-called q-Yangian. A new generic asymptotic limit of the same inductive systems is proposed, resulting in modules over the full quantum affine superalgebra. We derive generalized Baxter's relations in the sense of Frenkel-Hernandez for representations of the full quantum group.

  20. Affine Projection Algorithm Using Regressive Estimated Error


    Zhang, Shu; Zhi, Yongfeng


    An affine projection algorithm using regressive estimated error (APA-REE) is presented in this paper. By redefining the iterated error of the affine projection algorithm (APA), a new algorithm is obtained, and it improves the adaptive filtering convergence rate. We analyze the iterated error signal and the stability for the APA-REE algorithm. The steady-state weights of the APA-REE algorithm are proved to be unbiased and consist. The simulation results show that the proposed algorithm has a f...

  1. Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin (United States)

    Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia


    Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

  2. Purification treatment for underground water

    Energy Technology Data Exchange (ETDEWEB)

    Fonbershteyn, V.


    In order for underground water to be clean and to taste good, iron can be removed from it right underground, in the water-bearing stratum, before it is brought to the surface. G.M. Kommunar, V.S. Alekseyev, and V.T. Grebennikov, candidates of technical sciences and associates of the Moscow All-Union Hydrogeology Scientific Research Institute, developed the practical application of this beneficial technology, which makes it possible to do away with purification installations. With the new technology (Patent No. 985 214, 1 018 918) water saturated with oxygen is sent through an ejector and then pumped into a well. It passes through rocks that serve as a natural filter, and the filter is loaded with oxygen. The filter now becomes a barrier for mineral impurities contained in the artesian water. The amount of time needed to pump the oxidized water into the well is calculated beforehand, knowing the capacity of the water-bearing stratum, the porosity of the rocks, the expenditure of pumped oxidized water, and the radius of the zone of the filtering rocks. While the water is pumped out of the well, its properties are monitored periodically. If the concentration of iron exceeds the allowable norm-0.3 mg per liter-the extraction is halted, and oxidized water is once again pumped into the well. It is convenient and economical to combine several wells into one system, where each well will pump and accept water according to its own schedule. This new technology can also be used to remove manganese, heavy metals, and hydrogen sulfide from underground water.

  3. Analysis of Protein Target Interactions of Synthetic Mixtures by Affinity-LC/MS. (United States)

    Singh, Prachi; Madhaiyan, Kalaipriya; Duong-Thi, Minh-Dao; Dymock, Brian W; Ohlson, Sten


    Analysis of interactions between molecules is of fundamental importance in life science research. In this study, we applied weak affinity chromatography, based on high-performance liquid chromatography and mass spectrometry, as a powerful tool for direct analysis of the components of a chemical reaction mixture for their binding to a target protein. As a demonstration of the potential of this method, we analyzed the binding of the compounds of the reaction mixture to the chaperone heat shock protein 90 (Hsp90). It was possible to analyze quantitatively the binding of the components of the mixture to the target independently from each other without any preceding process such as purification. This feature has wide implications in biological sciences as crude mixtures, either natural or synthetic, can be analyzed directly for their possible binding to a target. This method could lead to savings in costs and labor through shortening chemical research project development time.

  4. Phospholipase C activity affinity purifies with the Torpedo nicotinic acetylcholine receptor. (United States)

    Labriola, Jonathan M; daCosta, Corrie J B; Wang, Shuzhi; Figeys, Daniel; Smith, Jeffrey C; Sturgeon, R Michel; Baenziger, John E


    Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function.

  5. Purification of foot-and-mouth disease virus by heparin as ligand for certain strains. (United States)

    Du, Ping; Sun, Shiqi; Dong, Jinjie; Zhi, Xiaoying; Chang, Yanyan; Teng, Zhidong; Guo, Huichen; Liu, Zaixin


    The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification. Copyright © 2016. Published by Elsevier B.V.

  6. Comparison of Methods for the Purification of Alpha-1 Acid Glycoprotein from Human Plasma

    Directory of Open Access Journals (Sweden)

    Teresa R. McCurdy


    Full Text Available Alpha-1 acid glycoprotein (AGP is a highly glycosylated, negatively charged plasma protein suggested to have anti-inflammatory and/or immunomodulatory activities. Purification of AGP could be simplified if methods that exploit its high solubility under chemically harsh conditions could be demonstrated to leave the protein in its native conformation. Procedures involving exposure of AGP to hot phenol or sulphosalicylic acid (SSA were compared to solely chromatographic methods. Hot phenol-purified AGP was more rapidly cleared from mice in vivo following intravenous injection than chromatographically purified AGP. In contrast, SSA-purified AGP demonstrated an identical in vivo clearance profile and circular dichroism spectrum to chromatographically purified AGP. Similarly, no differences in susceptibility to enzymatic deglycosylation or reactivity with Sambucus nigra lectin were detected between AGP purified via the two methods. Incorporation of the SSA step in the purification scheme for AGP eliminated the need for a large (4 mL resin/mL of plasma initial chromatographic step and simplified its purification without causing any detectable distortion in the conformation of the protein. Confirmation that this procedure is nondenaturing will simplify AGP purification and investigation of its possible biological roles in laboratory animals.

  7. Crossing Chris: Some Markerian Affinities

    Directory of Open Access Journals (Sweden)

    Adrian Martin


    -pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

    Abstract (E: This essay creatively explores a group of artists, writers, and other special individuals whose work or life story can be described as having an intriguing affinity with the protean career of Chris Marker. Avoiding the ‘usual suspects’ (such as Godard or Sebald, it discusses gossip columnist Milt Machlin, record collector Harry Smith, painter Gianfranco Baruchello, writer-filmmaker Edgardo Cozarinsky, and several others. From this constellation, a particular view of Markerian poetics emerges, touching upon the meanings of anonymity, storytelling, history and archiving.


    Abstract (F: Cet essai brosse de manière créative le portrait d’un groupe d'artistes, d'écrivains et d'autres personnes particulières dont le travail ou la biographie peuvent être décrits comme montrant une étrange mais certaine connivence avec la carrière protéiforme de Chris Marker. Evitant les lieux communs (comme Godard ou Sebald, cet article trace des références moins attendues :

  8. Secondary Mechanisms of Affinity Maturation in the Human Antibody Repertoire

    Directory of Open Access Journals (Sweden)

    Bryan S. Briney


    Full Text Available V(DJ recombination and somatic hypermutation (SHM are the primary mechanisms for diversification of the human antibody repertoire. These mechanisms allow for rapid humoral immune responses to a wide range of pathogenic challenges. V(DJ recombination efficiently generate a virtually limitless diversity through random recombination of variable (V, diversity (D and joining (J genes with diverse nontemplated junctions between the selected gene segments. Following antigen stimulation, affinity maturation by SHM produces antibodies with refined specificity mediated by mutations typically focused in complementarity determining regions (CDRs, which form the bulk of the antigen recognition site. While V(DJ recombination and SHM are responsible for much of the diversity of the antibody repertoire, there are several secondary mechanisms that, while less frequent, make substantial contributions to antibody diversity including V(DDJ recombination (or D-D fusion, somatic-hypermutation-associated insertions and deletions, and affinity maturation and antigen contact by non-CDR regions of the antibody. In addition to enhanced diversity, these mechanisms allow the production of antibodies that are critical to response to a variety of viral and bacterial pathogens but that would be difficult to generate using only the primary mechanisms of diversification.

  9. 21 CFR 876.5665 - Water purification system for hemodialysis. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Water purification system for hemodialysis. 876... SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665 Water purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is a...

  10. Purification of contaminated groundwater by membrane technology

    Energy Technology Data Exchange (ETDEWEB)

    Youn, In Soo; Chung, Chin Ki; Kim, Byoung Gon [Korea Institute of Geology Mining and Materials, Taejon (Korea, Republic of)


    The objective of this study is to apply the membrane separation technology to the purification of contaminated ground water in Korea. Under this scope, the purification was aimed to the drinking water level. The scale of the membrane system was chosen to a small filtration plant for local clean water supplies and/or heavy purifiers for buildings and public uses. The actual conditions of ground water contamination in Korea was surveyed to determine the major components to remove under the drinking water requirements. To set up a hybrid process with membrane methods, conventional purification methods were also investigated for the comparison purpose. The research results are summarized as follows : 1) Contamination of the groundwater in Korea has been found to be widespread across the country. The major contaminant were nitrate, bacteria, and organic chlorides. Some solvents and heavy metals are also supposed to exist in the ground water of industrial complexes, cities, and abandoned mines. 2) The purification methods currently used in public filtration plants appear not to be enough for new contaminants from recent industrial expanding. The advanced purification technologies generally adopted for this problem have been found to be unsuitable due to their very complicated design and operation, and lack of confidence in the purification performance. 3) The reverse osmosis tested with FilmTec FT30 membrane was found to remove nitrate ions in water with over 90 % efficiency. 4) The suitable membrane process for the contaminated groundwater in Korea has been found to be the treatments composed of activated carbon, microfiltration, reverse osmosis or ultrafiltration, and disinfection. The activated carbon treatment could be omitted for the water of low organic contaminants. The microfiltration and the reverse osmosis treatments stand for the conventional methods of filtration plants and the advanced methods for hardly removable components, respectively. It is recommended

  11. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Directory of Open Access Journals (Sweden)

    Hua-zhen Wang

    Full Text Available Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP could greatly increase the soluble expression level of Glucokinase (GlcK, α-Amylase (Amy and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

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

    Directory of Open Access Journals (Sweden)

    Gorbatiuk O. B.


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

  13. Capturing enveloped viruses on affinity grids for downstream cryo-electron microscopy applications. (United States)

    Kiss, Gabriella; Chen, Xuemin; Brindley, Melinda A; Campbell, Patricia; Afonso, Claudio L; Ke, Zunlong; Holl, Jens M; Guerrero-Ferreira, Ricardo C; Byrd-Leotis, Lauren A; Steel, John; Steinhauer, David A; Plemper, Richard K; Kelly, Deborah F; Spearman, Paul W; Wright, Elizabeth R


    Electron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

  14. Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success. (United States)

    Choi, Ryan; Kelley, Angela; Leibly, David; Hewitt, Stephen Nakazawa; Napuli, Alberto; Van Voorhis, Wesley


    The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure.

  15. On the Lp affine isoperimetric inequalities

    Indian Academy of Sciences (India)

    inequality. 1. Introduction. Projection bodies have a long and complicated history which goes back to Minkowski. [3]. The classical Petty projection inequality plays a central role in the framework of the affine isoperimetric inequalities (see the survey article by Lutwak [10]). It states that [19]:. If K is a convex body in Rn, then. V(.

  16. Classification of neocortical interneurons using affinity propagation

    Directory of Open Access Journals (Sweden)

    Roberto eSantana


    Full Text Available In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. Neuronal classification has been a difficult problem because it is unclear what a neuronal cell class actually is and what are the best characteristics are to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological or molecular characteristics, when applied to selected datasets, have provided quantitative and unbiased identification of distinct neuronal subtypes. However, better and more robust classification methods are needed for increasingly complex and larger datasets. We explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. In fact, using a combined anatomical/physiological dataset, our algorithm differentiated parvalbumin from somatostatin interneurons in 49 out of 50 cases. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  17. Fan Affinity Laws from a Collision Model (United States)

    Bhattacharjee, Shayak


    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  18. Affinity monolith preconcentrators for polymer microchip capillary electrophoresis (United States)

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


    Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate-co-ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a <25,000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples. PMID:18702050

  19. High-Affinity RGD-Knottin Peptide as a New Tool for Rapid Evaluation of the Binding Strength of Unlabeled RGD-Peptides to αvβ3, αvβ5, and α5β1 Integrin Receptors. (United States)

    Bernhagen, Dominik; De Laporte, Laura; Timmerman, Peter


    We describe a highly sensitive competition ELISA to measure integrin-binding of RGD-peptides in high-throughput without using cells, ECM-proteins, or antibodies. The assay measures (nonlabeled) RGD-peptides' ability to inhibit binding of a biotinylated "knottin"-RGD peptide to surface-immobilized integrins and, thus, enables quantification of the binding strength of high-, medium-, and low-affinity RGD-binders. We introduced the biotinylated knottin-RGD peptide instead of biotinylated cyclo[RGDfK] (as reported by Piras et al.), as integrin-binding was much stronger and clearly detectable for all three integrins. In order to maximize sensitivity and cost-efficiency, we first optimized several parameters, such as integrin-immobilization levels, knottin-RGD concentration, buffer compositions, type of detection tag (biotin, His- or cMyc-tag), and spacer length. We thereby identified two key factors, that is, (i) the critical spacer length (longer than Gly) and (ii) the presence of Ca2+ and Mg2+ in all incubation and washing buffers. Binding of knottin-RGD peptide was strongest for αvβ3 but also detectable for both αvβ5 and α5β1, while binding of biotinylated cyclo[RGDfK] was very weak and only detectable for αvβ3. For assay validation, we finally determined IC50 values for three unlabeled peptides, that is: (i) linear GRGDS, (ii) cyclo[RGDfK], and (iii) the knottin-RGD itself for binding to three different integrin receptors (αvβ3, αvβ5, α5β1). Major benefits of the novel assay are (i) the extremely low consumption of integrin (50 ng/peptide), (ii) the fact that neither antibodies/ECM-proteins nor integrin-expressing cells are required for detection, and (iii) its suitability for high-throughput screening of (RGD-)peptide libraries.

  20. Chemical looping integration with a carbon dioxide gas purification unit (United States)

    Andrus, Jr., Herbert E.; Jukkola, Glen D.; Thibeault, Paul R.; Liljedahl, Gregory N.


    A chemical looping system that contains an oxidizer and a reducer is in fluid communication with a gas purification unit. The gas purification unit has at least one compressor, at least one dryer; and at least one distillation purification system; where the gas purification unit is operative to separate carbon dioxide from other contaminants present in the flue gas stream; and where the gas purification unit is operative to recycle the contaminants to the chemical looping system in the form of a vent gas that provides lift for reactants in the reducer.

  1. Production, purification and characterization of antibodies to 1,25-dihydroxyvitamin D raised in chicken egg yolk. (United States)

    Bauwens, R M; Kint, J A; Devos, M P; Van Brussel, K A; De Leenheer, A P


    For this sensitive RIA for 1 alpha,25-dihydroxyvitamin D, we used antibodies to 1 alpha,25-dihydroxycholecalciferol-3-hemisuccinate conjugated to bovine serum albumin, raised in eggs by immunization of chickens. We describe an efficient method for purification of IgG from egg yolk. We characterized these antibodies with immunoelectrophoresis and by radioimmunoassay. These antibodies show a high affinity for 1 alpha,25-dihydroxyvitamin D3 but cross react with other vitamin D metabolites as well. Extraction and liquid chromatography are necessary to isolate the 1 alpha,25-dihydroxyvitamin D from human serum or plasma before determination by RIA. The sensitivity of the assay is estimated at 5 pg/tube.

  2. Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization. (United States)

    Subramanian, N; Adiga, P R


    Chicken egg yolk biotin-binding protein-I (BBP-I) has been purified to homogeneity along with the tetrameric BBP-II by a common protocol. The purification includes delipidation of egg yolk by butanol extraction, DEAE-Sephacel chromatography, treatment with guanidinium chloride and biotin-aminohexyl-Sepharose affinity chromatography. The identity of purified BBP-I was ascertained by its physicochemical properties as well as by its immunological cross-reactivity and precursor-product relationship with BBP-II.

  3. Radioiododestannylation. Convenient synthesis of a stable penicillin derivative for rapid penicillin binding protein (PBP) assay

    Energy Technology Data Exchange (ETDEWEB)

    Blaszczak, L.C.; Halligan, N.G. (Lilly (Eli) and Co., Indianapolis, IN (USA). Lilly Research Labs.); Seitz, D.E. (Indiana Univ.-Purdue Univ., Indianapolis, IN (USA). School of Medicine)


    Radioiodination of p-(trimethylstannyl)penicillin V with ({sup 125}I)Na using a modification of the chloramine-T method is simple, high yielding, and site-specific. The structure and penicillin binding protein (PBP) affinity of p-({sup 125}I)-penicillin V (IPV) are similar to penicillin G and the product can be used directly without purification in the PBP assay. Because of the high degree of stability toward autoradiolysis and equivalent PBP binding affinity, IPV can be used in place of ({sup 3}H)-penicillin G or ({sup 14}C)-penicillin G for these experiments. (author).

  4. Purification of tantalum by plasma arc melting (United States)

    Dunn, Paul S.; Korzekwa, Deniece R.


    Purification of tantalum by plasma arc melting. The level of oxygen and carbon impurities in tantalum was reduced by plasma arc melting the tantalum using a flowing plasma gas generated from a gas mixture of helium and hydrogen. The flowing plasma gases of the present invention were found to be superior to other known flowing plasma gases used for this purpose.

  5. Purification And Characterization Of Marine Bacillus Thuringiensis ...

    African Journals Online (AJOL)

    Urease was purified to homogeneity from Bacillus thuringiensis N2 using different purification steps namely, 55% acetone precipitation, DEAE-Sephadex A50 anion exchange column and Sephadex G120-200 gel filtration chromatography. The enzyme was purified 95.27 fold and showed a final specific activity of 10.48 ...

  6. Purification and biochemical properties of carboxylesterase from ...

    African Journals Online (AJOL)

    The order of inhibition on EII was β- mercaptoethanol > PMSF > DTNB > PCMB > iodoacetate. While the order of inhibition on EIII was β-mercaptoethanol > iodoacetate > DTNB> PCM > PMSF. Keywords: Carboxylesterase, Fasciola gigantica; Purification; Characterization; p-Nitrophenyl; α-Naphthyl; β-Naphthyl; Esters ...

  7. Isolation, partial purification and characterization of antifungal ...

    African Journals Online (AJOL)



    Jul 16, 2014 ... Full Length Research Paper. Isolation, partial purification and characterization of .... dried seeds were milled with coffee grinder and stored in air tight bags until required. Fifty grams of the matured ... was washed with 2.5% Triton-X 100 (3 × 20 min) with continual shaking to remove SDS followed by washing ...

  8. Isolation, purification and effects of hypoglycemic functional ...

    African Journals Online (AJOL)

    Isolation, purification and effects of hypoglycemic functional polysaccharides from Inonotus obliquus. Tao Hu, Ping Liu, Yuanying Ni, Chuntao Lu. Abstract. Inonotus obliquus is generally used for the treatment of diseases such as cancers, angiocardiopathy and diabetes. However, few studies are available on its functional ...

  9. Partial purification and biochemical characterization of acid ...

    African Journals Online (AJOL)


    Mung bean (Vigna radiata) is one of the important crops of the North Eastern Region of India. In the present study, acid phosphatase enzyme was isolated and partially purified from germinated local mung bean seeds. The sequential partial purification process was performed using ammonium sulphate precipitation method.

  10. Expression and Purification of Sperm Whale Myoglobin (United States)

    Miller, Stephen; Indivero, Virginia; Burkhard, Caroline


    We present a multiweek laboratory exercise that exposes students to the fundamental techniques of bacterial expression and protein purification through the preparation of sperm whale myoglobin. Myoglobin, a robust oxygen-binding protein, contains a single heme that gives the protein a reddish color, making it an ideal subject for the teaching…

  11. Partial purification and characterization of polygalacturonase ...

    African Journals Online (AJOL)



    Apr 3, 2012 ... purification of pearl millet [Pennisetum glaucum (L.) R.Br.] protein extract by cation exchange chromatography, which ... electrophoresis showed prominent bands between 29 and 43 kDa, consistent with the molecular weights of the ... EPGs (De Lorenzo et al., 2001; Federici et al., 2006), but are shown to be ...

  12. Purification and characterization of xylanase from Aspergillus ...

    African Journals Online (AJOL)

    Xylanase was subjected to a three-step purification scheme involving ammonium sulphate precipitation, gel filtration chromatography and anion exchange chromatography. Purity was verified by running the extracted protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single band was ...

  13. Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins. (United States)

    Blakeley, D; Sykes, D A; Ensor, P; Bertran, E; Aston, P J; Charlton, S J


    Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes. © 2015 The British Pharmacological Society.

  14. Affine Non-Local Means Image Denoising. (United States)

    Fedorov, Vadim; Ballester, Coloma


    This paper presents an extension of the Non-Local Means denoising method, that effectively exploits the affine invariant self-similarities present in the images of real scenes. Our method provides a better image denoising result by grounding on the fact that in many occasions similar patches exist in the image but have undergone a transformation. The proposal uses an affine invariant patch similarity measure that performs an appropriate patch comparison by automatically and intrinsically adapting the size and shape of the patches. As a result, more similar patches are found and appropriately used. We show that this image denoising method achieves top-tier performance in terms of PSNR, outperforming consistently the results of the regular Non-Local Means, and that it provides state-of-the-art qualitative results.

  15. Enhanced binding by dextran-grafting to Protein A affinity chromatographic media. (United States)

    Zhao, Lan; Zhu, Kai; Huang, Yongdong; Li, Qiang; Li, Xiunan; Zhang, Rongyue; Su, Zhiguo; Wang, Qibao; Ma, Guanghui


    Dextran-grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose-based matrix, followed by epoxy-activation and Protein A coupling site-directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran-grafted Protein A chromatographic medium. Its dynamic binding capacity was 61 mg immunoglobulin G/mL suction-dried gel, increased by 24% compared with that of the non-grafted medium. The binding capacity of dextran-grafted medium decreased about 7% after 40 cleaning-in-place cycles, much lower than that of the non-grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran-grafted medium faster than that of non-grafted one. Atomic force microscopy showed that this dextran-grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80 nm, while that of non-grafted one was ±10 nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high-performance dextran-grafted Protein A affinity chromatographic medium has promising applications in large-scale antibody purification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Staircase Models from Affine Toda Field Theory

    CERN Document Server

    Dorey, P; Dorey, Patrick; Ravanini, Francesco


    We propose a class of purely elastic scattering theories generalising the staircase model of Al. B. Zamolodchikov, based on the affine Toda field theories for simply-laced Lie algebras g=A,D,E at suitable complex values of their coupling constants. Considering their Thermodynamic Bethe Ansatz equations, we give analytic arguments in support of a conjectured renormalisation group flow visiting the neighbourhood of each W_g minimal model in turn.

  17. Excited state electron affinity calculations for aluminum (United States)

    Hussein, Adnan Yousif


    Excited states of negative aluminum ion are reviewed, and calculations of electron affinities of the states (3s^23p^2)^1D and (3s3p^3){^5}{S}° relative to the (3s^23p)^2P° and (3s3p^2)^4P respectively of the neutral aluminum atom are reported in the framework of nonrelativistic configuration interaction (CI) method. A priori selected CI (SCI) with truncation energy error (Bunge in J Chem Phys 125:014107, 2006) and CI by parts (Bunge and Carbó-Dorca in J Chem Phys 125:014108, 2006) are used to approximate the valence nonrelativistic energy. Systematic studies of convergence of electron affinity with respect to the CI excitation level are reported. The calculated value of the electron affinity for ^1D state is 78.675(3) meV. Detailed Calculations on the ^5S°c state reveals that is 1216.8166(3) meV below the ^4P state.

  18. Absolute proton affinity of some polyguanides (United States)

    Maksic; Kovacevic


    The problem of the absolute proton affinity (APA) of some polyguanides is addressed by the MP2(fc)/6-311+G//HF/6-31G theoretical model. It is shown that the linear chain polyguanides exhibit increased basicity as a function of the number of guanide subunits. However, the saturation effect yields an asymptotic APA value of 254 kcal/mol. Branched polyguanides on the other hand have higher APAs than their linear counterparts. The largest proton affinity is found in a doubly bifurcated heptaguanide, being as high as 285 kcal/mol, thus potentially representing one of the strongest organic bases. Finally, it is found that all polyguanides protonate at imino nitrogen atoms, since they are apparently susceptible the most to the proton attack. The origin of their very high intrinsic basicity is traced down to a dramatic increase in the resonance interaction of the corresponding conjugate bases. For instance, the increase in the resonance energy in the protonated guanidine is estimated to be in a range of 24-27 kcal/mol, which is higher than the aromatic stabilization in benzene. The proton affinity of some polycyclic guanides including Schwesinger proton sponge and porphine is briefly discussed.

  19. 2D Affine and Projective Shape Analysis. (United States)

    Bryner, Darshan; Klassen, Eric; Huiling Le; Srivastava, Anuj


    Current techniques for shape analysis tend to seek invariance to similarity transformations (rotation, translation, and scale), but certain imaging situations require invariance to larger groups, such as affine or projective groups. Here we present a general Riemannian framework for shape analysis of planar objects where metrics and related quantities are invariant to affine and projective groups. Highlighting two possibilities for representing object boundaries-ordered points (or landmarks) and parameterized curves-we study different combinations of these representations (points and curves) and transformations (affine and projective). Specifically, we provide solutions to three out of four situations and develop algorithms for computing geodesics and intrinsic sample statistics, leading up to Gaussian-type statistical models, and classifying test shapes using such models learned from training data. In the case of parameterized curves, we also achieve the desired goal of invariance to re-parameterizations. The geodesics are constructed by particularizing the path-straightening algorithm to geometries of current manifolds and are used, in turn, to compute shape statistics and Gaussian-type shape models. We demonstrate these ideas using a number of examples from shape and activity recognition.

  20. Production and Purification of a Polyclonal Antibody Against Purified Mouse IgG2b in Rabbits Towards Designing Mouse Monoclonal Isotyping Kits

    Directory of Open Access Journals (Sweden)

    Sadeq Eivazi


    Full Text Available Purpose: Mouse IgG subclasses containing IgG1, IgG2a, IgG2b and IgG3 have been defined and described both physiochemically and immunologically. Methods: Sepharose beads conjugated with protein A affinity chromatography was used for purification of mouse IgG2b. Sodium citrate buffer (0.1 M, pH: 3.5 was used for separation of mouse IgG2b. Verification of the purified fractions was monitored by SDS-PAGE (polyacrylamide gel electrophoresis in reducing condition. Immunized rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. After dialysis against tris-phosphate buffer (pH: 8.1 ion exchange chromatography column was used for purification of rabbit anti-mouse IgG2b. The periodate method was performed for conjugation with some variations. After conjugation, direct ELISA was used to determine the titer of HRP conjugated rabbit IgG against mouse IgG2b. Results: The titer of rabbit anti-mouse IgG2b that determined by ELISA was 32000. The purity of rabbit anti-mouse IgG2b was about 95%. The optimum dilution of prepared HRP conjugated IgG was 1:10000. This study showed that ion-exchange chromatography and affinity chromatography could be appropriate techniques for purification of mouse IgG and IgG subclasses respectively. Conclusion: This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies.

  1. Total chemical synthesis of proteins without HPLC purification. (United States)

    Loibl, S F; Harpaz, Z; Zitterbart, R; Seitz, O


    The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2-6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins

  2. A robust robotic high-throughput antibody purification platform. (United States)

    Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J


    Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Purification and Characterization of AsES Protein (United States)

    Chalfoun, Nadia R.; Grellet-Bournonville, Carlos F.; Martínez-Zamora, Martín G.; Díaz-Perales, Araceli; Castagnaro, Atilio P.; Díaz-Ricci, Juan C.


    In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2˙̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity. PMID:23530047

  4. Metal-conjugated affinity labels: A new concept to create enantioselective artificial metalloenzymes

    KAUST Repository

    Reiner, Thomas


    How to train a protein: Metal-conjugated affinity labels were used to selectively position catalytically active metal centers in the binding pocket of proteases. The resulting artificial metalloenzymes achieve up to 82% e.r. in the hydrogenation of ketones. The modular setup enables a rapid generation of artificial metalloenzyme libraries, which can be adapted to a broad range of catalytic conditions. 2013 The Authors.

  5. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.


    Milton, N G


    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

  6. Polyethyleneimine-grafted boronate affinity materials for selective enrichment of cis-diol-containing compounds. (United States)

    Xue, Yun; Shi, Wenjun; Zhu, Bangjie; Gu, Xue; Wang, Yan; Yan, Chao


    Polyethyleneimine (PEI)-grafted and 3-acrylamidophenylboronic acid (AAPBA)-functionalized SiO2 boronate affinity materials were synthesized for the selective enrichment of cis-diol-containing compounds. Characterization results of scanning electron microscopy, Fourier transform infrared spectroscopy, elemental analysis, zeta potential, and X-ray photoelectron spectroscopy indicated the successful fabrication of SiO2@PEI-AAPBA materials. Chromatographic separation of test mixtures reveals that SiO2@PEI-AAPBA has high selective enrichment ability for cis-diol-containing compounds. The binding pH between SiO2@PEI-AAPBA and catechol was found to be as low as pH 4.5, while that between SiO2@PEI-AAPBA and adenosine was only ~7.5. This difference might be attributed to the strong electrostatic repulsion between the solid phase and analytes at a low pH. Furthermore, a diphasic separation column was fabricated based on boronate affinity chromatography, C18-reversed-phase chromatography and applied in pressurized capillary electrochromatography (pCEC). Results showed that four polar nucleosides could be well captured by the boronate affinity chromatography (BAC) section and separated by reversed phase pCEC. Finally, SiO2@PEI600-AAPBA-based solid-phase extraction technology was applied to the purification of ribonucleosides in real urine samples, and results of UHPLC-MS/MS revealed that the intensities of the extracted ions (a neutral mass loss of m/z 132.04 Da) of the ribonucleosides were significantly enhanced after the enrichment. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Novel peptide VIP-TAT with higher affinity for PAC1 inhibited scopolamine induced amnesia. (United States)

    Yu, Rongjie; Yang, Yanxu; Cui, Zekai; Zheng, Lijun; Zeng, Zhixing; Zhang, Huahua


    A novel peptide VIP-TAT with a cell penetrating peptide TAT at the C-terminus of VIP was constructed and prepared using intein mediated purification with an affinity chitin-binding tag (IMPACT) system to enhance the brain uptake efficiency for the medical application in central nervous system. It was found by labeling VIP-TAT and VIP with fluorescein isothiocyanate (FITC) that the extension with TAT increased the brain uptake efficiency of VIP-TAT significantly. Then short-term and long-term treatment with scopolamine (Scop) was used to evaluate the effect of VIP-TAT or VIP on Scop induced amnesia. Both short-term and long-term administration of VIP-TAT inhibited the latent time reduction in step-through test induced by Scop significantly, but long-term administration of VIP aggravated the Scop induced amnesia. Long-term i.p. injection of VIP-TAT was shown to have positive effect by inhibiting the oxidative damage, apoptosis and the cholinergic system activity reduction that induced by Scop, while VIP exerted negative effect in brain opposite to that in periphery system. The in vitro data showed that VIP-TAT had not only protective but also proliferative effect on Neuro2a cells which was inhibited by PAC1 antagonist PACAP(6-38). Competition binding assay and cAMP assay confirmed that VIP-TAT had higher affinity and activation for PAC1 than VIP. So it was concluded that the significantly stronger protective effect of VIP-TAT against Scop induced amnesia than VIP was due to (1) the enhanced brain uptake efficiency of VIP-TAT and (2) the increased affinity and activation of VIP-TAT for receptor PAC1. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Adsorption of endotoxins on Ca2+ -iminodiacetic acid by metal ion affinity chromatography. (United States)

    Lopes, André Moreni; Romeu, Jorge Sánchez; Meireles, Rolando Páez; Perera, Gabriel Marquez; Morales, Rolando Perdomo; Pessoa, Adalberto; Cárdenas, Lourdes Zumalacárregui


    Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by-products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ -iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+ was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than 100 EU/mL and 100 000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.

  9. Affinity, Collaboration, and the Politics of Classroom Speaking. (United States)

    Hotelling, Kirstin; Schulteis, Alexandra


    Discusses using Donna Haraway's concept of affinity and affinity politics as a foundation for structuring collaborative pedagogy and feminist syllabi. Outlines the goals and assumptions of affinity-based pedagogy, and relates classroom experiences that illustrate its functioning. Notes the lessons that both students and teachers have taken away…

  10. Duals of Affine Grassmann Codes and Their Relatives

    DEFF Research Database (Denmark)

    Beelen, P.; Ghorpade, S. R.; Hoholdt, T.


    Affine Grassmann codes are a variant of generalized Reed-Muller codes and are closely related to Grassmann codes. These codes were introduced in a recent work by Beelen Here, we consider, more generally, affine Grassmann codes of a given level. We explicitly determine the dual of an affine Grassm...

  11. Affine fractal functions as bases of continuous funtions | Navascues ...

    African Journals Online (AJOL)

    The objective of the present paper is the study of affine transformations of the plane, which provide self-affine curves as attractors. The properties of these curves depend decisively of the coefficients of the system of affinities involved. The corresponding functions are continuous on a compact interval. If the scale factors are ...

  12. Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification (United States)

    Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.


    In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media

  13. Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

    Directory of Open Access Journals (Sweden)

    Kathryn E. Tiller


    Full Text Available The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease with the greatest gains in affinity (>5-fold have several (four to six CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability while other mutations enhance some of these properties (e.g., increased specificity and display trade-offs in others (e.g., reduced affinity and/or stability. Computational modeling reveals that improvements in affinity

  14. Purification and characterization of avian beta-defensin 11, an antimicrobial peptide of the hen egg. (United States)

    Hervé-Grépinet, Virginie; Réhault-Godbert, Sophie; Labas, Valérie; Magallon, Thierry; Derache, Chrystelle; Lavergne, Marion; Gautron, Joël; Lalmanach, Anne-Christine; Nys, Yves


    Natural antimicrobial peptides are present in different compartments (eggshell, egg white, and vitelline membranes) of the hen egg and are expected to be involved in the protection of the embryo during its development and to contribute to the production of pathogen-free eggs. In the present study, we used vitelline membranes from hen (Gallus gallus) eggs as a source of avian β-defensin 11 (AvBD11). A purification scheme using affinity chromatography and reverse-phase chromatography was developed. Purified AvBD11 was analyzed by a combination of mass spectrometry approaches to characterize its primary sequence and structure. A monoisotopic molecular species at [M + H](+) of 9,271.56 Da was obtained, and its N- and C-terminal sequences were determined. We also examined posttranslational modifications and identified the presence of 6 internal disulfide bonds. AvBD11 was found to exhibit antimicrobial activity toward both Gram-positive and Gram-negative bacteria.

  15. Purification and characterization of recombinant baculovirus-expressed mouse DNA methyltransferase. (United States)

    Glickman, J F; Flynn, J; Reich, N O


    DNA methylation is essential for normal embryonic development in mice. An understanding of how DNA methylation is controlled is largely dependent upon the isolation and characterization of the cellular components of the DNA methylation system. The enzyme which methylates DNA in eukaryotic cells is a C-5 cytosine DNA methyltransferase. Historically, the characterization of this enzyme has been limited by its availability and purity. Here, we present a single-step purification of 4 mg of baculovirus-expressed mouse DNA methyltransferase containing a nickel-affinity leader peptide. The recombinant DNA methyltransferase copurified with inhibitory RNA which was removed by treatment with ribonuclease A. Like its non-recombinant counterpart, the recombinant enzyme is activated by hemi-methylation. A direct steady-state kinetic comparison between the recombinant baculovirus-expressed enzyme with its MEL cell-derived counterpart is presented.

  16. Expression and Purification of Haemophilus influenzae Rhomboid Intramembrane Protease GlpG for Structural Studies. (United States)

    Panwar, Pankaj; Lemieux, M Joanne


    Rhomboid proteases are membrane-embedded proteases that cleave peptide bonds of transmembrane proteins. They play a variety of roles in cell signaling events. The rhomboid protease GlpG from Haemophilus influenzae (hiGlpG) is a canonical form of rhomboid protease having six transmembrane segments. In this unit, detailed protocols are presented for optimization of hiGlpG expression using the araBAD promotor system in the pBAD vector. The parameters for optimization include concentration of inducing agent, induction temperature, and time. Optimization of these key factors led to the development of a protocol yielding 1.6 to 2.5 mg/liter protein purified after ion metal affinity chromatography (IMAC). Further purification can include size exclusion chromatography (SEC). Copyright © 2014 John Wiley & Sons, Inc.


    Directory of Open Access Journals (Sweden)

    Dechechi E.C.


    Full Text Available A dynamic mathematical model, simulation and computer control of a Continuous Affinity Recycle Extraction (CARE process, a protein purification technique based on protein adsorption on solid-phase adsorbents is described in this work. This process, consisting of three reactors, is a multivariable process with considerable time delay in the on-line analyses of the controlled variable. An advanced predictive control configuration, specifically the Dynamic Matrix Control (DMC, was applied. The DMC algorithm was applied in process schemes where the aim was to maintain constant the enzyme concentration in the outlet of the third reactor. The performance of the DMC controller was analyzed in the feed-flow disturbances and the results are presented.

  18. Biotin-conjugated fusogenic liposomes for high-quality cell purification. (United States)

    Hersch, Nils; Wolters, Benjamin; Ungvari, Zoltan; Gautam, Tripti; Deshpande, Dhruva; Merkel, Rudolf; Csiszar, Anna; Hoffmann, Bernd; Csiszár, Agnes


    Purification of defined cell populations from mixed primary cell sources is essential for many biomedical and biotechnological applications but often very difficult to accomplish due to missing specific surface markers. In this study, we developed a new approach for efficient cell population separation based on the specific membrane fusion characteristics of distinct cell types upon treatment with fusogenic liposomes. When such liposomes are conjugated with biotin, specific cell populations can be efficiently surface functionalized by biotin after liposomal treatment while other populations remain unlabeled. Due to the high affinity of biotin for avidin-like proteins, biotin functionalized cells are ideal targets for conjugation of e.g. avidin tagged magnetic beads, fluorophores or antibodies with bioanalytical relevance. Here, based on the differential biotinylation of distinct cell populations high quality separation of cardiac fibroblasts from myocytes, and cerebromicrovascular endothelial cells from fibroblasts was successfully established. © The Author(s) 2015.

  19. Antihemorrhagin in the blood serum of king cobra (Ophiophagus hannah): purification and characterization. (United States)

    Chanhome, Lawan; Khow, Orawan; Omori-Satoh, Tamotsu; Sitprija, Visith


    King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS-PAGE. Its apparent molecular weight determined by SDS-PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.

  20. Rapid Prototyping (United States)


    Javelin, a Lone Peak Engineering Inc. Company has introduced the SteamRoller(TM) System as a commercial product. The system was designed by Javelin during a Phase II NASA funded small commercial product. The purpose of the invention was to allow automated-feed of flexible ceramic tapes to the Laminated Object Manufacturing rapid prototyping equipment. The ceramic material that Javelin was working with during the Phase II project is silicon nitride. This engineered ceramic material is of interest for space-based component.