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Sample records for gigas purification crystallization

  1. Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis

    PubMed Central

    Kladova, A. V.; Gavel, O. Yu.; Mukhopaadhyay, A.; Boer, D. R.; Teixeira, S.; Shnyrov, V. L.; Moura, I.; Moura, J. J. G.; Romão, M. J.; Trincão, J.; Bursakov, S. A.

    2009-01-01

    Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfo­vibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 Å resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 Å resolution, respectively. Zn2+–AK and Fe2+–AK crystallized in space group I222 with similar unit-cell parameters, whereas Co2+–AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn2+–AK and Fe2+–AK forms and a dimer was present for the Co2+–AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes. PMID:19724135

  2. Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas.

    PubMed

    Almendra, M J; Brondino, C D; Gavel, O; Pereira, A S; Tavares, P; Bursakov, S; Duarte, R; Caldeira, J; Moura, J J; Moura, I

    1999-12-01

    An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mössbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.

  3. Crystal structure of rubredoxin from Desulfovibrio gigas to ultra-high 0.68 A resolution

    SciTech Connect

    Chen, C.-J. . E-mail: cjchen@nsrrc.org.tw; Lin, Y.-H.; Huang, Y.-C.; Liu, M.-Y. . E-mail: mingliu@nsrrc.org.tw

    2006-10-13

    Rubredoxin (D.g. Rd) is a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas. The protein is generally purified from anaerobic bacteria in which it is thought to be involved in electron transfer or exchange processes. Rd transfers an electron to oxygen to form water as part of a unique electron transfer chain, composed by NADH:rubredoxin oxidoreductase (NRO), rubredoxin and rubredoxin:oxygen oxidoreductase (ROO) in D.g. The crystal structure of D.g. Rd has been determined by means of both a Fe single-wavelength anomalous dispersion (SAD) signal and the direct method, and refined to an ultra-high 0.68 A resolution, using X-ray from a synchrotron. Rd contains one iron atom bound in a tetrahedral coordination by the sulfur atoms of four cysteinyl residues. Hydrophobic and {pi}-{pi} interactions maintain the internal Rd folding. Multiple conformations of the iron-sulfur cluster and amino acid residues are observed and indicate its unique mechanism of electron transfer. Several hydrogen bonds, including N-H..., SG of the iron-sulfur, are revealed clearly in maps of electron density. Abundant waters bound to C-O peptides of residues Val8, Cys9, Gly10, Ala38, and Gly43, which may be involved in electron transfer. This ultrahigh-resolution structure allows us to study in great detail the relationship between structure and function of rubredoxin, such as salt bridges, hydrogen bonds, water structures, cysteine ligands, iron-sulfur cluster, and distributions of electron density among activity sites. For First time, this information will provide a clear role for this protein in a strict anaerobic bacterium.

  4. Purification of a Multidrug Resistance Transporter for Crystallization Studies

    PubMed Central

    Alegre, Kamela O.; Law, Christopher J.

    2015-01-01

    Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. PMID:27025617

  5. Large-scale crystallization of proteins for purification and formulation.

    PubMed

    Hekmat, Dariusch

    2015-07-01

    Since about 170 years, salts were used to create supersaturated solutions and crystallize proteins. The dehydrating effect of salts as well as their kosmotropic or chaotropic character was revealed. Even the suitability of organic solvents for crystallization was already recognized. Interestingly, what was performed during the early times is still practiced today. A lot of effort was put into understanding the underlying physico-chemical interaction mechanisms leading to protein crystallization. However, it was understood that already the solvation of proteins is a highly complex process not to mention the intricate interrelation of electrostatic and hydrophobic interactions taking place. Although many basic questions are still unanswered, preparative protein crystallization was attempted as illustrated in the presented case studies. Due to the highly variable nature of crystallization, individual design of the crystallization process is needed in every single case. It was shown that preparative crystallization from impure protein solutions as a capture step is possible after applying adequate pre-treatment procedures like precipitation or extraction. Protein crystallization can replace one or more chromatography steps. It was further shown that crystallization can serve as an attractive alternative means for formulation of therapeutic proteins. Crystalline proteins can offer enhanced purity and enable highly concentrated doses of the active ingredient. Easy scalability of the proposed protein crystallization processes was shown using the maximum local energy dissipation as a suitable scale-up criterion. Molecular modeling and target-oriented protein engineering may allow protein crystallization to become part of a platform purification process in the near future.

  6. Protein purification and crystallization artifacts: The tale usually not told.

    PubMed

    Niedzialkowska, Ewa; Gasiorowska, Olga; Handing, Katarzyna B; Majorek, Karolina A; Porebski, Przemyslaw J; Shabalin, Ivan G; Zasadzinska, Ewelina; Cymborowski, Marcin; Minor, Wladek

    2016-03-01

    The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non-reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building. PMID:26660914

  7. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  8. Purification and crystallization of Kokobera virus helicase

    SciTech Connect

    De Colibus, Luigi; Speroni, Silvia; Coutard, Bruno; Forrester, Naomi L.; Gould, Ernest; Canard, Bruno; Mattevi, Andrea

    2007-03-01

    Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3{sub 1}21 (or P3{sub 2}21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å.

  9. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    SciTech Connect

    White, Tommi A.; Tanner, John J.

    2005-08-01

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  10. Salt crystal purification by deliquescence/crystallization cycling

    NASA Astrophysics Data System (ADS)

    Desarnaud, J.; Shahidzadeh-Bonn, N.

    2011-08-01

    In this paper we show how by repetitive humidity cycling high-quality single crystals of salt (NaCl) can be obtained. The drying of droplets of saturated salt solution, leads to many individual microcrystallites that grow close to the contact line due to the "coffee stain effect". Subsequent humidity cycling leads to the growth of a smaller number of crystals by expulsing impurities. This allows us to obtain only one single crystal instead of several dozens of crystallites in as little as three cycles. The reduction in the number of cycles needed to obtain a single crystal can even be improved by the combination of two effects; firstly the deliquescence/recrystallisation cycling and secondly by controlling the wetting properties of the substrate with grafted monolayer treatments.

  11. From Egg to Crystal: A Practical on Purification, Characterization, and Crystallization of Lysozyme for Bachelor Students

    ERIC Educational Resources Information Center

    Olieric, Vincent; Schreiber, Angelique; Lorber, Bernard; Putz, Joern

    2007-01-01

    A practical hands-on course encompassing enzyme purification, biochemical characterization, and crystallization that completed the course work of 350 second-year bachelor students enrolled in molecular biology/biochemistry was given at the Universite Louis Pasteur of Strasbourg (France). The experimental part of the practical dealt entirely with…

  12. Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For

  13. Process of electrolysis and fractional crystallization for aluminum purification

    DOEpatents

    Dawless, Robert K.; Bowman, Kenneth A.; Mazgaj, Robert M.; Cochran, C. Norman

    1983-10-25

    A method for purifying aluminum that contains impurities, the method including the step of introducing such aluminum containing impurities to a charging and melting chamber located in an electrolytic cell of the type having a porous diaphragm permeable by the electrolyte of the cell and impermeable to molten aluminum. The method includes further the steps of supplying impure aluminum from the chamber to the anode area of the cell and electrolytically transferring aluminum from the anode area to the cathode through the diaphragm while leaving impurities in the anode area, thereby purifying the aluminum introduced into the chamber. The method includes the further steps of collecting the purified aluminum at the cathode, and lowering the level of impurities concentrated in the anode area by subjecting molten aluminum and impurities in said chamber to a fractional crystallization treatment wherein eutectic-type impurities crystallize and precipitate out of the aluminum. The eutectic impurities that have crystallized are physically removed from the chamber. The aluminum in the chamber is now suited for further purification as provided in the above step of electrolytically transferring aluminum through the diaphragm.

  14. Process of electrolysis and fractional crystallization for aluminum purification

    DOEpatents

    Dawless, R.K.; Bowman, K.A.; Mazgaj, R.M.; Cochran, C.N.

    1983-10-25

    A method is described for purifying aluminum that contains impurities, the method including the step of introducing such aluminum containing impurities to a charging and melting chamber located in an electrolytic cell of the type having a porous diaphragm permeable by the electrolyte of the cell and impermeable to molten aluminum. The method includes further the steps of supplying impure aluminum from the chamber to the anode area of the cell and electrolytically transferring aluminum from the anode area to the cathode through the diaphragm while leaving impurities in the anode area, thereby purifying the aluminum introduced into the chamber. The method includes the further steps of collecting the purified aluminum at the cathode, and lowering the level of impurities concentrated in the anode area by subjecting molten aluminum and impurities in said chamber to a fractional crystallization treatment wherein eutectic-type impurities crystallize and precipitate out of the aluminum. The eutectic impurities that have crystallized are physically removed from the chamber. The aluminum in the chamber is now suited for further purification as provided in the above step of electrolytically transferring aluminum through the diaphragm. 2 figs.

  15. Purification, characterization and crystallization of the human 80S ribosome

    PubMed Central

    Khatter, Heena; Myasnikov, Alexander G.; Mastio, Leslie; Billas, Isabelle M. L.; Birck, Catherine; Stella, Stefano; Klaholz, Bruno P.

    2014-01-01

    Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work. PMID:24452798

  16. Purification, crystallization and preliminary crystallographic analysis of the Hermes transposase

    SciTech Connect

    Perez, Zhanita N.; Musingarimi, Primrose; Craig, Nancy L.; Dyda, Fred; Hickman, Alison Burgess

    2005-06-01

    Upon purification, an N-terminally deleted version of the Hermes transposase exists in solution as a mixture of two species that are approximately hexameric and dimeric. Crystals have been obtained of the smaller species that diffract to 2.1 Å resolution. DNA transposition is the movement of a defined segment of DNA from one location to another. Although the enzymes that catalyze transposition in bacterial systems have been well characterized, much less is known about the families of transposase enzymes that function in higher organisms. Active transposons have been identified in many insect species, providing tools for gene identification and offering the possibility of altering the genotypes of natural insect populations. One of these active transposons is Hermes, a 2749-base-pair element from Musca domestica that encodes its own transposase. An N-terminally deleted version of the Hermes transposase (residues 79–612) has been overexpressed and purified, and crystals that diffract to 2.1 Å resolution have been obtained at 277 K by the hanging-drop method.

  17. Sequential Purification and Crystal Growth for the Production of Low Cost Silicon Substrates

    SciTech Connect

    Liaw, M; D'Aragona, F S

    1980-08-01

    The objective of this program is to identify and develop low cost precessing for fabricating large grain size polycrystalline silicon substrates. Metallurgical grade silicon (MG-Si) is chosen as the starting material for sequential purification and crystal growth. Several purification techniques have been studied. They include (1) acid leaching with HCl, (2) physical separation of insoluble impurities, (3) reactive gas treatment of molten silicon, and (4) slagging using a mixed-oxide slag. In this quarterly period purification by vaccum treatment and by impurity redistribution using ingot pulling has been studied. Procedures and results are reported.

  18. The Feasibility of Bulk Crystallization as an Industrial Purification and Production Technique for Proteins

    NASA Technical Reports Server (NTRS)

    Judge, Russell A.; Forsythe, Elizabeth L.; Johns, Michael R.; Pusey, Marc L.; White, Edward T.

    1998-01-01

    Bulk crystallization in stirred vessels is used industrially for the recovery and purification of many inorganic and organic materials. Although much has been written on the crystallization of proteins for X-ray diffraction analysis, very little has been reported on the application of bulk crystallization in stirred vessels. In this study, a 1-liter, seeded, stirred, batch crystallizer was used with ovalbumin as a model protein to test the feasibility of this crystallization method as a recovery and purification process for proteins. Results were obtained for ovalbumin solubility, nucleation thresholds, crystal breakage and crystal growth kinetics in bulk solution under a range of operating conditions of pH and ammonium sulphate concentration (Judge et al., 1996). Experiments were also performed to determine the degree of purification that can be achieved by the crystallization of ovalbumin from a mixture of proteins. The effect of the presence of these proteins upon the ovalbumin crystal growth kinetics was also investigated (Judge et al., 1995). All of these aspects are essential for the design of bulk crystallization processes which have not previously been reported for proteins. Results from a second study that investigated the effect of structurally different proteins on the solubility, crystal growth rates and crystal purity of chicken egg white lysozyme are also presented (Judge et al., 1997). In this case face growth rates were measured using lysozyme purified by liquid chromatography and the effect of the addition of specific protein impurities were observed on the (110) and (101) crystal faces. In these two studies the results are presented to show the feasibility and purifying ability of crystallization as a production process for proteins.

  19. Purification of precursors of Yb3+-doped YLF crystals by solvent extraction and electrochemical processing

    NASA Astrophysics Data System (ADS)

    Boncher, William L.; Judge, Elizabeth; Sansinena, Jose-Maria; Dirmyer, Matthew R.; Hehlen, Markus P.

    2015-03-01

    Optical refrigeration by laser irradiation of YLiF4:Yb3+ (YLF:Yb) crystals has been shown to be strongly deteriorated by impurities, which absorb energy at the laser wavelength, and relax non-radiatively, negating cooling produced from anti-Stokes fluorescence. We aim to increase the efficiency of optical refrigeration through materials purification. We start with the purest sources commercially available and process them in a cleanroom environment. Our method proceeds through electrochemical purification, separating out the transition metal impurities by their redox potentials, and can be scaled up to produce the amounts of material needed for crystal growth.

  20. High-throughput Protein Purification and Quality Assessment for Crystallization

    PubMed Central

    Kim, Youngchang; Babnigg, Gyorgy; Jedrzejczak, Robert; Eschenfeldt, William H.; Li, Hui; Maltseva, Natalia; Hatzos-Skintges, Catherine; Gu, Minyi; Makowska-Grzyska, Magdalena; Wu, Ruiying; An, Hao; Chhor, Gekleng; Joachimiak, Andrzej

    2012-01-01

    The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. “Structural biology-grade” proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; [1] the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are

  1. Fast and scalable purification of a therapeutic full-length antibody based on process crystallization.

    PubMed

    Smejkal, Benjamin; Agrawal, Neeraj J; Helk, Bernhard; Schulz, Henk; Giffard, Marion; Mechelke, Matthias; Ortner, Franziska; Heckmeier, Philipp; Trout, Bernhardt L; Hekmat, Dariusch

    2013-09-01

    The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies.

  2. A biosensor-based approach toward purification and crystallization of G protein-coupled receptors.

    PubMed

    Navratilova, Iva; Pancera, Marie; Wyatt, Richard T; Myszka, David G

    2006-06-15

    Biacore technology was used to develop an affinity purification method and screen cocrystallization conditions for the chemokine receptor CCR5. We characterized the binding of nine HIV gp120 variants and identified a truncated construct (YU2DV1V2) that bound CCR5 independent of CD4. This construct was used in an affinity purification step to improve the activity of detergent-solubilized receptor by approximately 300%. The biosensor was also used to screen receptor binding activity automatically under 50 different crystallization conditions. We found that high-molecular-weight polyethylene glycols (PEGs 4,000 and 8,000 Da) most often stabilized the receptor and improved complex formation with potential cocrystallization partners such as conformationally sensitive monoclonal antibodies and gp120. Our results show how biosensors can provide unique insights into receptor purification methods and reveal the effects of crystallization conditions on complex formation. Importantly, these methods can be readily applied to other systems.

  3. Purification, growth, and characterization of Zn(x)Cd(1-x)Se crystals

    NASA Technical Reports Server (NTRS)

    Silberman, E.; Burger, A.; Chen, W.; Henderson, D. O.; Morgan, S. H.; Springer, John M.; Yao, Y.

    1989-01-01

    The purification of starting materials which were used in the growth of Zn(x)Cd(1-x)Se (x = 0.2) single crystals using the traveling solution method (TSM) is reported. Up to 13 cm long single crystals and as grown resistivities of 6 x 10(exp 12) ohm/cm could be achieved. Infrared and Raman spectra of Zn(0.2)Cd(0.8)Se are also presented and discussed.

  4. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  5. TlBr purification and single crystal growth for the detector applications

    NASA Astrophysics Data System (ADS)

    Kozlov, Vasilij; Heikkilä, Mikko; Kostamo, Pasi; Lipsanen, Harri; Leskelä, Markku

    2011-05-01

    The combination of distillation, Bridgman-Stockbarger, hydrothermal recrystallisation and travelling molten zone (TMZ) methods were used for TlBr purification. Grown crystals were characterised by XRD rocking curve and FTIR spectroscopy methods, and by electrical measurements made from 200 to 300 K.

  6. Electromigration process for the purification of molten silicon during crystal growth

    NASA Technical Reports Server (NTRS)

    Shlichta, P. J. (Inventor)

    1982-01-01

    A process for the purification of molten materials during crystal growth by electromigration of impurities to localized dirty zones. In the Czochralski crystal growing process, the impurities are electromigrated away from the crystallization interface by applying a direct electrical current to the molten silicon for electromigrating the charged impurities away from the crystal growth interface. The edge-defined film-fed crystal growth process, a direct electrical current is applied between the two faces which are used in forming the molten silicon into a ribbon. The impurities, migrated to one side only of the crystal ribbon, may be removed or left in place. If left in place, they will not adversely affect the ribbon when used in solar collectors. The migration of the impurity to one side only of the silicon ribbon is especially suitable for use with asymmetric dies which preferentially crystallize uncharged impurities along one side or face of the ribbon.

  7. Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c from Mycobacterium tuberculosis

    SciTech Connect

    Lu, Feifei; Gao, Feng; Li, Honglin; Gong, Weimin; Zhou, Lin; Bi, Lijun

    2014-07-23

    The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv3705c from M. tuberculosis are described. The conserved protein Rv3705c from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 198.0, c = 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å.

  8. Effects of Purification on the Crystallization of Lysozyme

    NASA Technical Reports Server (NTRS)

    Ewing, Felecia L.; Forsythe, Elizabeth L.; Van Der Woerd, Mark; Pusey, Marc L.

    1996-01-01

    We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20 C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal- orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

  9. Effects of purification on the crystallization of lysozyme

    NASA Astrophysics Data System (ADS)

    Ewing, Felecia L.; Forsythe, Elizabeth L.; van der Woerd, Mark; Pusey, Marc L.

    1996-03-01

    We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20°C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal ↔ orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

  10. Expression, purification and crystallization of Streptococcus dysgalactiae-derived mitogen

    SciTech Connect

    Papageorgiou, Anastassios C. Saarinen, Susanna; Ramirez-Bartutis, Rosa; Kato, Hidehito; Uchiyama, Takehiko; Kirikae, Teruo; Miyoshi-Akiyama, Toru

    2006-03-01

    S. dysgalactiae-derived mitogen, a superantigen, was crystallized. Crystals diffract to 2.4 Å at a synchrotron-radiation source and belong to space group P3/P3{sub 1}/P3{sub 2}, with unit-cell parameters a = b = 52.7, c = 62.4 Å, γ = 120° and one molecule in the crystallographic asymmetric unit. Superantigens are bacterial or viral toxins with potent immunostimulatory properties. Streptococcus dysgalactiae-derived mitogen, a 25 kDa protein, is a recently discovered superantigen isolated from S. dysgalactiae culture supernatant. Sequence considerations suggest that it belongs to a new superantigen family distinct from other superantigens. The protein was expressed in Escherichia coli cells and purified to homogeneity. Crystals were grown at pH 4.2–4.4 in the presence of 18–20%(w/v) PEG 3350 and 0.4 M lithium nitrate. A complete data set to 2.4 Å resolution was collected from a single crystal at liquid-nitrogen temperatures using synchrotron radiation. The crystals belong to space group P3/P3{sub 1}/P3{sub 2}, with unit-cell parameters a = b = 52.7, c = 62.4 Å, γ = 120° and one molecule in the crystallographic asymmetric unit.

  11. Expression, purification and crystallization of Helicobacter pyloril-asparaginase

    SciTech Connect

    Dhavala, Prathusha; Krasotkina, Julya; Dubreuil, Christine; Papageorgiou, Anastassios C.

    2008-08-01

    l-Asparaginase from H. pylori was overexpressed in E. coli, purified and crystallized. The crystals belonged to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 Å and one molecule in the asymmetric unit. A complete data set to 1.6 Å resolution was collected using synchrotron radiation. The l-asparaginases from Escherichia coli and Erwinia chrysanthemi are effective drugs that have been used in the treatment of acute childhood lymphoblastic leukaemia for over 30 years. However, despite their therapeutic potential, they can cause serious side effects as a consequence of their intrinsic glutaminase activity, which leads to l-glutamine depletion in the blood. Consequently, new asparaginases with low glutaminase activity, fewer side effects and high activity towards l-asparagine are highly desirable as better alternatives in cancer therapy. l-Asparaginase from Helicobacter pylori was overexpressed in E. coli and purified for structural studies. The enzyme was crystallized at pH 7.0 in the presence of 16–19%(w/v) PEG 4000 and 0.1 M magnesium formate. Data were collected to 1.6 Å resolution at 100 K from a single crystal at a synchrotron-radiation source. The crystals belong to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 Å and one molecule of l-asparaginase in the asymmetric unit. Elucidation of the crystal structure will provide insight into the active site of the enzyme and a better understanding of the structure–activity relationship in l-asparaginases.

  12. The purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Clostridium botulinum

    SciTech Connect

    Dobson, Renwick C. J. Atkinson, Sarah C.; Gorman, Michael A.; Newman, Janet M.; Parker, Michael W.; Perugini, Matthew A.

    2008-03-01

    Dihydrodipicolinate synthase (DHDPS), an enzyme in the lysine-biosynthetic pathway, is a promising target for antibiotic development against pathogenic bacteria. Here, the expression, purification, crystallization and preliminary diffraction analysis of DHDPS from C. botulinum are reported. In recent years, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has received considerable attention from both mechanistic and structural viewpoints. This enzyme, which is part of the diaminopimelate pathway leading to lysine, couples (S)-aspartate-β-semialdehyde with pyruvate via a Schiff base to a conserved active-site lysine. In this paper, the expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from Clostridium botulinum, an important bacterial pathogen, are presented. The enzyme was crystallized in a number of forms, predominantly using PEG precipitants, with the best crystal diffracting to beyond 1.9 Å resolution and displaying P4{sub 2}2{sub 1}2 symmetry. The unit-cell parameters were a = b = 92.9, c = 60.4 Å. The crystal volume per protein weight (V{sub M}) was 2.07 Å{sup 3} Da{sup −1}, with an estimated solvent content of 41%. The structure of the enzyme will help guide the design of novel therapeutics against the C. botulinum pathogen.

  13. Purification, crystallization and preliminary X-ray characterization of the human GTP fucose pyrophosphorylase

    SciTech Connect

    Quirk, Stephen; Seley-Radtke, Katherine L.

    2006-04-01

    The human GTP fucose pyrophosphohydrolase protein has been crystallized via the hanging-drop technique over a reservoir of polyethylene glycol (MW 8000) and ethylene glycol. The orthorhombic crystals diffract to 2.8 Å resolution. The human nucleotide-sugar metabolizing enzyme GTP fucose pyrophosphorylase (GFPP) has been purified to homogeneity by an affinity chromatographic procedure that utilizes a novel nucleoside analog. This new purification regime results in a protein preparation that produces significantly better crystals than traditional purification methods. The purified 66.6 kDa monomeric protein has been crystallized via hanging-drop vapor diffusion at 293 K. Crystals of the native enzyme diffract to 2.8 Å and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. There is a single GFPP monomer in the asymmetric unit, giving a Matthews coefficient of 2.38 Å{sup 3} Da{sup −1} and a solvent content of 48.2%. A complete native data set has been collected as a first step in determining the three-dimensional structure of this enzyme.

  14. Expression, purification and crystallization of pecan (Carya illinoinensis) vicilin

    PubMed Central

    Lee, BoRam; Zhang, Renhao; Du, Wen-Xian; Grauke, Larry J.; McHugh, Tara H.; Zhang, Yu-Zhu

    2014-01-01

    Tree nuts are responsible for many cases of severe food allergies. The 7S seed storage protein vicilin has been identified as a food allergen in many kinds of tree nuts. The vicilin protein consists of an N-terminal low-complexity region with antimicrobial activity and a C-terminal domain that forms a trimeric structure that belongs to the cupin superfamily. In this study, vicilin from pecan (Carya illinoinensis) was isolated and was expressed in bacteria for the first time. The cupin structural core of the protein, residues 369–792, was purified by metal-affinity and gel-filtration chromatography to high purity. Vicilin crystals were obtained and the best crystal diffracted to 2.65 Å resolution in space group P212121. PMID:25084379

  15. Expression, purification and crystallization of pecan (Carya illinoinensis) vicilin.

    PubMed

    Lee, BoRam; Zhang, Renhao; Du, Wen-Xian; Grauke, Larry J; McHugh, Tara H; Zhang, Yu-Zhu

    2014-08-01

    Tree nuts are responsible for many cases of severe food allergies. The 7S seed storage protein vicilin has been identified as a food allergen in many kinds of tree nuts. The vicilin protein consists of an N-terminal low-complexity region with antimicrobial activity and a C-terminal domain that forms a trimeric structure that belongs to the cupin superfamily. In this study, vicilin from pecan (Carya illinoinensis) was isolated and was expressed in bacteria for the first time. The cupin structural core of the protein, residues 369-792, was purified by metal-affinity and gel-filtration chromatography to high purity. Vicilin crystals were obtained and the best crystal diffracted to 2.65 Å resolution in space group P212121. PMID:25084379

  16. Cloning, Expression, Purification, Crystallization and Preliminary X-ray Analysis of Mycoplasma Genitalium Protein MG289

    SciTech Connect

    Sippel, K.; Boehlein, S; Sakai, Y; Quirit, J; Agbandje-McKenna, M; Rosser, C; McKenna, R

    2009-01-01

    Mycoplasma genitalium is a human pathogen that is associated with nongonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 {angstrom} resolution. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 {angstrom}. The diffraction data after processing had an overall R{sub merge} of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.

  17. Expression, purification and crystallization of human 5-lipoxygenase-activating protein with leukotriene-biosynthesis inhibitors

    SciTech Connect

    Xu, Shihua; McKeever, Brian M.; Wisniewski, Douglas; Miller, Douglas K.; Spencer, Robert H.; Chu, Lin; Ujjainwalla, Feroze; Yamin, Ting-Ting; Evans, Jilly F.; Becker, Joseph W.; Ferguson, Andrew D.

    2007-12-01

    The expression, purification and crystallization of human 5-lipoxygenase-activating protein in complex with two leukotriene-biosynthesis inhibitors is decribed. The processes that were used to generate diffraction quality crystals are presented in detail. The nuclear membrane protein 5-lipoxygenase-activating protein (FLAP) plays an essential role in leukotriene synthesis. Recombinant full-length human FLAP with a C-terminal hexahistidine tag has been expressed and purified from the cytoplasmic membrane of Escherichia coli. Diffraction-quality crystals of FLAP in complex with leukotriene-synthesis inhibitor MK-591 and with an iodinated analogue of MK-591 have been grown using the sitting-drop vapor-diffusion method. The crystals exhibit tetragonal symmetry (P42{sub 1}2) and diffracted to a resolution limit of 4 Å.

  18. Electromigration process for the purification of molten silicon during crystal growth

    DOEpatents

    Lovelace, Alan M. Administrator of the National Aeronautics and Space; Shlichta, Paul J.

    1982-01-01

    A process for the purification of molten materials during crystal growth by electromigration of impurities to localized dirty zones. The process has particular applications for silicon crystal growth according to Czochralski techniques and edge-defined film-fed growth (EFG) conditions. In the Czochralski crystal growing process, the impurities are electromigrated away from the crystallization interface by applying a direct electrical current to the molten silicon for electromigrating the charged impurities away from the crystal growth interface. In the EFG crystal growth process, a direct electrical current is applied between the two faces which are used in forming the molten silicon into a ribbon. The impurities are thereby migrated to one side only of the crystal ribbon. The impurities may be removed or left in place. If left in place, they will not adversely affect the ribbon when used in solar collectors. The migration of the impurity to one side only of the silicon ribbon is especially suitable for use with asymmetric dies which preferentially crystallize uncharged impurities along one side or face of the ribbon.

  19. Purification, crystallization and preliminary X-ray diffraction analysis of royal palm tree (Roystonea regia) peroxidase

    SciTech Connect

    Watanabe, Leandra; Nascimento, Alessandro S.; Zamorano, Laura S.; Shnyrov, Valery L.; Polikarpov, Igor

    2007-09-01

    The purification, crystallization, X-ray diffraction data acquisition and molecular-replacement results of royal palm tree (R. regia) peroxidase are described. Royal palm tree peroxidase (RPTP), which was isolated from Roystonea regia leaves, has an unusually high stability that makes it a promising candidate for diverse applications in industry and analytical chemistry [Caramyshev et al. (2005 ▶), Biomacromolecules, 6, 1360–1366]. Here, the purification and crystallization of this plant peroxidase and its X-ray diffraction data collection are described. RPTP crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 Å. The crystals belong to the trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 116.83, c = 92.24 Å, and contain one protein molecule per asymmetric unit. The V{sub M} value and solvent content are 4.07 Å{sup 3} Da{sup −1} and 69.8%, respectively.

  20. The purification and contamination of liquid crystals by means of nanoparticles. The case of weakly ionized species

    NASA Astrophysics Data System (ADS)

    Garbovskiy, Yuriy

    2016-08-01

    This letter reports the effects of nanoparticles on the concentration of mobile ions in liquid crystals with weakly ionized species. Contrary to the case of fully ionized contaminants, the regimes of the purification and contamination achieved in liquid crystal nanocolloids with weakly ionized species depend on the interplay between the ion adsorption/ion desorption and ion generation/ion recombination. The competition between these processes results in the reduction of the magnitude of the purification and contamination levels in liquid crystals with weakly ionized species as compared to similar effects in the case of full ionization of contaminants.

  1. Cloning, purification, crystallization and preliminary crystallographic analysis of a hypothetical acetyltransferase from Pyrococcus furiosus

    PubMed Central

    Biarrotte-Sorin, Sabrina; Mayer, Claudine

    2005-01-01

    The GCN5-related N-acetyltransferase (GNAT) superfamily has a primordial role in cellular processes such as transcription initiation and regulation by histone acetylation, aminoglycoside resistance and melatonin metabolism. To date, no acetyltransferase from the archaeal domain of life has been studied. This paper describes the cloning, expression, purification and crystallization of a Pyrococcus furiosus hypothetical acetyltransferase PfGNAT (MW = 22 007 Da). The crystals belong to space group P622, with one molecule in the asymmetric unit and unit-cell parameters a = b = 82.6, c = 105.92 Å, α = β = 90, γ = 120°. Crystals diffract X-rays to 3.0 Å resolution on a synchrotron-radiation source. Determination of this structure will provide new insights into the substrate-specificity of this acetyltransferase and the thermal stability of the N-acetyltransferase domain. PMID:16511014

  2. Overexpression, purification, crystallization and crystallographic analysis of CopK of Cupriavidus metallidurans

    SciTech Connect

    Tricot, Catherine; Aelst, Sebastien van; Wattiez, Ruddy; Mergeay, Max; Stalon, Victor; Wouters, Johan

    2005-09-01

    Overexpression, purification and crystallization of C. metallidurans CopK allowed the collection of a complete data set to 2.2 Å resolution. CopK of Cupriavidus metallidurans is a 93-amino-acid protein whose mature form (73 amino acids) has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mM citrate pH 3.5, 200 mM Li{sub 2}SO{sub 4}, 20%(w/v) glycerol, 13%(w/v) PEG 8000. Crystals display orthorhombic symmetry, with unit-cell parameters a = 57.53, b = 128.65, c = 49.77 Å, and diffract to 2.2 Å resolution using synchrotron radiation.

  3. Expression, purification and crystallization of a novel nonstructural protein VP9 from white spot syndrome virus.

    SciTech Connect

    Liu,Y.; Sivaraman, J.; Hew, C.

    2006-01-01

    The nonstructural protein VP9 from white spot syndrome virus (WSSV) has been identified and expressed in Escherichia coli. To facilitate purification, a cleavable His{sub 6} tag was introduced at the N-terminus. The native protein was purified and crystallized by vapor diffusion against mother liquor containing 2 M sodium acetate, 100 mM MES pH 6.3, 25 mM cadmium sulfate and 3% glycerol. Crystals were obtained within 7 d and diffracted to 2.2 Angstroms; they belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 74.13, b = 78.21, c = 78.98 Angstroms and four molecules in the asymmetric unit. The selenomethionine-labeled protein produced isomorphous crystals that diffracted to approximately 3.3 Angstroms.

  4. Purification, crystallization and preliminary X-ray diffraction analysis of royal palm tree (Roystonea regia) peroxidase.

    PubMed

    Watanabe, Leandra; Nascimento, Alessandro S; Zamorano, Laura S; Shnyrov, Valery L; Polikarpov, Igor

    2007-09-01

    Royal palm tree peroxidase (RPTP), which was isolated from Roystonea regia leaves, has an unusually high stability that makes it a promising candidate for diverse applications in industry and analytical chemistry [Caramyshev et al. (2005), Biomacromolecules, 6, 1360-1366]. Here, the purification and crystallization of this plant peroxidase and its X-ray diffraction data collection are described. RPTP crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 A. The crystals belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 116.83, c = 92.24 A, and contain one protein molecule per asymmetric unit. The V(M) value and solvent content are 4.07 A3 Da(-1) and 69.8%, respectively. PMID:17768354

  5. Purification, crystallization, and characterization of peroxidase from Coprinus cinereus.

    PubMed

    Morita, Y; Yamashita, H; Mikami, B; Iwamoto, H; Aibara, S; Terada, M; Minami, J

    1988-04-01

    Peroxidase (donor: H2O2 oxidoreductase [EC 1.11.1.7]) was purified from a culture broth of an inkcap Basidiomycete, Coprinus cinereus S.F. Gray. A single component containing a low amount of carbohydrate was isolated by affinity chromatography on concanavalin A-Sepharose and crystallized from ammonium sulfate solution. The enzyme is an acidic protein (pI 3.5) and consists of a single polypeptide chain having the molecular weight of 41,600 daltons. The enzyme contains one protohemin per molecule and exhibits the characteristic absorption, circular dichroism, and magnetic circular dichroism spectra of a heme-protein. The Coprinus peroxidase forms two characteristic intermediate compounds, I and II, and the rate constants for hydrogen peroxide and guaiacol had similar values to those for higher plant peroxidases. The ferric enzyme formed a cyanide compound with a dissociation constant similar to those for higher plant enzyme, but the dissociation constant of the ferrous enzyme-cyanide was large. The chemical composition of Coprinus peroxidase showed 381 amino acid residues, 1 glucosamine, 3 true sugars, 3 calcium, and 1 non-heme iron other than 1 protohemin. The secondary structure of the fungal enzyme was very similar to that of horseradish peroxidase.

  6. Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase

    SciTech Connect

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2006-07-01

    Crystallization of a surface-localized acid phosphatase from Bacillus anthracis is reported. Flash annealing increased the high-resolution limit of usable data from 1.8 to 1.6 Å. Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 Å. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6° and a high-resolution limit of 1.8 Å. After flash-annealing, the apparent mosaicity decreased to 0.9° and the high-resolution limit of usable data increased to 1.6 Å. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

  7. GigaDB: announcing the GigaScience database

    PubMed Central

    2012-01-01

    With the launch of GigaScience journal, here we provide insight into the accompanying database GigaDB, which allows the integration of manuscript publication with supporting data and tools. Reinforcing and upholding GigaScience’s goals to promote open-data and reproducibility of research, GigaDB also aims to provide a home, when a suitable public repository does not exist, for the supporting data or tools featured in the journal and beyond. PMID:23587345

  8. Purification and crystallization of components of the protein-synthesizing system from Thermus thermophilus

    NASA Astrophysics Data System (ADS)

    Garber, M. B.; Agalarov, S. Ch.; Eliseikina, I. A.; Sedelnikova, S. E.; Tishchenko, S. V.; Shirokov, V. A.; Yusupov, M. M.; Reshetnikova, L. S.; Trakhanov, S. D.; Tukalo, M. A.; Yaremchuk, A. D.

    1991-03-01

    An extreme thermophilic bacterium Thermus thermophilus has been chosen as a source for the isolation of components of the protein-synthesizing system to investigate their structures by X-ray crystallographic methods. The scheme of simultaneous isolation of ribosomes, tRNA, three elongation factors, several aminoacyl-tRNA synthetases and several enzymes has been developed. Methods of purification of ribosomes and individual ribosomal proteins without denaturation were elaborated. Crystals of the elongation factor G, the 70S ribosome, the 30S ribosomal subunit, six ribosomal proteins and three aminoacyl-tRNA synthetases have been obtained. Structural investigations of EF-G and the 70S ribosome are underway.

  9. Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1.

    PubMed

    Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi; Fromme, Raimund; Doran, Jeffrey D; Grotjohann, Ingo; Mittman, Michele; Basu, Shibom; Deb, Arpan; Dörner, Katerina; Aquila, Andrew; Barty, Anton; Boutet, Sébastien; Chapman, Henry N; Doak, R Bruce; Hunter, Mark S; James, Daniel; Kirian, Richard A; Kupitz, Christopher; Lawrence, Robert M; Liu, Haiguang; Nass, Karol; Schlichting, Ilme; Schmidt, Kevin E; Seibert, M Marvin; Shoeman, Robert L; Spence, John C H; Stellato, Francesco; Weierstall, Uwe; Williams, Garth J; Yoon, Chunhong; Wang, Dingjie; Zatsepin, Nadia A; Hogue, Brenda G; Matoba, Nobuyuki; Fromme, Petra; Mor, Tsafrir S

    2014-09-01

    CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

  10. Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

    PubMed Central

    Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi; Fromme, Raimund; Doran, Jeffrey D.; Grotjohann, Ingo; Mittman, Michele; Basu, Shibom; Deb, Arpan; Dörner, Katerina; Aquila, Andrew; Barty, Anton; Boutet, Sébastien; Chapman, Henry N.; Doak, R. Bruce; Hunter, Mark S.; James, Daniel; Kirian, Richard A.; Kupitz, Christopher; Lawrence, Robert M.; Liu, Haiguang; Nass, Karol; Schlichting, Ilme; Schmidt, Kevin E.; Seibert, M. Marvin; Shoeman, Robert L.; Spence, John C. H.; Stellato, Francesco; Weierstall, Uwe; Williams, Garth J.; Yoon, Chunhong; Wang, Dingjie; Zatsepin, Nadia A.; Hogue, Brenda G.; Matoba, Nobuyuki; Fromme, Petra; Mor, Tsafrir S.

    2014-01-01

    CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before. PMID:25295172

  11. Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1.

    PubMed

    Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi; Fromme, Raimund; Doran, Jeffrey D; Grotjohann, Ingo; Mittman, Michele; Basu, Shibom; Deb, Arpan; Dörner, Katerina; Aquila, Andrew; Barty, Anton; Boutet, Sébastien; Chapman, Henry N; Doak, R Bruce; Hunter, Mark S; James, Daniel; Kirian, Richard A; Kupitz, Christopher; Lawrence, Robert M; Liu, Haiguang; Nass, Karol; Schlichting, Ilme; Schmidt, Kevin E; Seibert, M Marvin; Shoeman, Robert L; Spence, John C H; Stellato, Francesco; Weierstall, Uwe; Williams, Garth J; Yoon, Chunhong; Wang, Dingjie; Zatsepin, Nadia A; Hogue, Brenda G; Matoba, Nobuyuki; Fromme, Petra; Mor, Tsafrir S

    2014-09-01

    CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before. PMID:25295172

  12. Envelope protein VP24 from White spot syndrome virus: expression, purification and crystallization.

    PubMed

    Sun, Lifang; Wu, Yunkun

    2016-08-01

    White spot syndrome virus (WSSV) is a major shrimp pathogen known to infect penaeid shrimp and other crustaceans. VP24 is one of the major envelope proteins of WSSV. In order to facilitate purification, crystallization and structure determination, the predicted N-terminal transmembrane region of approximately 26 amino acids was truncated from VP24 and several mutants were prepared to increase the proportion of selenomethionine (SeMet) residues for subsequent structural determination using the SAD method. Truncated VP24, its mutants and the corresponding SeMet-labelled proteins were purified, and the native and SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of VP24 were obtained using a reservoir consisting of 0.1 M Tris-HCl pH 8.5, 2.75 M ammonium acetate with a drop volume ratio of two parts protein solution to one part reservoir solution. Notably, ATP was added as a critical additive to the drop with a final concentration of 10 mM. Crystals of SeMet-labelled VP24 mutant diffracted to 3.0 Å resolution and those of the native diffracted to 2.4 Å resolution; the crystals belonged to space group I213, with unit-cell parameters a = b = c = 140 Å. PMID:27487921

  13. Expression, purification and crystallization of the Crimean–Congo haemorrhagic fever virus nucleocapsid protein

    PubMed Central

    Carter, S. D.; Barr, J. N.; Edwards, T. A.

    2012-01-01

    Crimean–Congo haemorrhagic fever virus (CCHFV) is a member of the Nairovirus genus within the Bunyaviridae family of segmented negative-sense RNA viruses. This paper describes the expression, purification and crystallization of full-length CCHFV nucleocapsid (N) protein and the collection of a 2.1 Å resolution X-ray diffraction data set using synchrotron radiation. Crystals of the CCHFV N protein belonged to space group C2, with unit-cell parameters a = 150.38, b = 72.06, c = 101.23 Å, β = 110.70° and two molecules in the asymmetric unit. Circular-dichroism analysis provided insight into the secondary structure, whilst gel-filtration analysis revealed possible oligomeric states of the N protein. Structural determination is ongoing. PMID:22691790

  14. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    SciTech Connect

    Tang, Xuhua; Hew, Choy Leong

    2007-07-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.

  15. Expression purification crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa PelD

    SciTech Connect

    Marmont L. S.; Robinson H.; Whitney J. C.; Colvin K. M.; Parsek M. R.; Howell P. L.

    2012-02-01

    The production of the PEL polysaccharide in Pseudomonas aeruginosa requires the binding of bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) to the cytoplasmic GGDEF domain of the inner membrane protein PelD. Here, the overexpression, purification and crystallization of a soluble construct of PelD that encompasses the GGDEF domain and a predicted GAF domain is reported. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method. The crystals grew as flat plates, with unit-cell parameters a = 88.3, b = 114.0, c = 61.9 {angstrom}, {alpha} = {beta} = {gamma} = 90.0{sup o}. The PelD crystals exhibited the symmetry of space group P2{sub 1}2{sub 1}2 and diffracted to a minimum d-spacing of 2.2 {angstrom}. On the basis of the Matthews coefficient (V{sub M} = 2.29 {angstrom}{sup 3} Da{sup -1}), it was estimated that two molecules are present in the asymmetric unit.

  16. Purification and Crystallization of ZITB, A Zinc Transporter from Escherichia Coli

    SciTech Connect

    Kao, K.; Fu, D.

    2004-01-01

    Cellular zinc homeostasis is essential to human health. Zinc transporters transport zinc ions into and out of cells to maintain cellular zinc concentrations in a narrow range. Several membrane proteins have been shown to facilitate transmembrane fluxes of zinc ions, however, structures of these zinc transporters are unknown. The purpose of this work is to express, purify and crystallize a Zinc transporter, ZitB for crystallographic studies. ZitB was over-expressed as a His-tagged membrane protein using a pET15b expression vector hosted in E. coli BL21 cells. Purification of ZitB was achieved by preparation of ZitB-containing membrane vesicles, followed by detergent extraction, and completed with Ni-NTA metal affinity and size exclusion chromatography. The molecular identity of the purified ZitB was confirmed by mass spectrometry, which showed the expected molecular weight of 35.2kDa. Crystallization trials of ZitB were conducted at 20 oC, using a series of low molecular weight PEGs as precipitants. Micro-crystals were grown in 25% PEG 1K, whereas only amorphous precipitations were observed in PEG 400 and 600. In conclusion, this work yielded highly purified ZitB protein and defined an initial crystallization condition for ZitB.

  17. Expression, purification and crystallization of class C acid phosphatases from Francisella tularensis and Pasteurella multocida

    PubMed Central

    Singh, Harkewal; Felts, Richard L.; Ma, Li; Malinski, Thomas J.; Calcutt, Michael J.; Reilly, Thomas J.; Tanner, John J.

    2009-01-01

    Class C nonspecific acid phosphatases are bacterial enzymes that are secreted across the cytoplasmic membrane and hydrolyze a variety of phosphomono­esters at acidic pH. These enzymes are of interest for the development of improved vaccines and clinical diagnostic methods. In one case, the category A pathogen Francisella tularensis, the class C phosphatase plays a role in bacterial fitness. Here, the cloning, expression, purification and crystallization methods for the class C acid phosphatases from F. tularensis and Pasteurella multocida are reported. Crystals of the F. tularensis enzyme diffracted to 2.0 Å resolution and belonged to space group C2221, with one enzyme molecule in the asymmetric unit. Crystals of the P. multocida enzyme diffracted to 1.85 Å resolution and belonged to space group C2, with three molecules in the asymmetric unit. Diffraction patterns from crystals of the P. multocida enzyme exhibited multiple interpenetrating reciprocal-space lattices, indicating epitaxial twinning. Despite this aberrance, autoindexing was robust and the data could be satisfactorily processed to 1.85 Å resolution using MOSFLM and SCALA. PMID:19255471

  18. Expression, purification and crystallization of a novel nonstructural protein VP9 from white spot syndrome virus

    SciTech Connect

    Liu, Yang; Sivaraman, J.; Hew, Choy L.

    2006-08-01

    The nonstructural protein VP9 from white spot syndrome virus (WSSV) has been identified and expressed in Escherichia coli. Native protein was purified and crystallized by vapour diffusion. The nonstructural protein VP9 from white spot syndrome virus (WSSV) has been identified and expressed in Escherichia coli. To facilitate purification, a cleavable His{sub 6} tag was introduced at the N-terminus. The native protein was purified and crystallized by vapour diffusion against mother liquor containing 2 M sodium acetate, 100 mM MES pH 6.3, 25 mM cadmium sulfate and 3% glycerol. Crystals were obtained within 7 d and diffracted to 2.2 Å; they belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 74.13, b = 78.21, c = 78.98 Å and four molecules in the asymmetric unit. The selenomethionine-labelled protein produced isomorphous crystals that diffracted to approximately 3.3 Å.

  19. Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase

    SciTech Connect

    Qian, Kevin C.; Studts, Joey; Wang, Lian; Barringer, Kevin; Kronkaitis, Anthony; Peng, Charline; Baptiste, Alistair; LaFrance, Roger; Mische, Sheenah; Farmer, Bennett

    2005-01-01

    Pim kinases, belong to a distinctive serine/threonine protein-kinase family and are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Human Pim-1 kinase has been cloned, expressed and crystallized Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1–313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14–313) by thrombin digestion during purification. The Pim-1 (14–313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 Å resolution and belong to space group P6{sub 5}, with unit-cell parameters a = b = 95.9, c = 80.0 Å, β = 120° and one molecule per asymmetric unit.

  20. Expression, Purification, Crystallization of Two Major Envelope Proteins from White Spot Syndrome Virus

    SciTech Connect

    Tang,X.; Hew, C.

    2007-01-01

    White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapor-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 {angstrom} resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 {angstrom}. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 {angstrom}, and diffracts to 2.0 {angstrom} resolution.

  1. Expression, purification, crystallization and preliminary diffraction data characterization of Escherichia coli ribonuclease II (RNase II)

    SciTech Connect

    McVey, Colin E.; Amblar, Mónica; Barbas, Ana; Cairrão, Fátima; Coelho, Ricardo; Romão, Célia; Arraiano, Cecília M.; Carrondo, Maria A.; Frazão, Carlos

    2006-07-01

    Diffraction data from E. coli RNase II crystals of wild type and of an inactive mutant and its SeMet-derivative form were obtained to 2.44 and 2.74 Å resolution, providing a set of preliminary phases. An improved purification protocol allowed higher reproducibility in the crystallization of the mutant form. RNA degradation is important in the post-transcriptional control of gene expression. The processing, degradation and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a 643-amino-acid enzyme that degrades single-stranded RNA from its 3′-end, releasing ribonucleoside 5′-monophosphates. RNase II was expressed both as the wild type and as a D209N mutant form. The latter was also produced as an SeMet derivative. The various protein forms were crystallized using the vapour-diffusion method. Wild-type RNase II was crystallized in two crystal forms, both of which belonged to space group P2{sub 1}. X-ray diffraction data were collected to 2.44 and 2.75 Å resolution, with unit-cell parameters a = 56.8, b = 125.7, c = 66.2 Å, β = 111.9° and a = 119.6, b = 57.2, c = 121.2 Å, β = 99.7°, respectively. The RNase II D209N mutant gave crystals that belonged to space group P6{sub 5}, with unit-cell parameters a = b = 86.3, c = 279.2 Å, and diffracted to 2.74 Å. Diffraction data from the mutant and its SeMet derivative enabled the determination of a partial Se-atom substructure by SIRAS.

  2. Purification, crystallization and identification by X-ray analysis of a prostate kallikrein from horse seminal plasma.

    PubMed

    Carvalho, A L; Dias, J M; Sanz, L; Romero, A; Calvete, J J; Romão, M J

    2001-08-01

    The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purified from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42 A resolution were collected at the ESRF synchrotron-radiation source.

  3. Crystal Structure of the 30S Ribosomal Subunit from Thermus Thermophilus. Purification, Crystallization and Structure Determination

    SciTech Connect

    Clemons, William M.; Brodersen, Ditlev E.; McCutcheonn, John P.; May, Joanna L.C.; Carter, Andrew P.; Morgan-Warren, Robert J.; Wimberly, Brian T.; Ramakrishnan, Venki

    2009-10-07

    We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 {angstrom} resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 {angstrom} resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.

  4. Expression, purification and crystallization of human CD5 domain III, a nano-scale crystallization example.

    PubMed

    Rodamilans, Bernardo; Ibañez, Sonia; Bragado-Nilsson, Elisabeth; Sarrias, Maria Rosa; Lozano, Francisco; Blanco, Francisco J; Montoya, Guillermo

    2007-07-01

    The human lymphocyte receptor CD5, a key regulator of immune responses, is involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. CD5 is a membrane glycoprotein which belongs to the group B scavenger receptor cysteine-rich (SRCR) superfamily for which no structural information is available. The most conserved membrane-proximal SRCR domain of CD5 (domain III) has been expressed in HEK-EBNA-293 cells. Although the yield of the purified protein was at the level of micrograms, well diffracting crystals have been obtained. The crystals belong to a tetragonal space group P4(1)22 or P4(3)22. They contain two molecules per asymmetric unit and diffracted to 2.5A resolution using synchrotron radiation. The strategy shown here to produce, isolate and crystallize CD5 domain III can be used for other mammalian proteins difficult to produce for structural or other biophysical studies.

  5. HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies.

    PubMed

    Franken, P; Arold, S; Padilla, A; Bodeus, M; Hoh, F; Strub, M P; Boyer, M; Jullien, M; Benarous, R; Dumas, C

    1997-12-01

    Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.

  6. Expression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio carchariae

    SciTech Connect

    Songsiriritthigul, Chomphunuch; Yuvaniyama, Jirundon; Robinson, Robert C.; Vongsuwan, Archara; Prinz, Heino; Suginta, Wipa

    2005-10-01

    This article describes the high-level expression, purification and crystallization as well as preliminary X-ray diffraction study of a family 18 chitinase, chitinase A from V. carchariae. Chitinase A of Vibrio carchariae was expressed in Escherichia coli M15 host cells as a 575-amino-acid fragment with full enzymatic activity using the pQE60 expression vector. The yield of the highly purified recombinant protein was approximately 70 mg per litre of bacterial culture. The molecular mass of the expressed protein was determined by HPLC/ESI–MS to be 63 770, including the hexahistidine tag. Crystals of recombinant chitinase A were grown to a suitable size for X-ray structure analysis in a precipitant containing 10%(v/v) PEG 400, 0.1 M sodium acetate pH 4.6 and 0.125 M CaCl{sub 2}. The crystals belonged to the tetragonal space group P422, with two molecules per asymmetric unit and unit-cell parameters a = b = 127.64, c = 171.42 Å. A complete diffraction data set was collected to 2.14 Å resolution using a Rigaku/MSC R-AXIS IV{sup ++} detector system mounted on an RU-H3R rotating-anode X-ray generator.

  7. Control of crystal growth in water purification by directional freeze crystallization

    NASA Technical Reports Server (NTRS)

    Conlon, William M. (Inventor)

    1996-01-01

    A Directional Freeze Crystallization system employs an indirect contact heat exchanger to freeze a fraction of liquid to be purified. The unfrozen fraction is drained away and the purified frozen fraction is melted. The heat exchanger must be designed in accordance with a Growth Habit Index to achieve efficient separation of contaminants. If gases are dissolved in the liquid, the system must be pressurized.

  8. Cloning, expression, purification, crystallization and preliminary X-ray analysis of Thermus aquaticus succinyl-CoA synthetase

    SciTech Connect

    Joyce, Michael A.; Brownie, Edward R.; Hayakawa, Koto; Fraser, Marie E.

    2007-05-01

    Attempts to crystallize succinyl-CoA synthetase from the thermophile T. aquaticus were thwarted by proteolysis of the β-subunit and preferential crystallization of a truncated form. Crystals of the full-length enzyme were grown after the purification protocol was modified to include frequent additions of protease inhibitors. Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the β-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 Å resolution, are of the protein in complex with Mn{sup 2+}-GDP.

  9. Purification and Crystallization of Murine Myostatin: A Negative Regulator of Muscle Mass

    NASA Technical Reports Server (NTRS)

    Hong, Young S.; Adamek, Daniel; Bridge, Kristi; Malone, Christine C.; Young, Ronald B.; Miller, Teresa; Karr, Laurel

    2004-01-01

    Myostatin (MSTN) has been crystallized and its preliminary X-ray diffraction data were collected. MSTN is a negative regulator of muscle growt/differentiation and suppressor of fat accumulation. It is a member of TGF-b family of proteins. Like other members of this family, the regulation of MSTN is critically tied to its process of maturation. This process involves the formation of a homodimer followed by two proteolytic steps. The first proteolytic cleavage produces a species where the n-terminal portion of the dimer is covalently separated from, but remains non-covalently bound to, the c-terminal, functional, portion of the protein. The protein is activated upon removal of the n-terminal "pro-segment" by a second n-terminal proteolytic cut by BMP-1 in vivo, or by acid treatment in vitro. Understanding the structural nature and physical interactions involved in these regulatory processes is the objective of our studies. Murine MSTN was purified from culture media of genetically engineered Chinese Hamster Ovary cells by multicolumn purification process and crystallized using the vapor diffusion method.

  10. Overexpression, purification and crystallization of the response regulator NsrR involved in nisin resistance.

    PubMed

    Khosa, Sakshi; Hoeppner, Astrid; Kleinschrodt, Diana; Smits, Sander H J

    2015-10-01

    A number of Gram-positive bacteria produce a class of bacteriocins called `lantibiotics'. These lantibiotics are ribosomally synthesized peptides that possess high antimicrobial activity against Gram-positive bacteria, including clinically challenging pathogens, and are therefore potential alternatives to antibiotics. All lantibiotic producer strains and some Gram-positive nonproducer strains express protein systems to circumvent a suicidal effect or to become resistant, respectively. Two-component systems consisting of a response regulator and a histidine kinase upregulate the expression of these proteins. One of the best-characterized lantibiotics is nisin, which is produced by Lactococcus lactis and possesses bactericidal activity against various Gram-positive bacteria, including some human pathogenic strains. Within many human pathogenic bacterial strains inherently resistant to nisin, a response regulator, NsrR, has been identified which regulates the expression of proteins involved in nisin resistance. In the present study, an expression and purification protocol was established for the NsrR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method, resulting in crystals that diffracted X-rays to 1.4 Å resolution. PMID:26457525

  11. Overexpression, purification and crystallization of the response regulator NsrR involved in nisin resistance.

    PubMed

    Khosa, Sakshi; Hoeppner, Astrid; Kleinschrodt, Diana; Smits, Sander H J

    2015-10-01

    A number of Gram-positive bacteria produce a class of bacteriocins called `lantibiotics'. These lantibiotics are ribosomally synthesized peptides that possess high antimicrobial activity against Gram-positive bacteria, including clinically challenging pathogens, and are therefore potential alternatives to antibiotics. All lantibiotic producer strains and some Gram-positive nonproducer strains express protein systems to circumvent a suicidal effect or to become resistant, respectively. Two-component systems consisting of a response regulator and a histidine kinase upregulate the expression of these proteins. One of the best-characterized lantibiotics is nisin, which is produced by Lactococcus lactis and possesses bactericidal activity against various Gram-positive bacteria, including some human pathogenic strains. Within many human pathogenic bacterial strains inherently resistant to nisin, a response regulator, NsrR, has been identified which regulates the expression of proteins involved in nisin resistance. In the present study, an expression and purification protocol was established for the NsrR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method, resulting in crystals that diffracted X-rays to 1.4 Å resolution.

  12. Purification, crystallization and preliminary X-ray crystallographic analysis of TssL from Vibrio cholerae

    PubMed Central

    Jeong, Jae-Hee; Chang, Jeong Ho; Kim, Yeon-Gil

    2014-01-01

    The type VI secretion system (T6SS) is a macromolecular complex that is conserved in Gram-negative bacteria. The T6SS secretes effector proteins into recipient cells in a contact-dependent manner in order to accomplish cooperative and competitive interactions with the cells. Although the composition and mechanism of the T6SS have been intensively investigated across many Gram-negative bacteria, to date structural information on T6SS components from the important pathogen Vibrio cholerae has been rare. Here, the cloning, purification, crystallization and preliminary X-ray crystallographic analysis of the cytoplasmic domain of TssL, an inner membrane protein of the T6SS, from V. cholerae are reported. Diffraction data were collected to 1.5 Å resolution using synchrotron radiation. The crystal belonged to the hexagonal space group P61, with unit-cell parameters a = 78.4, b = 78.4, c = 49.5 Å. The successful structural characterization of TssL from V. cholerae will contribute to understanding the role of the membrane-associated subunits of the T6SS in more detail. PMID:25195905

  13. The quorum-quenching lactonase from Geobacillus caldoxylosilyticus: purification, characterization, crystallization and crystallographic analysis.

    PubMed

    Bergonzi, Celine; Schwab, Michael; Elias, Mikael

    2016-09-01

    Lactonases are enzymes that are capable of hydrolyzing various lactones such as aliphatic lactones or acyl-homoserine lactones (AHLs), with the latter being used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases therefore have the ability to quench the chemical communication, also known as quorum sensing, of numerous bacteria, and in particular to inhibit behaviors that are regulated by this system, such as the expression of virulence factors or the production of biofilms. A novel representative from the metallo-β-lactamase superfamily, dubbed GcL, was isolated from the thermophilic bacterium Geobacillus caldoxylosilyticus. Because of its thermophilic origin, GcL may constitute an interesting candidate for the development of biocontrol agents. Here, we show that GcL is a thermostable enzyme with a half-life at 75°C of 152.5 ± 10 min. Remarkably, it is also shown that GcL is among the most active lactonases characterized to date, with catalytic efficiencies (kcat/Km) against AHLs of greater than 10(6) M(-1) s(-1). The structure of GcL is expected to shed light on the catalytic mechanism of the enzyme and the molecular determinants for the substrate specificity in this class of lactonases. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.6 Å resolution of GcL are reported. PMID:27599858

  14. Purification, crystallization and preliminary crystallographic studies of the TLDc domain of oxidation resistance protein 2 from zebrafish

    PubMed Central

    Alsarraf, Husam M. A. B.; Laroche, Fabrice; Spaink, Herman; Thirup, Søren; Blaise, Mickael

    2011-01-01

    Cell metabolic processes are constantly producing reactive oxygen species (ROS), which have deleterious effects by triggering, for example, DNA damage. Numerous enzymes such as catalase, and small compounds such as vitamin C, provide protection against ROS. The TLDc domain of the human oxidation resistance protein has been shown to be able to protect DNA from oxidative stress; however, its mechanism of action is still not understood and no structural information is available on this domain. Structural information on the TLDc domain may therefore help in understanding exactly how it works. Here, the purification, crystallization and preliminary crystallographic studies of the TLDc domain from zebrafish are reported. Crystals belonging to the orthorhombic space group P21212 were obtained and diffracted to 0.97 Å resolution. Selenomethionine-substituted protein could also be crystallized; these crystals diffracted to 1.1 Å resolution and the structure could be solved by SAD/MAD methods. PMID:22102041

  15. The purification, crystallization and preliminary structural characterization of human MAWDBP, a member of the phenazine biosynthesis-like protein family

    SciTech Connect

    Herde, Petra; Blankenfeldt, Wulf

    2006-06-01

    The purification, crystallization and preliminary structural characterization of human MAWD-binding protein (MAWDBP) are described. MAWDBP is the only representative of the phenazine biosynthesis-like protein family in the human genome. Its expression is elevated in several disease processes, including insulin resistance, folate deficiency and hypotension, and it may also be involved in carcinogenesis. The exact molecular function of MAWDBP is unknown. Native and seleno-l-methionine-labelled MAWDBP were expressed in Escherichia coli and crystallized at room temperature from precipitants containing 10 mM KF, 14%(w/v) PEG 3350 and 0.1 M sodium citrate pH 5.4. Crystals belong to space group H32, with unit-cell parameters a = b = 187, c = 241 Å, indicative of three to five monomers per asymmetric unit. Crystals were cryoprotected with 15%(v/v) glycerol and data have been collected to 2.7 Å resolution.

  16. Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin.

    PubMed

    Jagadeesan, G; Malathy, P; Gunasekaran, K; Harikrishna Etti, S; Aravindhan, S

    2014-11-01

    Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3₁21, with unit-cell parameters a=b=55.64, c=153.38 Å, β=120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit. PMID:25372822

  17. Purification of Histone Lysine Methyltransferase SMYD2 and Co-Crystallization with a Target Peptide from Estrogen Receptor α.

    PubMed

    Jiang, Yuanyuan; Holcomb, Joshua; Spellmon, Nicholas; Yang, Zhe

    2016-01-01

    Methylation of estrogen receptor α by the histone lysine methyltransferase SMYD2 regulates ERα chromatin recruitment and its target gene expression. This protocol describes SMYD2 purification and crystallization of SMYD2 in complex with an ERα peptide. Recombinant SMYD2 is overexpressed in Escherichia coli cells. After release from the cells by French Press, SMYD2 is purified to apparent homogeneity with multiple chromatography methods. Nickel affinity column purifies SMYD2 based on specific interaction of its 6×His tag with the bead-immobilized nickel ions. Desalting column is used for protein buffer exchange. Gel filtration column purifies SMYD2 based on molecular size. The entire purification process is monitored and analyzed by SDS-polyacrylamide gel electrophoresis. Crystallization of SMYD2 is performed with the hanging drop vapor diffusion method. Crystals of the SMYD2-ERα peptide complex are obtained by microseeding using seeding bead. This method can give rise to large size of crystals which are suitable for X-ray diffraction data collection. X-ray crystallographic study of the SMYD2-ERα complex can provide structural insight into posttranslational regulation of ERα signaling.

  18. Expression, purification, crystallization and preliminary X-ray diffraction analysis of α-11 giardin from Giardia lamblia

    SciTech Connect

    Pathuri, Puja; Nguyen, Emily Tam; Luecke, Hartmut

    2006-11-01

    α-11 giardin from the intestinal protozoan parasite, G. lamblia has been cloned, expressed, purified and crystallized under two different conditions and in two different space groups. Crystals from the first condition diffracted to 1.1 Å and belong to a primitive orthorhombic space group and crystals obtained in the second condition diffracted to 2.93 Å and belong to a primitive monoclinic space group. α-11 Giardin, a protein from the annexin superfamily, is a 35.0 kDa protein from the intestinal protozoan parasite Giardia lamblia which triggers a form of diarrhea called giardiasis. Here, the cloning, expression, purification and the crystallization of α-11 giardin under two different conditions and in two different space groups is reported. Crystals from the first condition diffracted to 1.1 Å and belong to a primitive orthorhombic space group, while crystals from the second condition, which included calcium in the crystallization solution, diffracted to 2.93 Å and belong to a primitive monoclinic space group. Determination of the detailed atomic structure of α-11 giardin will provide a better insight into its biological function and might establish whether this class of proteins is a potential drug target against giardiasis.

  19. Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA.

    PubMed

    Gruss, Fabian; Hiller, Sebastian; Maier, Timm

    2015-01-01

    TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family.

  20. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    SciTech Connect

    DiMattia, Michael; Govindasamy, Lakshmanan; Levy, Hazel C.; Gurda-Whitaker, Brittney; Kalina, Amy; Kohlbrenner, Erik; Chiorini, John A.; McKenna, Robert; Muzyczka, Nicholas; Zolotukhin, Sergei; Agbandje-McKenna, Mavis

    2005-10-01

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  1. Sequential purification and crystal growth for the production of low cost silicon substrates. Annual report, 15 September 1979-14 September 1980

    SciTech Connect

    Liaw, M; D'Aragona, F S

    1980-01-01

    The objective of this program is to identify and develop low cost processing for fabricating large grain size polycrystalline silicon substrates. Metallurgical grade silicon (MG-Si) which is low cost and abundant for industrial usage was chosen as starting material. However, MG-Si cannot be used directly as substrates for solar cell fabrication for the following reasons: (1) it contains 1 to 2% metallic impurities, and (2) it is produced as irregular shapes with a fine grain structure. Various purification techniques have been reported. The techniques being studied under this program use direct methods for the purification of MG-Si. The process uses sequential steps of purification followed by crystal growth. The steps of sequential purification include: (1) leaching of MG-Si charge, (2) phase separation of non-soluble impurities from molten silicon, (3) reactive gas treatment of molten silicon, (4) liquid-liquid extraction (called slagging), and (5) impurity redistribution using ingot pulling. All the purification steps, with the exception of step (1), are performed in a consecutive manner using a crystal puller. The purified ingots will be produced in a desired ingot dimension and further recrystallization is not necessary. The theory and experimental results for each purification technique are presented. The relative effectiveness of the various steps are assessed and the most important step(s) are recommended. Finally the electrical characteristics of solar cells built on a thin epitaxial layer deposited on single pulled MG-Si substrates are discussed and compared to single crystal substrates. (WHK)

  2. Purification, crystallization and preliminary X-ray diffraction analysis of the kinase domain of human tousled-like kinase 2

    SciTech Connect

    Garrote, Ana M.; Redondo, Pilar; Montoya, Guillermo; Muñoz, Inés G.

    2014-02-19

    The C-terminal kinase domain of TLK2 (a human tousled-like kinase) has been cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in chromatin dynamics, including DNA replication and repair, transcription and chromosome segregation. The two members of the family reported in humans, namely TLK1 and TLK2, localize to the cell nucleus and are capable of forming homo- or hetero-oligomers by themselves. To characterize the role of TLK2, its C-terminal kinase domain was cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. The latter produced the best diffracting crystal (3.4 Å resolution using synchrotron radiation), with unit-cell parameters a = b = c = 126.05 Å, α = β = γ = 90°. The asymmetric unit contained one protein molecule, with a Matthews coefficient of 4.59 Å{sup 3} Da{sup −1} and a solvent content of 73.23%.

  3. Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

    SciTech Connect

    Gao, Jinlan; Li, Xiaolu; Feng, Yue; Zhang, Bo; Miao, Shiying; Wang, Linfang; Wang, Na

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We truncated the signal peptide of OppA{sub TTE0054} to make it express in Escherichia coli as a soluble protein. Black-Right-Pointing-Pointer Crystals of OppA{sub TTE0054} were grown by sitting-drop vapor diffusion method. Black-Right-Pointing-Pointer The crystal of OppA{sub TTE0054} diffracted to 2.25 A. -- Abstract: Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 A. The crystal belonged to space group C222{sub 1}, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 A, and {alpha} = {beta} = {gamma} = 90 Degree-Sign .

  4. Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin

    SciTech Connect

    Jagadeesan, G.; Malathy, P.; Gunasekaran, K.; Harikrishna Etti, S.; Aravindhan, S.

    2014-10-25

    The great cormorant hemoglobin has been isolated, purified and crystallized and the three dimensional structure is solved using molecular replacement technique. Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3{sub 1}21, with unit-cell parameters a = b = 55.64, c = 153.38 Å, β = 120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

  5. Overexpression, purification, crystallization and preliminary X-ray diffraction of the nisin resistance protein from Streptococcus agalactiae.

    PubMed

    Khosa, Sakshi; Hoeppner, Astrid; Kleinschrodt, Diana; Smits, Sander H J

    2015-06-01

    Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively. PMID:26057793

  6. Overexpression, purification, crystallization and preliminary X-ray diffraction of the nisin resistance protein from Streptococcus agalactiae.

    PubMed

    Khosa, Sakshi; Hoeppner, Astrid; Kleinschrodt, Diana; Smits, Sander H J

    2015-06-01

    Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively.

  7. Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization.

    PubMed

    Galián, Carmen; Manon, Florence; Dezi, Manuela; Torres, Cristina; Ebel, Christine; Lévy, Daniel; Jault, Jean-Michel

    2011-01-01

    Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)∼5 s(-1)). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment. PMID:21602923

  8. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the regulator AcrR from Escherichia coli

    SciTech Connect

    Li, Ming; Qiu, Xi; Su, Chih-Chia; Long, Feng; Gu, Ruoyu; McDermott, Gerry; Yu, Edward W.

    2006-11-01

    The transcriptional regulator AcrR from Escherichia coli has been cloned, overexpressed, purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.5 Å. This paper describes the cloning, expression, purification and preliminary X-ray data analysis of the AcrR regulatory protein. The Escherichia coli AcrR is a member of the TetR family of transcriptional regulators. It regulates the expression of the AcrAB multidrug transporter. Recombinant AcrR with a 6×His tag at the C-terminus was expressed in E. coli and purified by metal-affinity chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted to 2.5 Å. The space group was determined to be P3{sub 2}, with unit-cell parameters a = b = 46.61, c = 166.16 Å.

  9. Purification, crystallization and preliminary crystallographic analysis of the 16S rRNA methyltransferase RsmI from Escherichia coli.

    PubMed

    Zhao, Mohan; Wang, Li; Zhang, Heng; Dong, Yuhui; Gong, Yong; Zhang, Linbo; Wang, Jian

    2014-09-01

    RsmI and RsmH are AdoMet-dependent methyltransferases that are responsible for the 2'-O-methylation and N(4)-methylation of C1402 of Escherichia coli 16S rRNA, respectively. Modification of this site has been found to play a role in fine-tuning the shape and function of the P-site to increase the decoding fidelity. It is interesting to study the mechanism by which C1402 can be methylated by both RsmI and RsmH. The crystal structure of RsmH in complex with AdoMet and cytidine has recently been determined and provided some implications for N(4)-methylation of this site. Here, the purification and crystallization of RsmI as well as its preliminary crystallographic analysis are reported. Co-crystallization of RsmI with AdoMet was carried out by the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 2.60 Å resolution on beamline 1W2B at BSRF. The crystal contained three molecules per asymmetric unit and belonged to space group C2, with unit-cell parameters a = 121.9, b = 152.5, c = 54.2 Å, β = 93.4°. PMID:25195904

  10. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus.

    PubMed

    Ferrero, Diego; Buxaderas, Mònica; Rodriguez, José F; Verdaguer, Núria

    2012-10-01

    Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2(1)2(1)2 and diffracts up to 2.1 Å and the RdRp-Lu(3+) derivative co-crystals belong to the C222(1) space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses. PMID:23027763

  11. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus

    PubMed Central

    Ferrero, Diego; Buxaderas, Mònica; Rodriguez, José F.; Verdaguer, Núria

    2012-01-01

    Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P21212 and diffracts up to 2.1 Å and the RdRp-Lu3+ derivative co-crystals belong to the C2221 space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses. PMID:23027763

  12. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of bacterioferritin A from Mycobacterium tuberculosis

    SciTech Connect

    Gupta, Vibha; Gupta, Rakesh K.; Khare, Garima; Salunke, Dinakar M.; Tyagi, Anil K.

    2008-05-01

    The cloning, purification and crystallization of a bacterioferritin from M. tuberculosis together with preliminary X-ray characterization of its crystals are reported. Bacterioferritins (Bfrs) comprise a subfamily of the ferritin superfamily of proteins that play an important role in bacterial iron storage and homeostasis. Bacterioferritins differ from ferritins in that they have additional noncovalently bound haem groups. To assess the physiological role of this subfamily of ferritins, a greater understanding of the structural details of bacterioferritins from various sources is required. The gene encoding bacterioferritin A (BfrA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein product was purified by affinity chromatography on a Strep-Tactin column and crystallized with sodium chloride as a precipitant at pH 8.0 using the vapour-diffusion technique. The crystals diffracted to 2.1 Å resolution and belonged to space group P4{sub 2}, with unit-cell parameters a = 123.0, b = 123.0, c = 174.6 Å.

  13. Expression, purification, crystallization and preliminary X-ray crystallographic studies of a novel acetylcitrulline deacetylase from Xanthomonas campestris

    SciTech Connect

    Shi, Dashuang Yu, Xiaolin; Roth, Lauren; Morizono, Hiroki; Hathout, Yetrib; Allewell, Norma M.; Tuchman, Mendel

    2005-07-01

    The expression, purification and preliminary X-ray diffraction studies of a novel N-acetyl-l-citrulline deacetylase from X. campestris are reported. A novel N-acetyl-l-citrulline deacetylase that is able to catalyze the hydrolysis of N-acetyl-l-citrulline to acetate and citrulline was identified from Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the monoclinic space group C2 and diffract to 1.75 Å resolution, with unit-cell parameters a = 94.13, b = 95.23, c = 43.61 Å, β = 93.76°. Since attempts to use homologous structural models to solve the structure via molecular replacement were unsuccessful, the selenomethionine-substituted protein was prepared using an overnight auto-induction overexpression system. Selenomethionine incorporation into the protein was verified by MALDI–TOF/TOF mass-spectroscopic analysis after trypsin digestion. The crystals of the selenomethionine-substituted protein were prepared using crystallization conditions similar to those for the native protein. Multiple anomalous dispersion (MAD) data were collected at Brookhaven National Laboratory. Structure determination is under way using the MAD phasing method.

  14. Gigas Meets Ulysses

    NASA Technical Reports Server (NTRS)

    2003-01-01

    [figure removed for brevity, see original site]

    Released 9 July 2003

    Roughly halfway between the great volcanoes of Olympus Mons and Pavonis Mons, the graben (troughs) of Ulysses Fossae intersect with the furrows of Gigas (gigantic) Sulci. A clear time sequence is evident: first came the formation of the sulci terrain (to the left), which then was fractured by graben radial to Olympus Mons, followed by flooding of lava. All but the deepest graben are filled by lava in the topographic low between the two volcanic rises.

    Image information: VIS instrument. Latitude 11.8, Longitude 234.3 East (125.7 West). 19 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  15. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

    SciTech Connect

    Gowda, Giri; Sagurthi, Someswar Rao; Savithri, H. S.; Murthy, M. R. N.

    2008-02-01

    The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.

  16. Cloning Expression Purification Crystallization and Preliminary X-ray Diffractino Studies of a 12R-LOX-chaperone Complex

    SciTech Connect

    G Deb; K Boeshanes; W Idler; B Ahvazi

    2011-12-31

    Lipoxygenases are a family of nonheme iron-containing dioxygenases. An Escherichia coli expression system producing the bacterial chaperones GroES and GroEL was engineered and successfully used to produce large quantities of recombinant human 12R-LOX (LOXR; MW 80.34 kDa; 701 amino-acid residues). The co-overproduction of the two chaperones with 12R-LOX resulted in increased solubility of 12R-LOX and allowed the purification of milligram amounts of active enzyme for structural studies by X-ray diffraction. The lipoxygenase protein was purified on an affinity column and a gel-filtration column with chaperone protein (MW 57.16 kDa). The LOXR-chaperone complex was crystallized with ligand by the hanging-drop vapor-diffusion method using 1.5 M ammonium hydrogen phosphate as precipitant. The crystals belonged to the monoclinic system, space group P2{sub 1}, with unit-cell parameters a = 138.97, b = 266.11, c = 152.26 {angstrom}, {beta} = 101.07{sup o}. Based on the calculated Matthews coefficient (3.1 {angstrom}3 Da{sup -1}), it is estimated that one molecule of LOXR complexed with two molecules of chaperone is present in the asymmetric unit of the crystal lattice. X-ray diffraction data were collected to 4 {angstrom} resolution using synchrotron radiation.

  17. Expression, purification, crystallization and preliminary X-ray analysis of YaeQ (XAC2396) from Xanthomonas axonopodis pv. citri

    SciTech Connect

    Guzzo, Cristiane R.; Nagem, Ronaldo A. P.; Galvão-Botton, Leonor M. P.; Guimarães, Beatriz G.; Medrano, Francisco J.; Barbosa, João A. R. G.; Farah, Chuck S.

    2005-05-01

    The first crystallographic study of a member of the YaeQ family of proteins, which are conserved in a small group of Gram-negative bacteria, most of which are animal or plant pathogens, is reported. Diffraction data were collected to 1.9 Å resolution and an interpretable electron-density map was obtained. Xanthomonas axonopodis pv. citri YaeQ (XAC2396) is a member of a family of bacterial proteins conserved in several Gram-negative pathogens. Here, the cloning, expression, purification and crystallization of the 182-residue (20.6 kDa) YaeQ protein are described. Recombinant YaeQ containing selenomethionine was crystallized in space group P2{sub 1} and crystals diffracted to 1.9 Å resolution at a synchrotron source. The unit-cell parameters are a = 39.75, b = 91.88, c = 48.03 Å, β = 108.37°. The calculated Matthews coefficient suggests the presence of two YaeQ molecules in the asymmetric unit. Initial experimental phases were calculated by the multiple-wavelength anomalous dispersion technique and an interpretable electron-density map was obtained.

  18. Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

    SciTech Connect

    Levin, Inna; Kessler, Naama; Moor, Nina; Klipcan, Liron; Koc, Emine; Templeton, Paul; Spremulli, Linda; Safro, Mark

    2007-09-01

    The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55, b = 90, c = 96 Å.

  19. Purification of Crystallization-Grade RNA Polymerase I from S. cerevisiae.

    PubMed

    Engel, Christoph

    2016-01-01

    Purification of RNA polymerase (Pol) I is essential for functional as well as for structural studies. The product needs to be extremely pure in order to exclude secondary effects, e.g., caused by copurified nucleic acids in subsequent experiments. For this purpose, the method presented here was originally introduced nearly a decade ago but underwent constant optimization [1]. The polymerase is extracted from its endogenous source, since no overexpression system for the entire 590 kDa, 14-subunit complex is available thus far. Following yeast cultivation, a number of standard protein purification techniques are applied and combined to a robust but elaborate procedure that takes 3 days. In brief, a yeast strain with histidine-tagged RNA polymerase I is fermented, cells are broken by bead beating, and cell debris is removed by a two-step centrifugation. The lysate is then dialyzed, the Pol-I-containing pellet resuspended, and polymerase I enriched by a His-trap affinity step, followed by sequential purification via anion and cation exchange and a final size exclusion chromatography. PMID:27576712

  20. Sequential purification and crystal growth for the production of low cost silicon substrates. Quarterly technical progress report No. 2, 1 January 1980-31 March 1980

    SciTech Connect

    Liaw, M; Aragona, F S

    1980-05-01

    The objective of this program is to identify and develop low cost processing for fabricating large grain size polycrystalline silicon substrates. Metallurgical grade silicon (MG-Si) which is low cost and abundant for industrial usage was chosen as starting material. However, MG-Si cannot be used directly as substrates for solar cell fabrication; the further purification and recrystallization of MG-Si are needed. The conventional method of purifying MG-Si requires the conversion of Si to gaseous chlorosilanes. Chlorosilanes are purified by distallation. The purified chlorosilanes are then converted back to elemental silicon using a chemical vapor deposition process. Purified polysilicon requires recrystallization to become single crystals or large grain polycrystalline form for electronic device or solar cell applications. The techniques being studied under this program use direct methods for the purification of MG-Si. The process uses sequential steps of purification followed by crystal growth. The steps of sequential purification include: (1) leaching of MG-Si charge, 2) phase separation of non-soluble impurities from molten silicon, 3) reactive gas treatment of molten silicon, 4) liquid-liquid extraction (called slagging), and 5) impurity redistribution using ingot pulling. All the purification steps are performed in a consecutive manner using a crystal puller with the exception of step (1). The purified ingots will be in a desired ingot dimension and further recrystallization is not necessary. In this quarterly period the study of the purification by (1) reactive gas treatment, and 2) slagging was completed. Selection of reusable crucible has also been made. Progress is reported.

  1. Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis

    SciTech Connect

    Meining, Winfried; Scheuring, Johannes; Fischer, Markus; Weinkauf, Sevil

    2006-06-01

    SecA ATPase from E. faecalis has been cloned, overexpressed, purified and crystallized. Crystals belong to space group C2 and diffract to 2.4 Å resolution. The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 Å resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 Å, α = γ = 90.0, β = 119.1°. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.

  2. High-level Expression Purification Crystallization and Preliminary X-ray Crystallographic Studies of the Receptor Binding Domain of botulinum neurotoxin Serotype D

    SciTech Connect

    Y Zhang; X Gao; G Buchko; H Robinson; S Varnum

    2011-12-31

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.

  3. High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor-binding domain of botulinum neurotoxin serotype D

    SciTech Connect

    Zhang, Y.; Robinson, H.; Gao, X.; Qin, L.; Buchko, G. W.; Varnum, S. M.

    2010-12-01

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.

  4. Purification, crystallization and preliminary X-ray analysis of an acetylxylan esterase from Bacillus pumilus.

    PubMed

    Benini, S; Degrassi, G; Krastanova, I; Lamba, D; Venturi, V

    2001-12-01

    The gene encoding for acetylxylan esterase from Bacillus pumilus has been cloned and expressed in Escherichia coli. The recombinant protein has been purified to homogeneity and crystallized. The crystals obtained are of regular shape of dimensions 0.05 x 0.05 x 0.05 mm with R32 symmetry and diffract to 2.0 A using synchrotron radiation.

  5. Purification, crystallization and preliminary X-ray analysis of inositol dehydrogenase (IDH) from Bacillus subtilis

    SciTech Connect

    Van Straaten, K. E.; Hoffort, A.; Palmer, D. R. J.; Sanders, D. A. R.

    2008-02-01

    Selenomethionine-substituted IDH was crystallized using the microbatch method. The crystals diffracted to beyond 2.0 Å resolution using synchrotron radiation. Inositol dehydrogenase (IDH) is an enzyme that catalyses the NAD{sup +}-dependent oxidation of myo-inositol to scyllo-inosose. The enzyme has been purified to homogeneity by means of Ni{sup 2+}-affinity chromatography and was crystallized in both native and selenomethionine (SeMet) labelled forms using the microbatch method. SAD X-ray diffraction data were collected to 2.0 Å resolution from a SeMet-labelled crystal at the Advanced Photon Source (APS) and a MAD data set was collected to 1.75 Å resolution at the Canadian Light Source (CLS); this is the first reported anomalous diffraction experiment from the CLS. The crystals belong to space group I222 and contain one molecule per asymmetric unit.

  6. Isolation, purification, crystallization, and preliminary X-ray diffraction study of the crystals of HU protein from M. gallisepticum

    NASA Astrophysics Data System (ADS)

    Nikolaeva, A. Yu.; Timofeev, V. I.; Boiko, K. M.; Korzhenevskii, D. A.; Rakitina, T. V.; Dorovatovskii, P. V.; Lipkin, A. V.

    2015-11-01

    HU proteins are involved in bacterial DNA and RNA repair. Since these proteins are absent in cells of higher organisms, inhibitors of HU proteins can be used as effective and safe antibiotics. The crystallization conditions for the M. gallisepticum HU protein were found and optimized by the vapor-diffusion method. The X-ray diffraction data set was collected to 2.91 Å resolution from the crystals grown by the vapor-diffusion method on a synchrotron source. The crystals of the HU protein belong to sp. gr. P41212 and have the following unit-cell parameters: a = b = 97.94 Å, c = 77.92 Å, α = β = γ = 90°.

  7. Production, Purification, Crystallization and Preliminary X-ray Structural Studies of Adeno-Associated Virus Serotype 5

    SciTech Connect

    DiMattia,M.; Govindasamy, L.; Levy, H.; Whitaker-Gurda, B.; Kohlbrenner, E.; Chiorini, J.; McKenna, R.; Muzyczka, N.; Zolotukhin, S.; Agbandje-McKenna, M.

    2005-01-01

    Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Angstroms resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Angstroms. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  8. Purification, crystallization and preliminary crystallographic analysis of the catalytic core of cystathionine β-synthase from Saccharomyces cerevisiae

    PubMed Central

    Ereño-Orbea, June; Majtan, Tomas; Oyenarte, Iker; Kraus, Jan P.; Martínez-Cruz, Luis Alfonso

    2014-01-01

    Cystathionine β-synthase (CBS; EC 4.2.1.22) catalyzes the condensation of homocysteine and serine to form cystathionine, with the release of water. In humans, deficiency in CBS activity is the most common cause of hyperhomocysteinaemia and homocystinuria. More than 160 pathogenic mutations in the human CBS gene have been described to date. Here, the purification and preliminary crystallographic analysis of the catalytic core of CBS from Saccharomyces cerevisiae (ScCBS) is described which, in contrast to other eukaryotic CBSs, lacks the N-terminal haem-binding domain and is considered to be a useful model for investigation of the pyridoxal-5′-phosphate-mediated reactions of human CBS (hCBS). The purified protein yielded two different crystal forms belonging to space groups P41212 and P212121, with unit-cell parameters a = b = 72.390, c = 386.794 Å and a = 58.156, b = 89.988, c = 121.687 Å, respectively. Diffraction data were collected to 2.7 and 3.1 Å resolution, respectively, using synchrotron radiation. Preliminary analysis of the X-ray data suggests the presence of ScCBS homodimers in both types of crystals. PMID:24598918

  9. Purification, crystallization and preliminary crystallographic analysis of the CBS-domain pair of cyclin M2 (CNNM2)

    PubMed Central

    Gómez-García, Inmaculada; Stuiver, Marchel; Ereño, June; Oyenarte, Iker; Corral-Rodríguez, María Angeles; Müller, Dominik; Martínez-Cruz, Luis Alfonso

    2012-01-01

    This work describes the purification and preliminary crystallographic analysis of the CBS-domain pair of the murine CNNM2 magnesium transporter (formerly known as ancient domain protein 2; ACDP2), which consists of a pair of cystathionine β-synthase (CBS) motifs and has 100% sequence identity to its human homologue. CNNM proteins represent the least-studied members of the eight different types of magnesium transporters identified to date in mammals. In humans, the CNNM family is encoded by four genes: CNNM1–4. CNNM1 acts as a cytosolic copper chaperone, whereas CNNM2 and CNNM4 have been associated with magnesium handling. Interestingly, mutations in the CNNM2 gene cause familial dominant hypomagnesaemia (MIM:607803), a rare human disorder characterized by renal and intestinal magnesium (Mg2+) wasting, which may lead to symptoms of Mg2+ depletion such as tetany, seizures and cardiac arrhythmias. This manuscript describes the preliminary crystallographic analysis of two different crystal habits of a truncated form of the protein containing its regulatory CBS-domain pair, which has been reported to host the pathological mutation T568I in humans. The crystals belonged to space groups P21212 and I222 (or I212121) and diffracted X-­rays to 2.0 and 3.6 Å resolution, respectively, using synchrotron radiation. PMID:23027747

  10. Purification, crystallization and preliminary X-ray diffraction analysis of pyridoxal kinase from Plasmodium falciparum (PfPdxK)

    PubMed Central

    Kronenberger, Thales; Lunev, Sergey; Wrenger, Carsten; Groves, Matthew R.

    2014-01-01

    Pyridoxal kinases (PdxK) catalyze the phosphorylation of vitamin B6 precursors. Thus, these enzymes are an essential part of many metabolic processes in all organisms. The protozoan parasite Plasmodium falciparum (the main causative agent of Malaria tropica) possesses a unique de novo B6-biosynthesis pathway in addition to a interconversion pathway based on the activity of plasmodial PdxK (PfPdxK). The role of PdxK in B6 salvage has prompted previous authors to suggest PdxK as a promising target for structure-based antimalarial drug design. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of PfPdxK are reported. PfPdxK crystals have been grown in space group P21, with unit-cell parameters a = 52.7, b = 62.0, c = 93.7 Å, β = 95°. A data set has been collected to 2 Å resolution and an initial molecular-replacement solution is described. PMID:25372829

  11. Purification, crystallization and preliminary X-ray diffraction analysis of the plant Rho protein ROP5

    SciTech Connect

    Thomas, Christoph Berken, Antje

    2007-12-01

    Crystals of the plant Rho protein ROP5 from A. thaliana have been obtained that diffract to 1.53 Å resolution. The small G protein ROP5 from the model plant Arabidopsis thaliana was purified and crystallized using the hanging-drop vapour-diffusion method. ROP5 crystals were obtained using PEG 3000 as precipitant and belong to space group P2{sub 1}. A data set was collected to 1.53 Å resolution using synchrotron radiation at 100 K. A clear molecular-replacement solution was found using ROP4–GDP of the ROP4–GDP–PRONE8 complex as the search model.

  12. Expression, purification, crystallization and preliminary crystallographic analysis of the PAB0955 gene product

    PubMed Central

    Gras, Stéphanie; Fernandez, Bernard; Chaumont, Valérie; Carpentier, Philippe; Armengaud, Jean; Housset, Dominique

    2005-01-01

    PAB0955 from Pyrococcus abyssi is a prototype of a new Walker-type ATPase/GTPase conserved in archaea and eukaryota but not found in bacteria. PAB0955 has been expressed, purified and crystallized, and it has been shown that this thermostable protein is dimeric in reductive conditions. Crystals have been obtained either without nucleotide or in the presence of GDP or GTPγS. Preliminary X-ray crystallographic data up to 2.08 Å resolution have been collected from these crystals. PMID:16510996

  13. Purification, crystallization and initial crystallographic characterization of peanut major allergen Ara h 3

    SciTech Connect

    Jin, Tengchuan; Howard, Andrew; Zhang, Yu-Zhu

    2007-10-01

    The crystallization of peanut allergen Ara h 3 is reported. The peanut is a significant food source, but is responsible for many cases of anaphylaxis. The peanut 11S legumin-like seed storage protein Ara h 3 is one of the best characterized allergens. In this study, Ara h 3 was extracted from peanut kernels and purified by sequential anion-exchange, hydrophobic interaction and gel-filtration chromatography to very high purity to facilitate crystallization and structural studies. Well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained and refinement of the structure is currently under way.

  14. Expression, purification, crystallization and preliminary X-ray analysis of tannase from Lactobacillus plantarum.

    PubMed

    Wu, Mingbo; Peng, Xiaohong; Wen, Hua; Wang, Qin; Chen, Qianming; McKinstry, William J; Ren, Bin

    2013-04-01

    Tannase catalyses the hydrolysis of the galloyl ester bond of tannins to release gallic acid. It belongs to the serine esterases and has wide applications in the food, feed, beverage, pharmaceutical and chemical industries. The tannase from Lactobacillus plantarum was cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method with microseeding. The crystals belonged to space group P1, with unit-cell parameters a = 46.5, b = 62.8, c = 83.8 Å, α = 70.4, β = 86.0, γ = 79.4°. Although the enzyme exists mainly as a monomer in solution, it forms a dimer in the asymmetric unit of the crystal. The crystals diffracted to beyond 1.60 Å resolution using synchrotron radiation and a complete data set was collected to 1.65 Å resolution.

  15. Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

    SciTech Connect

    Abramchik, Yu. A. Timofeev, V. I. Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2015-07-15

    Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is the biological active form of E. coli. purine nucleoside phosphorylase.

  16. Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

    SciTech Connect

    Bajaj, Mamta; Moriyama, Hideaki

    2007-05-01

    The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal. The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V{sub M} of 1.8 Å{sup 3} Da{sup −1}.

  17. Overexpression, purification and crystallization of the tetrameric form of SorC sorbitol operon regulator

    SciTech Connect

    Sanctis, Daniele de; Rêgo, Ana T.; Marçal, David; McVey, Colin E.; Carrondo, Maria A.; Enguita, Francisco J.

    2008-01-01

    The sorbitol operon regulator from K. pneumoniae has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 3.2 Å. The sorbitol operon regulator (SorC) regulates the metabolism of l-sorbose in Klebsiella pneumonia. SorC was overexpressed in Escherichia coli and purified, and crystals were obtained of a tetrameric form. A single crystal showed X-ray diffraction to 3.20 Å. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 91.6, b = 113.3, c = 184.1 Å. Analysis of the molecular-replacement solution indicates the presence of four SorC molecules in the asymmetric unit.

  18. Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum

    SciTech Connect

    Lohkamp, Bernhard; Andersen, Birgit; Piškur, Jure; Dobritzsch, Doreen

    2006-01-01

    Dihydropyrimidinase from the slime mould D. discoideum was crystallized. A single crystal was shown to belong to space group I222 and diffracted anisotropically to better than 1.8 Å. Dihydropyrimidinase (EC 3.5.2.2) is the second enzyme in the reductive pyrimidine-degradation pathway and catalyses the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated β-amino acids. The recombinant enzyme from the slime mould Dictyostelium discoideum was overexpressed, purified and crystallized by the vapour-diffusion method. One crystal diffracted to better than 1.8 Å resolution on a synchrotron source and was shown to belong to space group I222, with unit-cell parameters a = 84.6, b = 89.6, c = 134.9 Å and one molecule in the asymmetric unit.

  19. Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan)

    SciTech Connect

    Balasubramanian, Anuradha; Ponnuraj, Karthe

    2008-07-01

    Urease from pigeon pea was purified and crystallized and X-ray diffraction data were collected at 2.5 Å resolution. Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized and the resulting crystals diffracted to 2.5 Å resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 Å.

  20. Purification and crystallization of α-amylases from mucoid and non-mucoid B. amyloliquefaciens strains

    NASA Astrophysics Data System (ADS)

    Sarikaya, Elif; Mikami, Bunzo

    2001-11-01

    The purified α-amylases from mucoid and non-mucoid B. amyloliquefaciens strains were crystallized in forms suitable for X-ray diffraction analysis. Crystals were grown by the hanging-drop vapor diffusion method. Very thin crystals of α-amylase of non-mucoid strain were obtained in the presence 15% propanol, 12% PEG 6000 and 0.1 M PIPES (pH: 7.1) at the end of 3 months and these were not suitable for X-ray diffraction analysis. Crystals of the mucoid strain of B. amyloliquefaciens α-amylase were obtained with 30% PEG 6000, 0.2 M (NH 4) 2SO 4 and 0.1 M PIPES (pH: 6.5) at the end of 3 months and these were suitable for X-ray diffraction analysis.

  1. Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6

    SciTech Connect

    Bäuerle, Bettina; Sandalova, Tatyana; Schneider, Gunter; Rieger, Paul-Gerhard

    2006-08-01

    This is the first report of the crystallization of an IDS-epimerase from A. tumefaciens BY6 and its l-selenomethionine derivative. The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH{sub 4}){sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 Å, β = 103.3°, and diffracted to 1.7 Å resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the l-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na{sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 8.5.

  2. Expression, purification and crystallization of the Atg5–Atg16 complex essential for autophagy

    SciTech Connect

    Matsushita, Minako; Suzuki, Nobuo N.; Fujioka, Yuko; Ohsumi, Yoshinori; Inagaki, Fuyuhiko

    2006-10-01

    S. cerevisiae Atg5 in complex with the N-terminal regions of Atg16 was expressed, purified and crystallized in four crystal forms. Atg5 is a novel 34 kDa protein that is covalently modified by Atg12, a ubiquitin-like modifier, and forms a complex with Atg16. The Atg12–Atg5–Atg16 complex localizes to autophagosome precursors and plays an essential role in autophagosome formation. Saccharomyces cerevisiae Atg5 in complex with the N-terminal regions of Atg16 was expressed, purified and crystallized in four crystal forms. Forms I, II and III belong to space group P2{sub 1}, with unit-cell parameters a = 66.3, b = 104.4, c = 112.1 Å, β = 92.1° (form I), a = 79.5, b = 101.4, c = 95.1 Å, β = 98.6° (form II) or a = 56.9, b = 101.2, c = 66.5 Å, β = 100.6° (form III). Form IV belongs to space group P4{sub 2}2{sub 1}2, with unit-cell parameters a = 73.3, c = 148.1 Å. Diffraction data were collected from all crystal forms and high-resolution data to beyond 2.0 Å resolution were obtained from a form IV crystal.

  3. Solutal Convection Around Growing Protein Crystal and Diffusional Purification in Space

    NASA Technical Reports Server (NTRS)

    Lee, Chun P.; Chernov, Alexander A.

    2004-01-01

    At least some protein crystals were found to preferentially trap microheterogeneous impurities. The latter are, for example, dimmer molecules of the crystallizing proteines (e.g. ferritin, lysozyme), or the regular molecules on which surfaces small molecules or ions are adsorbed (e.g. acetilated lysozyme) and modi@ molecular charge. Impurities may induce lattice defects and deteriorate structural resolution. Distribution of impurities between mother solution and gorwing crystal is defined by two interrelated distribution coefficients: kappa = rho(sup c, sub 2) and K = (rho(sup c, sub 2)/rho(sup c, sub 1)/rho(sub 2)/rho(sub 1). Here, rho(sub 2), rho(sub 1) and rho(sup c, sub 2) are densities of impurity (2) and regular protein (1) in solution at the growing interface and within the crystal ("c"). For the microheterogeneous impurities studied, K approx. = 2 - 4, so that kappa approx. - 10(exp 2) - 10(exp 3), since K = kappa (rho(sub 1)/rho(sup c, sub 1) and protein solubility ratio rho(sub 1)/rho(sub=p c, sub 2) much less than 1. Therefore, a crystal growing in absence of convection purifies mother solution around itself, grows cleaner and, probably, more perfect. If convection is present, the solution flow permanently brings new impurities to the crystal. This work theoretically addressed two subjects: 1) onset of convection, 2) distribution of impurities.

  4. Purification, crystallization and preliminary X-ray diffraction analysis of human enolase-phosphatase E1

    SciTech Connect

    Wang, Hui; Pang, Hai; Ding, Yi; Li, Yi; Wu, Xiao’ai; Rao, Zihe

    2005-05-01

    Recombinant human E1 enzyme has been crystallized using the hanging-drop vapour-diffusion method and diffraction-quality crystals were grown at 291 K using PEG 4000 as precipitant. Enolase-phosphatase E1 (MASA) is a bifunctional enzyme in the ubiquitous methionine-salvage pathway and catalyzes the continuous reaction of 2,3-diketo-5-methylthio-1-phosphopentane to yield the acireductone metabolite. Recombinant human E1 enzyme has been crystallized using the hanging-drop vapour-diffusion method and diffraction-quality crystals were grown at 291 K using PEG 4000 as precipitant. Diffraction data were collected to 1.7 Å resolution from SeMet-derivative crystals at 100 K using synchrotron radiation. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 54.02, b = 57.55, c = 87.32 Å. The structure was subsequently solved by the multi-wavelength anomalous diffraction (MAD) phasing method.

  5. Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans.

    PubMed

    Morales-Rios, Edgar; Watt, Ian N; Zhang, Qifeng; Ding, Shujing; Fearnley, Ian M; Montgomery, Martin G; Wakelam, Michael J O; Walker, John E

    2015-09-01

    The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.

  6. Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain

    SciTech Connect

    Hall, Troii; Emmons, Thomas L.; Chrencik, Jill E.; Gormley, Jennifer A.; Weinberg, Robin A.; Leone, Joseph W.; Hirsch, Jeffrey L.; Saabye, Matthew J.; Schindler, John F.; Day, Jacqueline E.; Williams, Jennifer M.; Kiefer, James R.; Lightle, Sandra A.; Harris, Melissa S.; Guru, Siradanahalli; Fischer, H. David; Tomasselli, Alfredo G.

    2012-05-29

    Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69 mg per 20 L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3 mg per 10 L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K{sub m} and k{sub cat}) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4 {angstrom}. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

  7. Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

    PubMed Central

    Cheng, Hao; Fan, Chen; Zhang, Si-wei; Wu, Zhong-shan; Cui, Zhi-cheng; Melcher, Karsten; Zhang, Cheng-hai; Jiang, Yi; Cong, Yao; Xu, H Eric

    2015-01-01

    Aim: To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target. Methods: α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis. Results: Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel. Conclusion: We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis. PMID:26073323

  8. Purification, Crystallization, and Preliminary X-ray Analysis of Native Canavalin

    NASA Technical Reports Server (NTRS)

    Pusey, Marc; Dowell, Jennifer; Ng, Joseph; Gavira, Jose A.

    2003-01-01

    The protein canavalin is a 7S vicilin, from the Jack Bean, Canavalis ensfomis. Canavalin is described as a seed storage protein, an energy source for a developing seed, as no other known activity or function has been found. The protein was first isolated and crystallized by Sumner and Howell (J. Biol. Chem. 113, 607-610, 1936). Canavalin spontaneously crystallizes after proteolytic cleavage at neutral pH, which removes residues 1-46, 224-245, and 325-330 and produces peptides of approximately 25, 13, and 12 kDa. Preliminary gel filtration experiments indicated the presence of nucleic acid with the uncleaved protein. We developed a dual column procedure, ion exchange followed by hydroxy apatite chromatography, that effectively removes the nucleic acid and yields an essentially pure uncut canavalin preparation with an OD 280/260 ratio of approximately 1.9-2.0. Standard crystallization screens using this material gave a number of positive results having a common requirement for alcohols and Mg(2+) ion, with crystals typically appearing within a day or less. Optimization experiments to date have shown that we can obtain crystals from pH 6.5 to pH 8.2, using MPD from 5 to 20% and 0.05 to 0.2M Mg(2+) (sulfate or acetate). The crystals are of space group P2(sub 1)2(sub 1)2(sub 1), unit cell dimensions, and a complete data set to 1.5 Angstroms, resolution has now been collected at a synchrotron source. Most importantly, the crystals are not twinned, a persistent problem with the most commonly obtained rhombohedral form of proteolytically cleaved canavalin.

  9. Cloning, overexpression, purification and crystallization of malate dehydrogenase from Thermus thermophilus

    PubMed Central

    Chang, Yu-Yung; Hung, Chih-Hung; Hwang, Tzann-Shun; Hsu, Chun-Hua

    2013-01-01

    Malate dehydrogenase (MDH) has been used as a conjugate for enzyme immunoassay of a wide variety of compounds, such as drugs of abuse, drugs used in repetitive therapeutic application and hormones. In consideration of the various biotechnological applications of MDH, investigations of MDH from Thermus thermophilus were carried out to further understand the properties of this enzyme. The DNA fragment containing the open reading frame of mdh was amplified from the genomic DNA of T. thermophilus and cloned into the expression vector pET21b(+). The protein was expressed in a soluble form in Escherichia coli strain BL21(DE3). Homogeneous protein was obtained using a three-step procedure consisting of thermal treatment, Ni2+-chelating chromatography and size-exclusion chromatography. The purified MDH was crystallized and the crystals diffracted to a resolution of 1.80 Å on the BL13C1 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 71.3, b = 86.1, c = 118.2 Å. The unit-cell volume of the crystal is compatible with the presence of two monomers in the asymmetric unit, with a corresponding Matthews coefficient V M of 2.52 Å3 Da−1 and a solvent content of 51.2%. The crystal structure of MDH has been solved by molecular replacement and is currently under refinement. PMID:24192361

  10. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of pantothenate kinase from Mycobacterium tuberculosis

    SciTech Connect

    Das, Satyabrata; Kumar, Parimal; Bhor, Vikrant; Surolia, A. Vijayan, M.

    2005-01-01

    Pantothenate kinase, the first enzyme of the universal coenzyme A biosynthetic pathway, from M. tuberculosis H37Rv has been cloned, expressed, purified and X-ray analysed in two different crystal forms. Pantothenate kinase is an essential enzyme in the bacterial life cycle. It catalyzes the phosphorylation of pantothenate (vitamin B{sub 5}) to 4′-phosphopantothenate, the first step in the coenzyme A biosynthetic pathway. The enzyme from Mycobacterium tuberculosis, MW 35.7 kDa, has been cloned, expressed, purified and crystallized in two different trigonal crystal forms, both belonging to space group P3{sub 1}21. Two complete data sets of resolution 2.5 Å (form I) and 2.9 Å (form II) from crystals with unit-cell parameters a = b = 78.3, c = 115.45 Å and a = b = 107.63, c = 89.85 Å, respectively, were collected at room temperature on a home X-ray source. Structures of both crystal forms were solved for one subunit in the asymmetric unit by molecular replacement.

  11. Purification, crystallization and preliminary crystallographic investigation of FrnE, a disulfide oxidoreductase from Deinococcus radiodurans

    PubMed Central

    Panicker, Lata; Misra, Hari Sharan; Bihani, Subhash Chandra

    2014-01-01

    In prokaryotes, Dsb proteins catalyze the formation of native disulfide bonds through an oxidative folding pathway and are part of the cell machinery that protects proteins from oxidative stress. Deinococcus radiodurans is an extremophile which shows unparalleled resistance to ionizing radiation and oxidative stress. It has a strong mechanism to protect its proteome from oxidative damage. The genome of Deinococcus shows the presence of FrnE, a Dsb protein homologue that potentially provides the bacterium with oxidative stress tolerance. Here, crystallization and preliminary X-ray crystallographic analysis of FrnE from D. radiodurans are reported. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method with reservoir solution consisting of 100 mM sodium acetate pH 5.0, 10% PEG 8000, 15–20% glycerol. Diffraction data were collected on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 1.8 Å resolution at 100 K. The space group of the crystal was found to be P21221, with unit-cell parameters a = 47.91, b = 62.94, c = 86.75 Å, α = β = γ = 90°. Based on Matthews coefficient analysis, one monomer per asymmetric unit is present in the crystal, with a solvent content of approximately 45%. PMID:25372826

  12. Purification, crystallization and preliminary crystallographic investigation of FrnE, a disulfide oxidoreductase from Deinococcus radiodurans.

    PubMed

    Panicker, Lata; Misra, Hari Sharan; Bihani, Subhash Chandra

    2014-11-01

    In prokaryotes, Dsb proteins catalyze the formation of native disulfide bonds through an oxidative folding pathway and are part of the cell machinery that protects proteins from oxidative stress. Deinococcus radiodurans is an extremophile which shows unparalleled resistance to ionizing radiation and oxidative stress. It has a strong mechanism to protect its proteome from oxidative damage. The genome of Deinococcus shows the presence of FrnE, a Dsb protein homologue that potentially provides the bacterium with oxidative stress tolerance. Here, crystallization and preliminary X-ray crystallographic analysis of FrnE from D. radiodurans are reported. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method with reservoir solution consisting of 100 mM sodium acetate pH 5.0, 10% PEG 8000, 15-20% glycerol. Diffraction data were collected on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 1.8 Å resolution at 100 K. The space group of the crystal was found to be P2₁22₁, with unit-cell parameters a=47.91, b=62.94, c=86.75 Å, α=β=γ=90°. Based on Matthews coefficient analysis, one monomer per asymmetric unit is present in the crystal, with a solvent content of approximately 45%.

  13. Purification, crystallization and preliminary crystallographic analysis of a Thermostable Endonuclease IV from Thermotoga maritima

    SciTech Connect

    Coates, Leighton; Tomanicek, Stephen J; Hughes, Ronny C; NG, Joseph D; Demarse, Neil A

    2009-01-01

    The DNA repair enzyme Endonuclease IV from the thermophilic bacterium Thermotoga Maritima MSB8 (reference sequence: NC_000853) has been expressed in Escherichia coli and crystallized for X ray analysis. Thermotoga maritima Endonuclease IV is a 287 amino acid protein with 32% sequence identity to the Escherichia coli Endonuclease IV. The protein was purified to homogeneity and was crystallized using the sitting drop vapor diffusion method. The protein crystallized in the space group P61, with a composition of one biological molecule in the asymmetric unit corresponding to a Mathew s coefficient of 2.39 and a 47% solvent fraction. The unit cell parameters for the crystals are a = 123.23 , b = 123.23 , c = 35.34 , = = 90 , = 120 . Microseeding and further optimization yielded crystals with an X ray diffraction limit of 2.4 . A single 70 data set was collected and processed resulting in an overall Rmerge and completeness of 9.5% and 99.3% respectively.

  14. Expression, purification and crystallization of the SARS-CoV macro domain

    SciTech Connect

    Malet, Hélène; Dalle, Karen; Brémond, Nicolas; Tocque, Fabienne; Blangy, Stéphanie; Campanacci, Valérie; Coutard, Bruno; Grisel, Sacha; Lichière, Julie; Lantez, Violaine; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre

    2006-04-01

    The SARS-CoV macro domain was expressed, purified and crystallized. Selenomethionine-labelled crystals diffracted to 1.8 Å resolution. Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1′′-phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macro domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182–355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 37.5, b = 55.6, c = 108.9 Å, β = 91.4°, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8 Å.

  15. Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp

    SciTech Connect

    Linke, Christian; Caradoc-Davies, Tom T.; Proft, Thomas; Baker, Edward N.

    2008-02-01

    The S. pyogenes laminin-binding protein Lbp, which is essential for adhesion to human laminin, has been expressed, purified and crystallized. The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution.

  16. Purification, crystallization and preliminary crystallographic characterization of the caspase-recruitment domain of human Nod1

    SciTech Connect

    Srimathi, Thiagarajan; Robbins, Sheila L.; Dubas, Rachel L.; Seo, Jang-Hoon; Park, Young Chul

    2007-01-01

    The caspase-recruitment domain of the cytosolic pathogen receptor Nod1 was crystallized. X-ray diffraction data were collected to 1.9 Å resolution. The caspase-recruitment domain (CARD) is known to play an important role in apoptosis and inflammation as an essential protein–protein interaction domain. The CARD of the cytosolic pathogen receptor Nod1 was overexpressed in Escherichia coli and purified by affinity chromatography and gel filtration. The purified CARD was crystallized at 277 K using the microseeding method. X-ray diffraction data were collected to 1.9 Å resolution. The crystals belong to space group P3{sub 1} or P3{sub 2}, with unit-cell parameters a = b = 79.1, c = 80.9 Å. Preliminary analysis indicates that there is one dimeric CARD molecule in the asymmetric unit.

  17. Purification, crystallization and preliminary diffraction studies of an ectromelia virus glutaredoxin

    SciTech Connect

    Bacik, John-Paul; Brigley, Angela M.; Channon, Lisa D.; Audette, Gerald F.; Hazes, Bart

    2005-06-01

    Ectromelia virus glutaredoxin has been crystallized in the presence of the reducing agent DTT. A diffraction data set has been collected and processed to 1.8 Å resolution. Ectromelia, vaccinia, smallpox and other closely related viruses of the orthopoxvirus genus encode a glutaredoxin gene that is not present in poxviruses outside of this genus. The vaccinia glutaredoxin O2L has been implicated as the reducing agent for ribonucleotide reductase and may thus play an important role in viral deoxyribonucleotide synthesis. As part of an effort to understand nucleotide metabolism by poxviruses, EVM053, the O2L ortholog of the ectromelia virus, has been crystallized. EVM053 crystallizes in space group C222{sub 1}, with unit-cell parameters a = 61.98, b = 67.57, c = 108.55 Å. Diffraction data have been processed to 1.8 Å resolution and a self-rotation function indicates that there are two molecules per asymmetric unit.

  18. Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX

    SciTech Connect

    Weadge, J.T.; Robinson, H.; Yip, P. P.; Arnett, K.; Tipton, P. A.; Howell, P. L.

    2010-05-01

    AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method. The crystals grew as flat plates with unit-cell parameters a = 46.4, b = 120.6, c = 86.9 {angstrom}, {beta} = 95.7{sup o}. The crystals exhibited the symmetry of space group P2{sub 1} and diffracted to a minimum d-spacing of 2.1 {angstrom}. On the basis of the Matthews coefficient (V{sub M} = 2.25 {angstrom}{sup 3} Da{sup -1}), two molecules were estimated to be present in the asymmetric unit.

  19. Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgL.

    PubMed

    Wolfram, Francis; Arora, Kritica; Robinson, Howard; Neculai, Ana Mirela; Yip, Patrick; Howell, P Lynne

    2012-05-01

    The periplasmic alginate lyase AlgL is essential for the synthesis and export of the exopolysaccharide alginate in Pseudomonas sp. and also plays a role in its depolymerization. P. aeruginosa PAO1 AlgL has been overexpressed and purified and diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method. The crystals grew as thin plates, with unit-cell parameters a = 56.4, b = 59.6, c = 102.1 Å, α = β = γ = 90°. The AlgL crystals exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to a minimum d-spacing of 1.64 Å. Based on the Matthews coefficient (V(M) = 2.20 Å(3) Da(-1)), one molecule is estimated to be present in the asymmetric unit.

  20. Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto.

    PubMed

    Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

    2010-12-01

    Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27,724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a=74.3, b=49.9, c=56.3 Å, β=95.2°. Diffraction images were processed to a resolution of 1.74 Å with an Rmerge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase.

  1. Eukaryotic expression, purification, crystallization and preliminary X-ray analysis of murine Manic Fringe

    SciTech Connect

    Jinek, Martin; Conti, Elena

    2006-08-01

    The catalytic domain of the murine glycosyltransferase Manic Fringe was expressed in insect cells. Removal by site-directed mutagenesis of two N-glycosylation sites present in the protein was essential to obtain crystals that diffracted to 1.8 Å resolution. Fringe proteins are Golgi-resident β1,3-N-acetylglucosaminyltransferases that regulate development in metazoa through glycosylation of the Notch receptor and its ligands. The catalytic domain of murine Manic Fringe was expressed in the baculovirus/insect-cell system as a secreted protein. Mass-spectrometric analysis of the purified protein indicated the presence of two N-linked glycans. Abolishing the glycosylation sites by site-directed mutagenesis was necessary in order to obtain orthorhombic crystals that diffracted to 1.8 Å resolution. For phasing, a highly redundant data set was collected using a crystal soaked with halide salts.

  2. Purification, crystallization and initial crystallographic characterization of the Ginkgo biloba 11S seed globulin ginnacin

    SciTech Connect

    Jin, Tengchuan; Chen, Yu-Wei; Howard, Andrew; Zhang, Yu-Zhu

    2008-07-01

    The crystallization of ginnacin, the 11S seed storage protein from G. biloba, is reported. Ginkgo biloba, a well known ‘living fossil’ native to China, is grown worldwide as an ornamental shade plant. Medicinal and nutritional uses of G. biloba in Asia have a long history. However, ginkgo seed proteins have not been well studied at the biochemical and molecular level. In this study, the G. biloba 11S seed storage protein ginnacin was purified by sequential anion-exchange and gel-filtration chromatography. A crystallization screen was performed and well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained. There are six protomers in an asymmetric unit. Structure refinement is currently in progress.

  3. Purification and crystallization of dengue and West Nile virus NS2B–NS3 complexes

    SciTech Connect

    D’Arcy, Allan Chaillet, Maxime; Schiering, Nikolaus; Villard, Frederic; Lim, Siew Pheng; Lefeuvre, Peggy; Erbel, Paul

    2006-02-01

    Crystals of dengue serotype 2 and West Nile virus NS2B–NS3 protease complexes have been obtained and the crystals of both diffract to useful resolution. Sample homogeneity was essential for obtaining X-ray-quality crystals of the dengue protease. Controlled proteolysis produced a crystallizable fragment of the apo West Nile virus NS2B–NS3 and crystals were also obtained in the presence of a peptidic inhibitor. Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B–NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.

  4. Purification of Restriction Endonuclease EcoRII and its Co-Crystallization

    NASA Technical Reports Server (NTRS)

    Karpova, E. A.; Chen, L.; Meehan, E.; Pusey, M.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Restriction endonuclease EcoRII (EcoRII) is a homodimeric DNA-binding protein. It belongs to the type II family of restriction-modification enzymes (subclass IIe). EcoRII recognizes the nucleotide sequence 5'-CCWGG (W=A or T) and cleaves the phosphodiester bond preceding the first cytosine. Methylation at C5 of the second cytosine inhibits cleavage. The enzyme has a unique ability to search for the presence of two substrate sites before cleavage. To the best of our knowledge no other subclass IIe restriction endonuclease has been crystallized yet, without or with a DNA-substrate. We have recently grown and characterized the crystals of this enzyme (1) Here we report on the result of co-crystallization experiments of EcoRII with an 11 b.p. oligonucleotide substrate. The dissociation constant (Kd) EcoRII: 11 b.p. was determined earlier (unpublished results). The needle-like crystals of oligonucleotide-EcoRII protein complex were obtained with this substrate by the technique of vapor diffusion hanging drops. The crystals obtained were washed and dissolved in an aliquot of 10 mM Tris-HCl buffer, pH=7.5. Running a portion of this solution on the SDS-get indicated the presence of endonuclease in the solution. A UV-spectrophotometric test of a second portion confirmed the presence of DNA. We are now working on improvement of the DNA-EcoRII protein crystals. Results obtained from these and ongoing efforts will be reported.

  5. Expression, purification, crystallization and preliminary X-ray studies of Lactobacillus jensenii enolase

    SciTech Connect

    Harris, Paul T.; Raghunathan, Kannan; Spurbeck, Rachel R.; Arvidson, Cindy G.; Arvidson, Dennis N.

    2010-09-02

    Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25 {angstrom} resolution. The crystals belonged to space group I4, with unit-cell parameters a = b = 145.31, c = 99.79 {angstrom}. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient V{sub M} of 2.8 {angstrom}{sup 3} Da{sup -1}, corresponding to 55.2% solvent content.

  6. Isolation, purification, crystallization and preliminary crystallographic studies of chitinase from tamarind (Tamarindus indica) seeds.

    PubMed

    Patil, Dipak N; Datta, Manali; Chaudhary, Anshul; Tomar, Shailly; Sharma, Ashwani Kumar; Kumar, Pravindra

    2009-04-01

    A protein with chitinase activity has been isolated and purified from tamarind (Tamarindus indica) seeds. N-terminal amino-acid sequence analysis of this protein confirmed it to be an approximately 34 kDa endochitinase which belongs to the acidic class III chitinase family. The protein was crystallized by the vapour-diffusion method using PEG 4000. The crystals belonged to the tetragonal space group P4(1), with two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.6 A.

  7. Cloning, purification, crystallization and preliminary crystallographic analysis of a ribokinase from Staphylococcus aureus

    PubMed Central

    Wang, Lin; Wang, Haipeng; Ruan, Jianbin; Tian, Changlin; Sun, Baolin; Zang, Jianye

    2009-01-01

    The gene SA239 from Staphylococcus aureus encodes a ribokinase that catalyzes the phosphorylation of d-ribose to produce ribose-5-phosphate. Sa239 was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.9 Å resolution and belonged to space group P6122 or P6522, with unit-cell parameters a = b = 91.8, c = 160.7 Å. Preliminary crystallographic analysis revealed that the Matthews coefficient V M was 3.01 Å3 Da−1, indicating the presence of one molecule in the asymmetric unit. PMID:19478434

  8. Expression, purification and crystallization of a fungal type III polyketide synthase that produces the csypyrones

    PubMed Central

    Yang, Dengfeng; Mori, Takahiro; Matsui, Takashi; Hashimoto, Makoto; Morita, Hiroyuki; Fujii, Isao; Abe, Ikuro

    2014-01-01

    CsyB from Aspergillus oryzae is a novel type III polyketide synthase that catalyzes the formation of csypyrone B1 [4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid] from fatty acyl-CoA, malonyl-CoA and acetoacetyl-CoA. Recombinant CsyB expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space P21, with unit-cell parameters a = 70.0, b = 104.8, c = 73.5 Å, β = 114.4°. PMID:24915080

  9. Expression, purification and crystallization of an indole prenyltransferase from Aspergillus fumigatus

    PubMed Central

    Chen, Jing; Morita, Hiroyuki; Kato, Ryohei; Noguchi, Hiroshi; Sugio, Shigetoshi; Abe, Ikuro

    2012-01-01

    CdpNPT from Aspergillus fumigatus is a dimethylallyltryptophan synthase/indole prenyltransferase that catalyzes reverse prenylation at position N1 of tryptophan-containing cyclic dipeptides. Residues 38–440 of CdpNPT were expressed in Escherichia coli and crystallized using the sitting-drop vapour-diffusion and microseeding techniques. The crystals belonged to space group P212121, with unit-cell parameters a = 84.4, b = 157.1, c = 161.8 Å, α = β = γ = 90.0°. PMID:22442243

  10. Purification, crystallization and preliminary X-ray analysis of glutathione peroxidase Gpx3 from Saccharomyces cerevisiae

    SciTech Connect

    Yang, Zhu; Zhou, Cong-Zhao

    2006-06-01

    The glutathione peroxidase Gpx3 from the yeast S. cerevisiae has been overexpressed, purified, crystallized and diffracted to 2.6 Å resolution. Gpx3 is a monomer in solution which is different from its counterparts in mammals. The glutathione peroxidase Gpx3 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. Both gel-filtration and dynamic light-scattering (DLS) results indicate that Gpx3 is a monomer in solution at a concentration of about 2 mg ml{sup −1}, whereas glutathione peroxidases are normally tetrameric or dimeric. X-ray diffraction data from a single crystal of Gpx3 have been collected to 2.6 Å resolution. The crystals are triclinic and belong to space group P1, with unit-cell parameters a = 38.187, b = 43.372, c = 56.870 Å, α = 71.405, β = 73.376, γ = 89.633°. There are two Gpx3 monomers in a crystallographic asymmetric unit. Preliminary analyses show that the yeast Gpx3 is quite different from those of mammals.

  11. Overexpression, purification and crystallization of a choline-binding protein CbpI from Streptococcus pneumoniae

    SciTech Connect

    Paterson, Neil G. Riboldi-Tunicliffe, Alan; Mitchell, Timothy J.; Isaacs, Neil W.

    2006-07-01

    The choline-binding protein CbpI from S. pneumoniae has been purified and crystallized and diffraction data have been collected to 3.5 Å resolution. The choline-binding protein CbpI from Streptococcus pneumoniae is a 23.4 kDa protein with no known function. The protein has been successfully purified initially using Ni–NTA chromatography and to homogeneity using Q-Sepharose ion-exchange resin as an affinity column. CbpI was crystallized using PEG 3350 as a precipitant and X-ray crystallographic analysis showed that the crystals belonged to the tetragonal space group P4, with unit-cell parameters a = b = 83.31, c = 80.29 Å, α = β = γ = 90°. The crystal contains two molecules in the asymmetric unit with a solvent content of 55.7% (V{sub M} = 2.77 Å{sup 3} Da{sup −1}) and shows a diffraction limit of 3.5 Å.

  12. Refolding, purification and crystallization of the FrpB outer membrane iron transporter from Neisseria meningitidis.

    PubMed

    Saleem, Muhammad; Prince, Stephen M; Patel, Hema; Chan, Hannah; Feavers, Ian M; Derrick, Jeremy P

    2012-02-01

    FrpB is an integral outer membrane protein from the human pathogen Neisseria meningitidis. It is a member of the TonB-dependent transporter family and promotes the uptake of iron across the outer membrane. There is also evidence that FrpB is an antigen and hence a potential component of a vaccine against meningococcal meningitis. FrpB incorporating a polyhistidine tag was overexpressed in Escherichia coli into inclusion bodies. The protein was then solubilized in urea, refolded and purified to homogeneity. Two separate antigenic variants of FrpB were crystallized by sitting-drop vapour diffusion. Crystals of the F5-1 variant diffracted to 2.4 Å resolution and belonged to space group C2, with unit-cell parameters a = 176.5, b = 79.4, c = 75.9 Å, β = 98.3°. Crystal-packing calculations suggested the presence of a monomer in the asymmetric unit. Crystals of the F3-3 variant also diffracted to 2.4 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 85.3, b = 104.6, c = 269.1 Å. Preliminary analysis suggested the presence of an FrpB trimer in the asymmetric unit.

  13. Purification, characterization, and crystallization of single molecular species of beta-conglycinin from soybean seeds.

    PubMed

    Morita, S; Fukase, M; Yamaguchi, M; Fukuda, Y; Morita, Y

    1996-05-01

    Four major molecular species of beta-conglycinin, alpha 3, alpha 2 beta, alpha beta 2, and beta 3, were isolated and purified from seeds of an alpha' subunit-deficient strain of soybeans (Glycine max). All components were found to be homogeneous by high pressure liquid chromatography, SDS-polyacrylamide gel electrophoresis, and amino acid and amino terminal sequence analyses. The amino acid compositions of the alpha 3 and beta 3 components agreed fairly well with the compositions deduced from the cDNA sequences, and all of the components were highly glycosylated. The alpha 3 and beta 3 components were compared regarding their secondary structures. The secondary structure of the alpha 3 component deduced from CD measurements showed a higher alpha-helix content than that of the beta 3 component. The beta 3 component was crystallized by decreasing the ionic strength from 0.5 to 0.14 in phosphate buffer, pH 7.3, and the crystals grew to a size (1.0 mm x 0.2 mm x 0.2 mm) suitable for X-ray crystallographic analysis. A preliminary X-ray analysis showed that the crystal belonged to an orthorhombic crystal system having the space group P2(1)2(1)2(1) and unit cell dimensions of a = 185.1 A, b = 107.9 A, and c = 97.6 A.

  14. Purification, crystallization and preliminary crystallographic analysis of Arabidopsis thaliana imidazoleglycerol-phosphate dehydratase

    SciTech Connect

    Glynn, Steven E.; Baker, Patrick J.; Sedelnikova, Svetlana E.; Levy, Colin W.; Rodgers, H. Fiona; Blank, Jutta; Hawkes, Timothy R.; Rice, David W.

    2005-08-01

    Imidazoleglycerol-phosphate dehydratase from A. thaliana has been overexpressed, purified and crystallized and data have been collected to 3 Å resolution. Imidazoleglycerol-phosphate dehydratase catalyses the sixth step of the histidine-biosynthesis pathway in plants and microorganisms and has been identified as a possible target for the development of novel herbicides. Arabidopsis thaliana IGPD has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized in the presence of manganese. Under these conditions, the inactive trimeric form of the metal-free enzyme is assembled into a fully active species consisting of a 24-mer exhibiting 432 symmetry. X-ray diffraction data have been collected to 3.0 Å resolution from a single crystal at 293 K. The crystal belongs to space group R3, with approximate unit-cell parameters a = b = 157.9, c = 480.0 Å, α = β = 90, γ = 120° and with either 16 or 24 subunits in the asymmetric unit. A full structure determination is under way in order to provide insights into the mode of subunit assembly and to initiate a programme of rational herbicide design.

  15. The purification, crystallization and preliminary diffraction of a glycerophosphodiesterase from Enterobacter aerogenes

    SciTech Connect

    Jackson, Colin J.; Carr, Paul D.; Kim, Hye-Kyung; Liu, Jian-Wei; Ollis, David L.

    2006-07-01

    The metallo-glycerophosphodiesterase from E. aerogenes (GpdQ) has been cloned, expressed in E. coli and purified. Initial screening of crystallization conditions for this enzyme resulted in the identification of needles from one condition in a sodium malonate grid screen. Removal of the metals from the enzyme and subsequent optimization of these conditions led to crystals. The metallo-glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) has been cloned, expressed in Escherichia coli and purified. Initial screening of crystallization conditions for this enzyme resulted in the identification of needles from one condition in a sodium malonate grid screen. Removal of the metals from the enzyme and subsequent optimization of these conditions led to crystals that diffracted to 2.9 Å and belonged to space group P2{sub 1}3, with unit-cell parameter a = 164.1 Å. Self-rotation function analysis and V{sub M} calculations indicated that the asymmetric unit contains two copies of the monomeric enzyme, corresponding to a solvent content of 79%. It is intended to determine the structure of this protein utilizing SAD phasing from transition metals or molecular replacement.

  16. Expression, purification, crystallization and crystallographic study of Lutzomyia longipalpis LJL143

    SciTech Connect

    Kelleher, Alan; Liu, Zhuyun; Seid, Christopher A.; Zhan, Bin; Asojo, Oluwatoyin A.

    2015-06-27

    LJL143, a salivary protein from L. longipalpis, was produced using P. pastoris and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. Leishmaniasis is a neglected vector-borne disease with a global prevalence of over 12 million cases and 59 000 annual deaths. Transmission of the parasite requires salivary proteins, including LJL143 from the New World sandfly Lutzomyia longipalpis. LJL143 is a known marker of sandfly exposure in zoonotic hosts. LJL143 was crystallized from soluble protein expressed using Pichia pastoris. X-ray data were collected to 2.6 Å resolution from orthorhombic crystals belonging to space group P2{sub 1}2{sub 1}2{sub 1}, with average unit-cell parameters a = 57.39, b = 70.24, c = 79.58 Å. The crystals are predicted to have a monomer in the asymmetric unit, with an estimated solvent content of 48.5%. LJL143 has negligible homology to any reported structures, so the phases could not be determined by molecular replacement. All attempts at S-SAD failed and future studies include experimental phase determination using heavy-atom derivatives.

  17. Protein purification in multicompartment electrolyzers for crystal growth of r-DNA products in microgravity

    NASA Technical Reports Server (NTRS)

    Righetti, Pier Giorgio; Casale, Elena; Carter, Daniel; Snyder, Robert S.; Wenisch, Elisabeth; Faupel, Michel

    1990-01-01

    Recombinant-DNA (deoxyribonucleic acid) (r-DNA) proteins, produced in large quantities for human consumption, are now available in sufficient amounts for crystal growth. Crystallographic analysis is the only method now available for defining the atomic arrangements within complex biological molecules and decoding, e.g., the structure of the active site. Growing protein crystals in microgravity has become an important aspect of biology in space, since crystals that are large enough and of sufficient quality to permit complete structure determinations are usually obtained. However even small amounts of impurities in a protein preparation are anathema for the growth of a regular crystal lattice. A multicompartment electrolyzer with isoelectric, immobiline membranes, able to purify large quantities of r-DNA proteins is described. The electrolyzer consists of a stack of flow cells, delimited by membranes of very precise isoelectric point (pI, consisting of polyacrylamide supported by glass fiber filters containing Immobiline buffers and titrants to uniquely define a pI value) and very high buffering power, able to titrate all proteins tangent or crossing such membranes. By properly selecting the pI values of two membranes delimiting a flow chamber, a single protein can be kept isoelectric in a single flow chamber and thus, be purified to homogeneity (by the most stringent criterion, charge homogeneity).

  18. Overexpression, purification, crystallization and preliminary structural studies of catabolic ornithine transcarbamylase from Lactobacillus hilgardii

    PubMed Central

    de las Rivas, Blanca; Rodríguez, Héctor; Angulo, Iván; Muñoz, Rosario; Mancheño, José M.

    2007-01-01

    The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His6 tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8%(w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%(v/v) 2-methyl-2,4-pentanediol, 7.5%(w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, β = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å3 Da−1, respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code 1dxh) as the search model. PMID:17620711

  19. The complex between SOS3 and SOS2 regulatory domain from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis

    PubMed Central

    Sánchez-Barrena, María José; Moreno-Pérez, Sandra; Angulo, Iván; Martínez-Ripoll, Martín; Albert, Armando

    2007-01-01

    The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P212121, with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 Å. PMID:17620712

  20. Purification, crystallization and preliminary X-ray diffraction studies to near-atomic resolution of dihydrodipicolinate synthase from methicillin-resistant Staphylococcus aureus

    SciTech Connect

    Burgess, Benjamin R.; Dobson, Renwick C. J. Dogovski, Con; Jameson, Geoffrey B.; Parker, Michael W.; Perugini, Matthew A.

    2008-07-01

    Dihydrodipicolinate synthase (DHDPS), an enzyme of the lysine-biosynthetic pathway, is a promising target for antibiotic development against pathogenic bacteria. Here, the expression, purification, crystallization and preliminary diffraction analysis to 1.45 Å resolution of DHDPS from methicillin-resistant S. aureus is reported. In recent years, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has received considerable attention from both mechanistic and structural viewpoints. DHDPS is part of the diaminopimelate pathway leading to lysine, coupling (S)-aspartate-β-semialdehyde with pyruvate via a Schiff base to a conserved active-site lysine. In this paper, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from methicillin-resistant Staphylococcus aureus, an important bacterial pathogen, are reported. The enzyme was crystallized in a number of forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.45 Å resolution. The space group was P1 and the unit-cell parameters were a = 65.4, b = 67.6, c = 78.0 Å, α = 90.1, β = 68.9, γ = 72.3°. The crystal volume per protein weight (V{sub M}) was 2.34 Å{sup 3} Da{sup −1}, with an estimated solvent content of 47% for four monomers per asymmetric unit. The structure of the enzyme will help to guide the design of novel therapeutics against the methicillin-resistant S. aureus pathogen.

  1. Overexpression, purification, crystallization and preliminary structural studies of catabolic ornithine transcarbamylase from Lactobacillus hilgardii

    SciTech Connect

    Rivas, Blanca de las; Rodríguez, Héctor; Angulo, Iván; Muñoz, Rosario; Mancheño, José M.

    2007-07-01

    The catabolic ornithine transcarbamylase (cOTC) from L. hilgardii has been overexpressed in E. coli, purified and crystallized under two different experimental conditions. The structure has been solved by the molecular-replacement method using the atomic coordinates of catabolic ornithine transcarbamylase from P. aeruginosa as the search model. The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His{sub 6} tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8%(w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%(v/v) 2-methyl-2,4-pentanediol, 7.5%(w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, β = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å{sup 3} Da{sup −1}, respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code) as the search model.

  2. Purification, crystallization and preliminary X-ray diffraction of human S100A15

    SciTech Connect

    Boeshans, Karen M.; Wolf, Ronald; Voscopoulos, Christopher; Gillette, William; Esposito, Dominic; Mueser, Timothy C.; Yuspa, Stuart H.; Ahvazi, Bijan

    2006-05-01

    S100 proteins are differentially expressed during epithelial cell maturation, tumorigenesis and inflammation. The novel human S100A15 protein has been cloned, expressed, purified and crystallized in two crystal forms, a triclinic and a monoclinic form, which diffract to 1.7 and 2.0 Å, respectively. Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 Å, α = 71.2, β = 68.1, γ = 67.8° and an estimated two molecules in the asymmetric unit, and diffract to 1.7 Å resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 Å, β = 128.2° and an estimated one molecule in the asymmetric unit, and diffract to 2.0 Å resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.

  3. Expression, purification, enzymatic characterization and crystallization of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa.

    PubMed

    Machado, Agnes Thiane Pereira; Silva, Marcio; Iulek, Jorge

    2016-11-01

    Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characterization and crystallization of N. gruberi GAPDH, the first one for an organism belonging to phylum Percolozoa. The results indicated that 10 mM, 8.0 and 25 °C are the optimum arsenate concentration, pH and temperature, respectively. The enzyme presents allosteric positive cooperativity for substrates NAD(+) and G3P as indicated by the Hill coefficients. The phylogenetic proximity between N. fowleri and N. gruberi suggests that contributions from the study of the latter might provide information to assist the search for treatments of Primary Amebic Meningoencephalitis, especially, in this work, taking into account that GAPDH is identified as a therapeutic target. PMID:27426132

  4. SOS3 (salt overly sensitive 3) from Arabidopsis thaliana: expression, purification, crystallization and preliminary X-ray analysis.

    PubMed

    Sánchez-Barrena, M J; Martínez-Ripoll, M; Zhu, J K; Albert, A

    2004-07-01

    The salt-tolerance gene SOS3 (salt overly sensitive 3) of Arabidopsis thaliana encodes a calcium-binding protein that is able to sense the cytosolic calcium signal elicited by salt stress. SOS3 activates the SOS2 protein kinase, which activates various ion transporters. SOS3 was cloned into a plasmid and expressed in Escherichia coli, allowing purification of the protein to homogeneity. Two crystals with different additive contents were grown. Both diffract to 3.2 A resolution and belong to space group I4(1), with unit-cell parameters a = 93.65, c = 80.08 A and a = 91.79, c = 85.78 A, respectively. A promising molecular-replacement solution has been found using neuronal calcium-sensor 1 as the search model. Interestingly, no solution was found using AtCBL2 (A. thaliana calcineurin B-like protein) structure as a search model, although this protein belongs to the same family and displays 50% sequence identity.

  5. Studies on the "Aerobic" Acetyl-Coenzyme A Synthetase of Saccharomyces Cerevisiae: Purification, Crystallization, and Physical Properties of the Enzyme

    NASA Technical Reports Server (NTRS)

    Satyanarayana, T.; Klein, Harold P.

    1976-01-01

    A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis. Sedimentation velocity runs revealed a single symmetric peak with an s(sub (20,w)) value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 +/- 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 +/- 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic Saccharomyces cerevisiae is composed of three subunits of identical or nearly identical size.

  6. Expression, purification, enzymatic characterization and crystallization of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa.

    PubMed

    Machado, Agnes Thiane Pereira; Silva, Marcio; Iulek, Jorge

    2016-11-01

    Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characterization and crystallization of N. gruberi GAPDH, the first one for an organism belonging to phylum Percolozoa. The results indicated that 10 mM, 8.0 and 25 °C are the optimum arsenate concentration, pH and temperature, respectively. The enzyme presents allosteric positive cooperativity for substrates NAD(+) and G3P as indicated by the Hill coefficients. The phylogenetic proximity between N. fowleri and N. gruberi suggests that contributions from the study of the latter might provide information to assist the search for treatments of Primary Amebic Meningoencephalitis, especially, in this work, taking into account that GAPDH is identified as a therapeutic target.

  7. Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutS[beta

    SciTech Connect

    Tseng, Quincy; Orans, Jillian; Hast, Michael A.; Iyer, Ravi R.; Changela, Anita; Modrich, Paul L.; Beese, Lorena S.

    2012-03-16

    MutS{beta} is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutS{alpha} (MSH2-MSH6). Although mismatch recognition by MutS{alpha} has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutS{beta}. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification of recombinant human MutS{beta} and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.

  8. Expression, Purification, Crystallization And Preliminary X-Ray Studies of Histamine Dehydrogenase From Nocardioides Simplex

    SciTech Connect

    Reed, T.M.; Hirakawa, H.; Mure, M.; Scott, E.E.; Limburg, J.

    2009-05-21

    Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 {angstrom} resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 {angstrom}.

  9. Purification, crystallization and preliminary X-ray analysis of a hexameric beta-glucosidase from wheat.

    PubMed

    Sue, Masayuki; Yamazaki, Kana; Kouyama, Jun-ichi; Sasaki, Yasuyuki; Ohsawa, Kanju; Miyamoto, Toru; Iwamura, Hajime; Yajima, Shunsuke

    2005-09-01

    The wheat beta-glucosidase TaGlu1b, which is only active in a hexameric form, was tagged with 6xHis at the N-terminus, overexpressed in Escherichia coli and purified in two steps. The protein complexed with a substrate aglycone was crystallized at 293 K from a solution containing 10 mM HEPES pH 7.2, 1 M LiSO4 and 150 mM NaCl using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.7 A at the Photon Factory. The crystal belongs to space group P4(1)32, with unit-cell parameters a = b = c = 194.65 A, alpha = beta = gamma = 90 degrees. The asymmetric unit was confirmed by molecular-replacement solution to contain one monomer, giving a solvent content of 72.1%. PMID:16511181

  10. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Bifidobacterium adolescentis xylose isomerase

    PubMed Central

    dos Reis, Caio Vinicius; Bernardes, Amanda; Polikarpov, Igor

    2013-01-01

    Xylose isomerase (EC 5.3.1.5) is a key enzyme in xylose metabolism which is industrially important for the transformation of glucose and xylose into fructose and xylulose, respectively. The Bifidobacterium adolescentis xylA gene (NC_008618.1) encoding xylose isomerase (XI) was cloned and the enzyme was overexpressed in Escherichia coli. Purified recombinant XI was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as the precipitating agent. A complete native data set was collected to 1.7 Å resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 88.78, b = 123.98, c = 78.63 Å. PMID:23695585

  11. Purification and crystallization of the human EF-hand tumour suppressor protein S100A2

    PubMed Central

    Koch, Michael; Diez, Joachim; Fritz, Günter

    2006-01-01

    S100A2 is a Ca2+-binding EF-hand protein that is mainly localized in the nucleus. There, it acts as a tumour suppressor by binding and activating p53. Wild-type S100A2 and a S100A2 variant lacking cysteines have been purified. CD spectroscopy showed that there are no changes in secondary-structure composition. The S100A2 mutant was crystallized in a calcium-free form. The crystals, with dimensions 30 × 30 × 70 µm, diffract to 1.7 Å and belong to space group P212121, with unit-cell parameters a = 43.5, b = 57.8, c = 59.8 Å, α = β = γ = 90°. Preliminary analysis of the X-ray data indicates that there are two subunits per asymmetric unit. PMID:17077493

  12. Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsG

    SciTech Connect

    Jens, Jason; Raghunathan, Kannan; Vago, Frank; Arvidson, Dennis

    2010-01-12

    EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 {angstrom} and belonged to space group P2{sub 1}, with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 {angstrom}.

  13. Purification and lipid-layer crystallization of yeast RNA polymerase II.

    PubMed Central

    Edwards, A M; Darst, S A; Feaver, W J; Thompson, N E; Burgess, R R; Kornberg, R D

    1990-01-01

    Yeast RNA polymerase II was purified to homogeneity by a rapid procedure involving immunoaffinity chromatography. The purified enzyme contained 10 subunits, as reported for conventional preparations, but with no detectable proteolysis of the largest subunit. In assays of initiation of transcription at the yeast CYC1 promoter, the enzyme complemented the deficiency of an extract from a strain that produces a temperature-sensitive polymerase II. Mammalian RNA polymerase II was inactive in this initiation assay. The purified yeast enzyme formed two-dimensional crystals on positively charged lipid layers, as previously found for Escherichia coli RNA polymerase holoenzyme. Image analysis of electron micrographs of crystals in negative stain, which diffracted to about 30-A resolution, showed protein densities of dimensions consistent with those of single polymerase molecules. Images PMID:2179949

  14. Expression, purification and crystallization of the ammonium transporter Amt-1 from Archaeoglobus fulgidus

    SciTech Connect

    Andrade, Susana L. A. Dickmanns, Antje; Ficner, Ralf; Einsle, Oliver

    2005-09-01

    The ammonium transporter Amt-1 from the cytoplasmic membrane of the hyperthermophilic archaeon A. fulgidus has been purified and crystallized. Ammonium transporters (Amts) are a class of membrane-integral transport proteins found in organisms from all kingdoms of life. Their key function is the transport of nitrogen in its reduced bioavailable form, ammonia, across cellular membranes, a crucial step in nitrogen assimilation for biosynthetic purposes. The genome of the hyperthermophilic archaeon Archaeoglobus fulgidus has been annotated with three individual genes for ammonium transporters, amt1–3, the roles of which are as yet unknown. The amt1 gene product has been produced by heterologous overexpression in Escherichia coli and the resulting protein has been purified to electrophoretic homogeneity. Crystals of Amt-1 have been obtained by sitting-drop vapour diffusion and diffraction data have been collected.

  15. Cloning, purification, crystallization and preliminary crystallographic analysis of acylphosphatase from Pyrococcus horikoshii OT3.

    PubMed

    Miyazono, Ken Ichi; Kudo, Norio; Tanokura, Masaru

    2004-06-01

    Acylphosphatase is one of the smallest enzymes and catalyzes the hydrolysis of the carboxy-phosphate bond. An extremely thermostable acylphosphatase from a hyperthermophilic archaea, Pyrococcus horikoshii OT3, has been cloned, expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with potassium/sodium tartrate as the precipitant at pH 5.5. X-ray diffraction data have been collected to a highest resolution of 1.72 angstroms on a synchrotron-radiation source. The crystals belong to space group P3(2)21, with approximate unit-cell parameters a = b = 86.6, c = 75.4 angstroms and two monomers in the asymmetric unit.

  16. Purification, crystallization and initial crystallographic characterization of brazil-nut allergen Ber e 2

    SciTech Connect

    Guo, Feng; Jin, Tengchuan; Howard, Andrew; Zhang, Yu-Zhu

    2007-11-01

    The crystallization of the brazil nut allergen Ber e 2 is reported. Peanut and tree-nut allergies have attracted considerable attention because of their frequency and their lifelong persistence. Brazil-nut (Bertholletia excelsa) allergies have been well documented and the 11S legumin-like seed storage protein Ber e 2 (excelsin) is one of the two known brazil-nut allergens. In this study, Ber e 2 was extracted from brazil-nut kernels and purified to high purity by crystalline precipitation and gel-filtration chromatography. Well diffracting single crystals were obtained using the hanging-drop vapour-diffusion method. A molecular-replacement structural solution has been obtained. Refinement of the structure is currently under way.

  17. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Zucchini from Drosophila melanogaster.

    PubMed

    Fukuhara, Satoshi; Nishimasu, Hiroshi; Bonnefond, Luc; Matsumoto, Naoki; Ishitani, Ryuichiro; Nureki, Osamu

    2012-11-01

    PIWI-interacting RNAs (piRNAs) bind PIWI proteins and silence transposons to maintain the genomic integrity of germ cells. Zucchini (Zuc), a phospholipase D superfamily member, is conserved among animals and is implicated in piRNA biogenesis. However, the underlying mechanism by which Zuc participates in piRNA biogenesis remains elusive. Drosophila melanogaster Zuc (DmZuc) was expressed in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 1.75 Å resolution. The crystal belonged to space group P2(1), with unit-cell parameters a=55.0, b=71.2, c=56.3 Å, β=107.9°.

  18. Purification, crystallization and preliminary crystallographic analysis of recombinant Lac15 from a marine microbial metagenome.

    PubMed

    Ge, Honghua; Xu, Peisong; Xu, Ying; Fang, Zemin; Xiao, Yazhong

    2011-08-01

    Laccases are members of the blue multi-copper oxidase family that can oxidize a wide range of aromatic compounds. A new bacterial laccase (Lac15) has recently been obtained from a marine microbial metagenome from the South China Sea and characterized. In this work, recombinant Lac15 was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.2 Å resolution. The crystal belonged to space group C121, with unit-cell parameters a = 123.41, b = 91.36, c = 86.157 Å, β = 112.10°.

  19. Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    SciTech Connect

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E.

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2{sub 1}; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3{sub 2}. The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.

  20. Cloning, sequencing, purification, and crystal structure of Grenache (Vitis vinifera) polyphenol oxidase.

    PubMed

    Virador, Victoria M; Reyes Grajeda, Juan P; Blanco-Labra, Alejandro; Mendiola-Olaya, Elizabeth; Smith, Gary M; Moreno, Abel; Whitaker, John R

    2010-01-27

    The full-length cDNA sequence (P93622_VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C222(1). The structure was obtained at 2.2 A resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1 ) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and alpha, beta, and gamma) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed. PMID:20039636

  1. Solutal Convection Around Growing Protein Crystal and Diffusional Purification in Space

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.; Lee, C. P.

    2002-01-01

    This work theoretically addressed two subjects: 1) onset of convection, 2) distribution of impurities. Onset of convection was considered analytically and numerically. Crystal growth was characterized by slow surface incorporation kinetics, i.e. growth kinetic coefficient beta (cm/s) small as compared to the typical bulk diffusion rate, D(sub 1)/h, where D(sub 1) is diffusivity of major crystallizing protein and h is the crystal size. Scaling type analysis predicted two laws on how the convection rate, v, essentially the Peclet number, Pe exactly equal to vh/D(sub 1), depends on dimensionless kinetic coefficient a exactly equal to beta h/D(sub 1). Namely: Pe = C(sub 2/5)(aRa(sup 2/5)) and Pe = C(sub 1) aRa. Here, Reynolds number Ra = rho(sub 1)(sup 0)gh(sup 3)(rho(sub p) - rho(sub w))/rho(sup p)rho(sub 1)vD(sub 1), v being solution viscosity. The constants C(sub 2/5), exactly equal to 0.28 and C(sub 1) exactly equal to 10(exp -2) found from the full scale computer simulation for a cylindrical crystal inside big cylindrical vessel. The linear boundary conditions connecting protein and impurity concentration at the interface with the flux to/from the interface was applied. No-slip condition for Navier-Shocker equations was employed. With these conditions, flow and concentration distributions were calculated. Validity of the Pe(Ra) dependencies follows for wide range of parameters for which numerical calculations have been accomplished and presented by various points.

  2. Cloning, Sequencing, Purification, and Crystal Structure of Grenache (Vitis vinifera) Polyphenol Oxidase

    SciTech Connect

    Virador, V.; Reyes Grajeda, J; Blanco-Labra, A; Mendiola-Olaya, E; Smith, G; Moreno, A; Whitaker, J

    2010-01-01

    The full-length cDNA sequence (P93622{_}VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C2221. The structure was obtained at 2.2 {angstrom} resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and {alpha}, {beta}, and {gamma}) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed.

  3. Purification, crystallization and preliminary X-ray analysis of a hexameric β-glucosidase from wheat

    SciTech Connect

    Sue, Masayuki; Yamazaki, Kana; Kouyama, Jun-ichi; Sasaki, Yasuyuki; Ohsawa, Kanju; Miyamoto, Toru; Iwamura, Hajime; Yajima, Shunsuke

    2005-09-01

    Recombinant β-glucosidase from wheat seedlings complexed with a substrate aglycone has been crystallized in a hexameric active form. A diffraction data set has been collected at 1.7 Å. The wheat β-glucosidase TaGlu1b, which is only active in a hexameric form, was tagged with 6×His at the N-terminus, overexpressed in Escherichia coli and purified in two steps. The protein complexed with a substrate aglycone was crystallized at 293 K from a solution containing 10 mM HEPES pH 7.2, 1 M LiSO{sub 4} and 150 mM NaCl using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.7 Å at the Photon Factory. The crystal belongs to space group P4{sub 1}32, with unit-cell parameters a = b = c = 194.65 Å, α = β = γ = 90°. The asymmetric unit was confirmed by molecular-replacement solution to contain one monomer, giving a solvent content of 72.1%.

  4. Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum

    SciTech Connect

    Rodríguez, Héctor; Rivas, Blanca de las; Muñoz, Rosario; Mancheño, José M.

    2007-04-01

    The enzyme p-coumaric acid decarboxylase (PDC) from L. plantarum has been recombinantly expressed, purified and crystallized. The structure has been solved at 2.04 Å resolution by the molecular-replacement method. The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His{sub 6}-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris–HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 Å resolution. Crystals belonged to the tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 43.15, c = 231.86 Å. The estimated Matthews coefficient was 2.36 Å{sup 3} Da{sup −1}, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism.

  5. Expression, purification and crystallization of a human tau-tubulin kinase 2 that phosphorylates tau protein

    SciTech Connect

    Kitano-Takahashi, Michiko; Morita, Hiroyuki; Kondo, Shin; Tomizawa, Kayoko; Kato, Ryohei; Tanio, Michikazu; Shirota, Yoshiko; Takahashi, Hiroshi; Sugio, Shigetoshi; Kohno, Toshiyuki

    2007-07-01

    The kinase domain (residues 1–331) of human tau-tubulin kinase 2 was expressed in insect cells, purified and crystallized. Diffraction data have been collected to 2.9 Å resolution. Tau-tubulin kinase 2 (TTBK2) is a Ser/Thr kinase that putatively phosphorylates residues Ser208 and Ser210 (numbered according to a 441-residue human tau isoform) in tau protein. Functional analyses revealed that a recombinant kinase domain (residues 1–331) of human TTBK2 expressed in insect cells with a baculovirus overexpression system retains kinase activity for tau protein. The kinase domain of TTBK2 was crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55.6, b = 113.7, c = 117.3 Å, α = β = γ = 90.0°. Diffraction data were collected to 2.9 Å resolution using synchrotron radiation at BL24XU of SPring-8.

  6. Expression, purification and crystallization of l-methionine γ-lyase 2 from Entamoeba histolytica

    SciTech Connect

    Sato, Dan; Yamagata, Wataru; Kamei, Kaeko; Nozaki, Tomoyoshi; Harada, Shigeharu

    2006-10-01

    l-Methionine γ-lyase 2 from E. histolytica, a key enzyme in sulfur-containing amino-acid degradation in this protozoan parasite, has been crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 Å. The crystal diffracted to a resolution of 2.0 Å. The presence of a tetramer in the asymmetric unit (4 × 43.1 kDa) gives a Matthews coefficient of 2.2 Å{sup 3} Da{sup −1}. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

  7. Expression, purification, crystallization and crystallographic study of Lutzomyia longipalpis LJL143.

    PubMed

    Kelleher, Alan; Liu, Zhuyun; Seid, Christopher A; Zhan, Bin; Asojo, Oluwatoyin A

    2015-07-01

    Leishmaniasis is a neglected vector-borne disease with a global prevalence of over 12 million cases and 59,000 annual deaths. Transmission of the parasite requires salivary proteins, including LJL143 from the New World sandfly Lutzomyia longipalpis. LJL143 is a known marker of sandfly exposure in zoonotic hosts. LJL143 was crystallized from soluble protein expressed using Pichia pastoris. X-ray data were collected to 2.6 Å resolution from orthorhombic crystals belonging to space group P2(1)2(1)2(1), with average unit-cell parameters a = 57.39, b = 70.24, c = 79.58 Å. The crystals are predicted to have a monomer in the asymmetric unit, with an estimated solvent content of 48.5%. LJL143 has negligible homology to any reported structures, so the phases could not be determined by molecular replacement. All attempts at S-SAD failed and future studies include experimental phase determination using heavy-atom derivatives.

  8. Purification, Crystallization and Preliminary X-ray Crystallographic Studies of RAIDD Death-Domain (DD)

    SciTech Connect

    Jang, T.; Park, H

    2009-01-01

    Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3121 (or its enantiomorph P3221) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 and ? = 120 degrees. The crystals were obtained at room temperature and diffracted to 2.0 A resolution.

  9. Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis

    SciTech Connect

    Rathinaswamy, Priya; Pundle, Archana V.; Prabhune, Asmita A.; SivaRaman, Hepzibah; Brannigan, James A. Dodson, Guy G.; Suresh, C. G.

    2005-07-01

    An unannotated protein reported from B. subtilis has been expressed in E. coli and identified as possessing penicillin V acylase activity. The crystallization and preliminary crystallographic analysis of this penicillin V acylase is presented. Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their β-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris–HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 Å. The crystals belonged to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 Å. The estimated Matthews coefficient was 3.23 Å{sup 3} Da{sup −1}, corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.

  10. Cloning, overexpression, purification and crystallization of the CRN12 coiled-coil domain from Leishmania donovani

    PubMed Central

    Srivastava, Vijay Kumar; Rana, Ajay Kumar; Sahasrabuddhe, Amogh A.; Gupta, C. M; Pratap, J. V.

    2013-01-01

    Leishmania donovani coronin CRN12 is an actin-binding protein which consists of two domains: an N-terminal WD repeat domain and a C-terminal coiled-coil domain. The coiled-coil domain is 53 residues in length. Helix–helix interactions in general and coiled coils in particular are ubiquitous in the structure of proteins and play a significant role in the association among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signalling, transport and actin dynamics. The L. donovani coronin CRN12 coiled-coil domain (5.8 kDa) was cloned, overexpressed, purified to homogeneity and the N-terminal 6×His tag was successfully removed by thrombin cleavage. Crystals of recombinant L. donovani coronin CRN12 coiled-coil domain were grown by vapour diffusion using a hanging-drop setup. Diffraction-quality crystals were obtained and data extending to 2.46 Å resolution were collected at 100 K on BM14, ESRF, Grenoble, France. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 118.0, b = 50.6, c = 46.0 Å, β = 111.0°. Matthews coefficient (V M) calculations suggested the presence of 4–6 molecules in the asymmetric unit, corresponding to a solvent content of ∼33–55%, and are consistent with self-rotation function calculations. PMID:23695571

  11. Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin.

    PubMed

    Lu, Shuangshuang; Yao, Shugang; Chen, Rong; Zhang, Nianzhi; Chen, Jianmin; Gao, Feng; Xia, Chun

    2012-04-01

    β(2)-Microglobulin (β(2)m) is an essential subunit of the major histocompatibility complex (MHC) class I molecule that helps to stabilize the structure of peptide-MHC I (pMHC I). It is also one of the typical immunoglobulin superfamily (IgSF) molecules in the adaptive immune system (AIS). Sharks belong to the cartilaginous fish, which are the oldest jawed vertebrate ancestors with an AIS to exist in the world. Thus, the study of cartilaginous fish β(2)m would help in understanding the evolution of IgSF molecules. In order to demonstrate this, β(2)m from a cartilaginous fish, nurse shark (Ginglymostoma cirratum), was expressed, refolded, purified and crystallized. Diffraction data were collected to a resolution of 2.3 Å. The crystal belonged to space group P3(2)21, with unit-cell parameters a = b = 88.230, c = 67.146 Å. The crystal structure contained two molecules in the asymmetric unit. The results will provide structural information for study of the evolution of β(2)m and IgSF in the AIS.

  12. Expression, purification, crystallization and preliminary X-ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase

    SciTech Connect

    Obiero, Josiah; Bonderoff, Sara A.; Goertzen, Meghan M.; Sanders, David A. R.

    2006-08-01

    Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. Deinococcus radiodurans, a Gram-positive bacterium capable of withstanding extreme ionizing radiation, contains two thioredoxins (Trx and Trx1) and a single thioredoxin reductase (TrxR) as part of its response to oxidative stress. Thioredoxin reductase is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. X-ray diffraction data were collected on a cryocooled crystal to a resolution of 1.9 Å using a synchrotron-radiation source. The space group was determined to be P3{sub 2}21, with unit-cell parameters a = b = 84.33, c = 159.88 Å. The structure of the enzyme has been solved by molecular-replacement methods and structure refinement is in progress.

  13. Cloning, overexpression, purification and crystallization of the CRN12 coiled-coil domain from Leishmania donovani.

    PubMed

    Srivastava, Vijay Kumar; Rana, Ajay Kumar; Sahasrabuddhe, Amogh A; Gupta, C M; Pratap, J V

    2013-05-01

    Leishmania donovani coronin CRN12 is an actin-binding protein which consists of two domains: an N-terminal WD repeat domain and a C-terminal coiled-coil domain. The coiled-coil domain is 53 residues in length. Helix-helix interactions in general and coiled coils in particular are ubiquitous in the structure of proteins and play a significant role in the association among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signalling, transport and actin dynamics. The L. donovani coronin CRN12 coiled-coil domain (5.8 kDa) was cloned, overexpressed, purified to homogeneity and the N-terminal 6×His tag was successfully removed by thrombin cleavage. Crystals of recombinant L. donovani coronin CRN12 coiled-coil domain were grown by vapour diffusion using a hanging-drop setup. Diffraction-quality crystals were obtained and data extending to 2.46 Å resolution were collected at 100 K on BM14, ESRF, Grenoble, France. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 118.0, b = 50.6, c = 46.0 Å, β = 111.0°. Matthews coefficient (VM) calculations suggested the presence of 4-6 molecules in the asymmetric unit, corresponding to a solvent content of ∼33-55%, and are consistent with self-rotation function calculations. PMID:23695571

  14. Purification of receptor complexes of interleukin-10 stoichiometry and the importance of deglycosylation in their crystallization.

    PubMed

    Hoover, D M; Schalk-Hihi, C; Chou, C C; Menon, S; Wlodawer, A; Zdanov, A

    1999-05-01

    Interleukin-10 (IL-10) is a pleiotropic immunosuppressive cytokine that has a wide range of effects in controlling inflammatory responses. Viral IL-10 (vIL-10) is a homologue of human IL-10 (hIL-10) produced by Epstein-Barr virus (EBV). Both hIL-10 and vIL-10 bind to the soluble extracellular fragment of the cytokine receptor IL-10R1 (shIL-10R1). The stoichiometry of the vIL-10 : shIL-10R1 complex has been found to be the same as hIL-10 : shIL-10R1, with two vIL-10 dimers binding to four shIL-10R1 monomers. Complexes of both hIL-10 and vIL-10 with glycosylated shIL-10R1 could not be crystallized. Controlled deglycosylation using peptide : N-glycosidase F and endo-beta-N-acetylglucosaminidase F3 resulted in the formation of crystals of both hIL-10 : shIL-10R1 and vIL-10 : shIL-10R1 complexes, indicating that the difficulty in the crystal formation was largely due to the presence of complex carbohydrate side chains. The availability of the structure of the ligand-receptor complexes should facilitate our understanding of the basis of the interaction between IL-10 and the IL-10 receptor.

  15. Expression, purification and crystallization of two endonuclease III enzymes from Deinococcus radiodurans.

    PubMed

    Sarre, Aili; Ökvist, Mats; Klar, Tobias; Moe, Elin; Timmins, Joanna

    2014-12-01

    Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Å resolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å, β = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å, β = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.

  16. Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto

    PubMed Central

    Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

    2010-01-01

    Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27 724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a = 74.3, b = 49.9, c = 56.3 Å, β = 95.2°. Diffraction images were processed to a resolution of 1.74 Å with an R merge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase. PMID:21139221

  17. Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA

    SciTech Connect

    Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2006-01-01

    The P. rubrum sucrose isomerase SmuA, a key enzyme in the industrial production of isomaltulose, was crystallized and diffraction data were collected to 1.95 Å resolution. Palatinose (isomaltulose, α-d-glucosylpyranosyl-1,6-d-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 Å, and diffract to 1.95 Å resolution on a synchrotron-radiation source.

  18. Purification and Crystal Growth of Lead Iodide by Physical Vapor Transport Method

    NASA Technical Reports Server (NTRS)

    Wright, G. W.; Cole, M.; Chen, Y.-F.; Chen, K.-T.; Chen, H.; Chattopadhyay, K.; Burger, A.

    1998-01-01

    Lead iodide (PbI2) is a layered compound semiconductor being developed as room temperature x- and gamma-ray detector. Compared to the more studied material, mercuric iodide, PbI2 has a higher melting temperature and no phase transition until liquid phase which are indications of better mechanical properties. In this study, the source material was purified by the zone-refining process, and the purest section was extracted from center of the the zone-refined ingot to be grown by physical vapor transport (PVT) method. The zone-refined material and as-grown crystals were characterized by optical microscopy and differential scanning calorimetry (DSC) to reveal the surface morphology, purity and stoichiometry. The results shows that both materials are near-stoichiometric composition, with the purity of the as-grown crystals higher than zone-refined materials. The resistivity of the as-grown crystal (10" Omega-cm) was derived from current-voltage (I-V) measurement, and is 10 times higher than the zone-refined materials. Detail results will be presented and discussed.

  19. Identification of succinic semialdehyde reductases from Geobacter: expression, purification, crystallization, preliminary functional, and crystallographic analysis

    SciTech Connect

    Zhang, Yanfeng; Gao, Xiaoli; Zheng, Yi; Garavito, R. Michael

    2012-04-30

    Succinic semialdehyde reductase (SSAR) is an important enzyme involved in {gamma}-aminobutyrate (GABA) metabolism. By converting succinic semialdehyde (SSA) to {gamma}-hydroxybutyrate (GHB), the SSAR facilitates an alternative pathway for GABA degradation. In this study, we identified SSARs from Geobacter sulfurreducens and Geobacter metallireducens (GsSSAR and GmSSAR, respectively). The enzymes were over-expressed in Escherichia coli and purified to near homogeneity. Both GsSSAR and GmSSAR showed the activity of reducing SSA using nicotinamide adenine dinucleotide phosphate as a co-factor. The oligomeric sizes of GsSSAR and GmSSAR, as determined by analytical size exclusion chromatography, suggest that the enzymes presumably exist as tetramers in solution. The recombinant GsSSAR and GmSSAR crystallized in the presence of NADP{sup +}, and the resulting crystals diffracted to 1.89 {angstrom} (GsSSAR) and 2.25 {angstrom} (GmSSAR) resolution. The GsSSAR and GmSSAR crystals belong to the space groups P2{sub 1}22{sub 1} (a = 99.61 {angstrom}, b = 147.49 {angstrom}, c = 182.47 {angstrom}) and P1 (a = 75.97 {angstrom}, b = 79.14 {angstrom}, c = 95.47 {angstrom}, {alpha} = 82.15{sup o}, {beta} = 88.80{sup o}, {gamma} = 87.66{sup o}), respectively. Preliminary crystallographic data analysis suggests the presence of eight protein monomers in the asymmetric units for both GsSSAR and GmSSAR.

  20. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    PubMed

    Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2015-12-01

    Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

  1. Purification, crystallization and preliminary X-ray diffraction studies of the archaeal virus resolvase SIRV2

    SciTech Connect

    Ennifar, Eric; Basquin, Jerôme; Birkenbihl, Rainer; Suck, Dietrich

    2005-05-01

    The Holliday junction resolvase of the archaeal virus SIRV2 infecting the archaeon Sulfolobus islandicus has been crystallized and a full data set has been collected at 3.4 Å resolution. Analysis of the self-rotation function suggests the presence of two dimers in the asymmetric unit with a solvent content of 77%. The Holliday junction (or four-way junction) is the universal DNA intermediate whose interaction with resolving proteins is one of the major events in the recombinational process. These proteins, called DNA junction-resolving enzymes or resolvases, bind to the junction and catalyse DNA cleavage, promoting the release of two DNA duplexes. SIRV2 Hjc, a viral resolvase infecting a thermophylic archaeon, has been cloned, expressed and purified. Crystals have been obtained in space group C2, with unit-cell parameters a = 147.8, b = 99.9, c = 87.6, β = 109.46°, and a full data set has been collected at 3.4 Å resolution. The self-rotation function indicates the presence of two dimers in the asymmetric unit and a high solvent content (77%). Molecular-replacement trials using known similar resolvase structures have so far been unsuccessful, indicating possible significant structural rearrangements.

  2. Purification, Refolding, and Crystallization of the Outer Membrane Protein OmpG from Escherichia coli.

    PubMed

    Köster, Stefan; van Pee, Katharina; Yildiz, Özkan

    2015-01-01

    OmpG is a pore-forming protein from E. coli outer membranes. Unlike the classical outer membrane porins, which are trimers, the OmpG channel is a monomeric β-barrel made of 14 antiparallel β-strands with short periplasmic turns and longer extracellular loops. The channel activity of OmpG is pH dependent and the channel is gated by the extracellular loop L6. At neutral/high pH, the channel is open and permeable for substrate molecules with a size up to 900 Da. At acidic pH, loop L6 folds across the channel and blocks the pore. The channel blockage at acidic pH appears to be triggered by the protonation of a histidine pair on neighboring β-strands, which repel one another, resulting in the rearrangement of loop L6 and channel closure. OmpG was purified by refolding from inclusion bodies and crystallized in two and three dimensions. Crystallization and analysis by electron microscopy and X-ray crystallography revealed the fundamental mechanisms essential for the channel activity.

  3. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of tomato β-galactosidase 4.

    PubMed

    Eda, Masahiro; Ishimaru, Megumi; Tada, Toshiji

    2015-02-01

    Plant β-galactosidases play important roles in carbohydrate-reserve mobilization, cell-wall expansion and degradation, and turnover of signalling molecules during ripening. Tomato β-galactosidase 4 (TBG4) not only has β-galactosidase activity but also has exo-β-(1,4)-galactanase activity, and prefers β-(1,4)-galactans longer than pentamers as its substrates; most other β-galactosidases only have the former activity. Recombinant TBG4 protein expressed in the yeast Pichia pastoris was crystallized by the sitting-drop vapour-diffusion method using PEG 10,000 as a precipitant. The crystals belonged to the orthorhombic space group P212121, with unit-parameters a = 92.82, b = 96.30, c = 159.26 Å, and diffracted to 1.65 Å resolution. Calculation of the Matthews coefficient suggested the presence of two monomers per asymmetric unit (VM = 2.2 Å(3) Da(-1)), with a solvent content of 45%.

  4. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    PubMed

    Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2015-12-01

    Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC. PMID:26625288

  5. Expression, Purification, Assay, and Crystal Structure of Perdeuterated Human Arginase I⋆, ⋆⋆

    PubMed Central

    Di Costanzo, Luigi; Moulin, Martine; Haertlein, Michael; Meilleur, Flora; Christianson, David W.

    2007-01-01

    Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to yield L-ornithine and urea. In order to establish a foundation for future neutron diffraction studies that will provide conclusive structural information regarding proton/deuteron positions in enzyme-inhibitor complexes, we have expressed, purified, assayed, and determined the X-ray crystal structure of perdeuterated (i.e., fully deuterated) human arginase I complexed with 2-amino-6-boronohexanoic acid (ABH) at 1.90 Å resolution. Prior to the neutron diffraction experiment, it is important to establish that perdeutaration does not cause any unanticipated structural or functional changes. Accordingly, we find that perdeuterated human arginase I exhibits catalytic activity essentially identical to that of the unlabeled enzyme. Additionally, the structure of the perdeuterated human arginase I-ABH complex is identical to that of the corresponding complex with the unlabeled enzyme. Therefore, we conclude that crystals of the perdeuterated human arginase I-ABH complex are suitable for neutron crystallographic study. PMID:17562323

  6. Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase from Deinococcus radiodurans.

    PubMed

    Are, Venkata Narayana; Ghosh, Biplab; Kumar, Ashwani; Yadav, Pooja; Bhatnagar, Deepak; Jamdar, Sahayog N; Makde, Ravindra D

    2014-09-01

    Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of an N-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the β-amyloid peptides implicated in Alzheimer's disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium, Deinococcus radiodurans (APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space group P21, with unit-cell parameters a = 77.6, b = 189.6, c = 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å(3) Da(-1) corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme in D. radiodurans.

  7. Expression, purification, crystallization and preliminary X-ray analysis of the polysaccharide lyase RB5312 from the marine planctomycete Rhodopirellula baltica

    PubMed Central

    Dabin, Jérôme; Jam, Murielle; Czjzek, Mirjam; Michel, Gurvan

    2008-01-01

    Polysaccharide lyases belonging to family PL1 act on pectins. These anionic polymers are usually produced by terrestrial plants and therefore pectinolytic enzymes are not frequently observed in marine microorganisms. The protein RB5312 from the marine bacterium Rhodopirellula baltica is distantly related to family PL1 pectate lyases, but its exact function is unclear. In this study, the expression and purification of a recombinant form of RB5312 are described. This protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belongs to space group P212121, with unit-cell parameters a = 39.05, b = 144.05, c = 153.97 Å, α = β = γ = 90°. A complete data set was collected to 1.8 Å resolution from a native crystal. PMID:18323615

  8. Expression, purification, crystallization and preliminary X-ray analysis of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    SciTech Connect

    Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.; Moody, Peter C. E.

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase B from H. pylori has been cloned, expressed, purified and crystallized in the presence of NAD. Crystals of GAPDHB diffracted to 2.8 Å resolution and belonged to space group P6{sub 5}22, with unit-cell parameters a = b = 166.1, c = 253.1 Å. Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X-ray diffraction of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from H. pylori are reported.

  9. Expression, purification, crystallization and preliminary X-ray analysis of the polysaccharide lyase RB5312 from the marine planctomycete Rhodopirellula baltica.

    PubMed

    Dabin, Jérôme; Jam, Murielle; Czjzek, Mirjam; Michel, Gurvan

    2008-03-01

    Polysaccharide lyases belonging to family PL1 act on pectins. These anionic polymers are usually produced by terrestrial plants and therefore pectinolytic enzymes are not frequently observed in marine microorganisms. The protein RB5312 from the marine bacterium Rhodopirellula baltica is distantly related to family PL1 pectate lyases, but its exact function is unclear. In this study, the expression and purification of a recombinant form of RB5312 are described. This protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 39.05, b = 144.05, c = 153.97 A, alpha = beta = gamma = 90 degrees. A complete data set was collected to 1.8 A resolution from a native crystal. PMID:18323615

  10. Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex

    PubMed Central

    Pata, Janice D.; King, Bradford R.; Steitz, Thomas A.

    2002-01-01

    Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates DNA synthesis from the 3′ end of human tRNALys3. We have used cis-acting hammerhead ribozymes to produce homogeneous-length transcribed tRNALys3 and have developed conditions for purifying highly structured RNAs on a modified tube-gel apparatus. Titration experiments show that this RNA can assemble into an initiation complex that contains equimolar amounts of HIV-1 RT, transcribed tRNALys3, and chemically synthesized template RNA. We have purified this complex using gel-filtration chromatography and have found that it is homogeneous with respect to molecular weight, demonstrating that the initiation complex forms a single discrete species at micromolar concentrations. When this initiation complex is supplied with deoxynucleotides, essentially all of the tRNA is used as a primer by HIV-1 RT and is fully extended to the 5′ end of the template. Thus, in vitro transcribed tRNA can be used efficiently as a primer by HIV-1 RT. We have also obtained crystals of the HIV-1 initiation complex that require the precisely defined ends of this in vitro transcribed tRNALys3 to grow. PMID:12433988

  11. Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

    PubMed

    Izumori, K; Rees, A W; Elbein, A D

    1975-10-25

    D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative. PMID:240851

  12. Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

    PubMed

    Izumori, K; Rees, A W; Elbein, A D

    1975-10-25

    D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative.

  13. Expression, purification, crystallization and preliminary X-ray analysis of the polysaccharide lyase RB5312 from the marine planctomycete Rhodopirellula baltica

    SciTech Connect

    Dabin, Jérôme; Jam, Murielle; Czjzek, Mirjam Michel, Gurvan

    2008-03-01

    This study describes the crystallization and preliminary X-ray analysis of the family PL1 polysaccharide lyase RB5312 from the marine bacterium R. baltica. Purified recombinant protein was crystallized; the crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1} and diffracted X-rays to a resolution of 1.8 Å. Polysaccharide lyases belonging to family PL1 act on pectins. These anionic polymers are usually produced by terrestrial plants and therefore pectinolytic enzymes are not frequently observed in marine microorganisms. The protein RB5312 from the marine bacterium Rhodopirellula baltica is distantly related to family PL1 pectate lyases, but its exact function is unclear. In this study, the expression and purification of a recombinant form of RB5312 are described. This protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 39.05, b = 144.05, c = 153.97 Å, α = β = γ = 90°. A complete data set was collected to 1.8 Å resolution from a native crystal.

  14. Expression, purification, crystallization and preliminary X-ray analysis of the PaaI-like thioesterase SAV0944 from Staphylococcus aureus.

    PubMed

    Khandokar, Yogesh B; Roman, Noelia; Smith, Kate M; Srivastava, Parul; Forwood, Jade K

    2014-02-01

    Staphylococcus aureus is the causative agent of many diseases, including meningitis, bacteraemia, pneumonia, food poisoning and toxic shock syndrome. Structural characterization of the PaaI-like thioesterase SAV0944 (SaPaaI) from S. aureus subsp. aureus Mu50 will aid in understanding its potential as a new therapeutic target by knowledge of its molecular details and cellular functions. Here, the recombinant expression, purification and crystallization of SaPaaI thioesterase from S. aureus are reported. This protein initially crystallized with the ligand coenzyme A using the hanging-drop vapour-diffusion technique with condition No. 40 of Crystal Screen from Hampton Research at 296 K. Optimal final conditions consisting of 24% PEG 4000, 100 mM sodium citrate pH 6.5, 12% 2-propanol gave single diffraction-quality crystals. These crystals diffracted to beyond 2 Å resolution at the Australian Synchrotron and belonged to space group P12(1)1, with unit-cell parameters a = 44.05, b = 89.05, c = 60.74 Å, β = 100.5°. Initial structure determination and refinement gave an R factor and R(free) of 17.3 and 22.0%, respectively, confirming a positive solution in obtaining phases using molecular replacement.

  15. A putative siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIMB 400: cloning, expression, purification, crystallization and X-ray diffraction analysis.

    PubMed

    Trindade, Inês B; Fonseca, Bruno M; Matias, Pedro M; Louro, Ricardo O; Moe, Elin

    2016-09-01

    Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacterium Shewanella frigidimarina NCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 48.04, b = 78.31, c = 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing. PMID:27599855

  16. Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin

    SciTech Connect

    Hillier, Brian J.; Sundaresan, Vidyasankar; Stout, C. David; Vacquier, Victor D.

    2006-01-01

    The olfactomedin (OLF) domain from the sea urchin cell-adhesion protein amassin has been crystallized. A native data set extending to 2.7 Å has been collected using an in-house X-ray source. A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein–protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 Å under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain.

  17. A putative siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIMB 400: cloning, expression, purification, crystallization and X-ray diffraction analysis

    PubMed Central

    Trindade, Inês B.; Fonseca, Bruno M.; Matias, Pedro M.; Louro, Ricardo O.; Moe, Elin

    2016-01-01

    Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacterium Shewanella frigidimarina NCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 48.04, b = 78.31, c = 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing. PMID:27599855

  18. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    SciTech Connect

    Kraschnefski, Mark J.; Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von; Blanchard, Helen

    2005-11-01

    The carbohydrate-binding component (VP8*{sub 64–223}) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8*{sub 64–223} structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3{sub 2}21 and monoclinic P2{sub 1}) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*{sub 64–223} structure by molecular replacement.

  19. Expression, purification, crystallization and preliminary X-ray diffraction analysis of an essential lipoprotein implicated in cell-wall biosynthesis in Mycobacteria

    SciTech Connect

    Marland, Zara; Beddoe, Travis; Zaker-Tabrizi, Leyla; Coppel, Ross L.; Crellin, Paul K.; Rossjohn, Jamie

    2005-12-01

    A lipoprotein implicated in mycobacterial cell-wall biosynthesis, LpqW, was expressed in E. coli. Crystals were obtained that diffracted to 2.4 Å resolution. Mycobacterium tuberculosis is a renewed cause of devastation in the developing world. Critical to the success of this re-emerging pathogen is its unusual waxy cell wall, which is rich in rare components including lipoarabinomannan (LAM) and its precursors, the phosphatidylinositol mannosides (PIMs). Balanced synthesis of these related glycolipids is intrinsic to both cell-wall integrity and virulence in M. tuberculosis and presents a promising, albeit poorly defined, therapeutic target. Here, the expression, purification and crystallization of an essential 600-amino-acid lipoprotein, LpqW, implicated in this process are reported. Crystals of LpqW were grown using 20–24%(w/v) PEG 4000, 8–16%(v/v) 2-propanol, 100 mM sodium citrate pH 5.5 and 10 mM DTT. A complete data set was collected at 2.4 Å using synchrotron radiation on a crystal belonging to space group C222, with unit-cell parameters a = 188.57, b = 312.04, c = 104.15 Å. Structure determination is under way.

  20. High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor binding domain of botulinum neurotoxin serotype D

    SciTech Connect

    Zhang, Yanfeng; Gao, Xiaoli; Qin, Lin; Buchko, Garry W.; Robinson, Howard; Varnum, Susan M.

    2010-12-01

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and can cause neuroparalytic disease botulism. Due to the limitations of production and manipulation of holoenzymes, expressing non-toxic heavy chain receptor binding domains (HCR) has become a common strategy for vaccine and antibody development. Meanwhile, large quantities and highly purified soluble proteins are required for research areas such as antibody maturation and structural biology. We present high level expression and purification of the BoNT serotype D HCR in E. coli using a codon-optimized cDNA. By varying expression conditions, especially at low temperature, the protein was expressed at a high level with high solubility. About 150-200 mg protein was purified to >90% purity from 1 L cell culture. The recombinant D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belong to space group P212121 with unit cell dimensions a = 60.8 Å, b = 89.7 Å, c = 93.9 Å. Preliminary crystallographic data analysis revealed one molecule in asymmetric unit.

  1. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of universal stress protein F (YnaF) from Salmonella typhimurium

    SciTech Connect

    Sagurthi, Someswar Rao; Panigrahi, Rashmi Rekha; Gowda, Giri; Savithri, H. S.; Murthy, M. R. N.

    2007-11-01

    The cloning, purification and crystallization of YnaF from S. typhimurium are reported along with preliminary X-ray crystallographic studies. The universal stress protein UspF (YnaF) is a small cytoplasmic bacterial protein. The expression of stress proteins is enhanced when cells are exposed to heat shock, nutrition starvation and certain other stress-inducing agents. YnaF promotes cell survival during prolonged exposure to stress and may activate a general mechanism for stress endurance. This manuscript reports preliminary crystallographic studies on YnaF from Salmonella typhimurium. The gene coding for YnaF was cloned and overexpressed and the protein was purified by Ni–NTA affinity chromatography. Purified YnaF was crystallized using vapour-diffusion and microbatch methods. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 37.51, b = 77.18, c = 56.34 Å, β = 101.8°. A data set was collected to 2.5 Å resolution with 94.6% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. Attempts to determine the structure are in progress.

  2. Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA–HasR from Serratia marcescens

    SciTech Connect

    Huché, Frédéric; Delepelaire, Philippe; Wandersman, Cécile; Welte, Wolfram

    2006-01-01

    The expression, purification, and crystallization in space group P2{sub 1}2{sub 1}2{sub 1} of the complex HasA-HasR from S. marcescens are reported. Diffraction data have been collected and processed to 6.8 Å. Serratia marcescens is able to acquire iron using its haem-acquisition system (‘has’), which contains an outer membrane receptor HasR and a soluble haemophore HasA. After secretion, HasA binds free haem in the extracellular medium or extracts it from haemoproteins and delivers it to the receptor. Here, the crystallization of a HasA–HasR complex is reported. HasA and HasR have been overexpressed in Escherichia coli and the complex formed and crystallized. Small platelets and bunches of needles of dimensions 0.01 × 0.1 × 1 mm were obtained. A native data set has been collected to 6.8 Å.

  3. Purification, crystallization and preliminary X-ray diffraction of wild-type and mutant recombinant human transforming growth factor β-induced protein (TGFBIp)

    PubMed Central

    Runager, Kasper; García-Castellanos, Raquel; Valnickova, Zuzana; Kristensen, Torsten; Nielsen, Niels Chr.; Klintworth, Gordon K.; Gomis-Rüth, F. Xavier; Enghild, Jan J.

    2009-01-01

    Transforming growth factor β-induced protein (TGFBIp) has been linked to several corneal dystrophies as certain point mutations in the protein may give rise to a progressive accumulation of insoluble protein material in the human cornea. Little is known about the biological functions of this extracellular protein, which is expressed in various tissues throughout the human body. However, it has been found to interact with a number of extracellular matrix macromolecules such as collagens and proteoglycans. Structural information about TGFBIp might prove to be a valuable tool in the elucidation of its function and its role in corneal dystrophies caused by mutations in the TGFBI gene. A simple method for the purification of wild-type and mutant forms of recombinant human TGFBIp from human cells under native conditions is presented here. Moreover, the crystallization and preliminary X-ray analysis of TGFBIp are reported. PMID:19255489

  4. Expression, purification, crystallization and preliminary X-ray structure analysis of wild-type and L(M196)H-mutant Rhodobacter sphaeroides reaction centres

    PubMed Central

    Gabdulkhakov, A. G.; Fufina, T. Y.; Vasilieva, L. G.; Mueller, U.; Shuvalov, V. A.

    2013-01-01

    The electron and proton transport mediated by protein-bound cofactors in photosynthesis have been investigated by various methods in order to determine the energetics, the dynamics and the pathway of this process. In purple bacteria, primary photosynthetic charge separation and the build-up of a proton gradient across the periplasmic membrane are catalyzed by the photosynthetic reaction centre (RC). Here, the purification, crystallization and preliminary X-ray analysis of wild-type and L(M196)H-mutant RCs of Rhodobacter sphaeroides are presented, enabling study of the influence of the protein environment of the primary electron donor on the spectral properties and photochemical activity of the RC. PMID:23695564

  5. Cloning, purification, crystallization and preliminary X-ray analysis of the catalytic domain of human receptor-like protein tyrosine phosphatase [gamma] in three different crystal forms

    SciTech Connect

    Kish, Kevin; McDonnell, Patricia A.; Goldfarb, Valentina; Gao, Mian; Metzler, William J.; Langley, David R.; Bryson, James W.; Kiefer, Susan E.; Carpenter, Brian; Kostich, Walter A.; Westphal, Ryan S.; Sheriff, Steven

    2013-03-07

    Protein tyrosine phosphatase {gamma} is a membrane-bound receptor and is designated RPTP{gamma}. RPTP{gamma} and two mutants, RPTP{gamma}(V948I, S970T) and RPTP{gamma}(C858S, S970T), were recombinantly expressed and purified for X-ray crystallographic studies. The purified enzymes were crystallized using the hanging-drop vapor-diffusion method. Crystallographic data were obtained from several different crystal forms in the absence and the presence of inhibitor. In this paper, a description is given of how three different crystal forms were obtained that were used with various ligands. An orthorhombic crystal form and a trigonal crystal form were obtained both with and without ligand, and a monoclinic crystal form was only obtained in the presence of a particularly elaborated inhibitor.

  6. The effect of O-methylated flavonoids and other co-metabolites on the crystallization and purification of artemisinin.

    PubMed

    Suberu, John O; Yamin, Peyman; Leonhard, Kai; Song, Lijiang; Chemat, Smain; Sullivan, Neil; Barker, Guy; Lapkin, Alexei A

    2014-02-10

    Methoxylated flavonoids casticin, artemetin and retusin were identified as putative causative factors for low crystallization yields of artemisinin from extracts. Comparative profiling of biomass grown in different countries found elevated levels (∼60% higher) of artemetin in the East African biomass, which also demonstrates poor crystallization yields. The single compound and the combined doping experiments at 0, 25 and 50 μg mL⁻¹ doping levels showed that artemetin (50 μg mL⁻¹) caused a reduction in the amount of artemisinin crystallized by ca. 60%. A combination of the three flavonoids at 50 μg mL⁻¹ almost completely inhibited crystallization, reducing the yield by 98%. Treatment of extracts by adsorbents efficiently resolves the problem of low crystallization yield.

  7. Purification, crystallization and preliminary X-ray diffraction analysis of water-soluble chlorophyll-binding protein from Chenopodium album

    SciTech Connect

    Ohtsuki, Takayuki; Ohshima, Shigeru; Uchida, Akira

    2007-09-01

    A water-soluble chlorophyll-binding protein with photoconvertibility from C. album was extracted, purified and crystallized in a darkroom. The crystal diffracted to around 2.0 Å resolution. A water-soluble chlorophyll-binding protein (WSCP) with photoconvertibility from Chenopodium album was extracted, purified and crystallized in a darkroom. Green crystals suitable for data collection appeared in about 10 d. A native data set was collected to 2.0 Å resolution at 100 K. The space group of the crystal was determined to be orthorhombic I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 48.13, b = 60.59, c = 107.21 Å. Preliminary analysis of the X-ray data indicated that there is one molecule per asymmetric unit.

  8. Purification, crystallization and preliminary X-ray analysis of the fumarylacetoacetase family member TTHA0809 from Thermus thermophilus HB8

    SciTech Connect

    Mizutani, Hisashi; Kunishima, Naoki

    2007-09-01

    The putative fumarylacetoacetase TTHA0809 from T. thermophilus HB8 has been overexpressed, purified and crystallized. The crystals diffracted X-rays to 2.2 Å resolution using synchrotron radiation. Fumarylacetoacetase catalyzes the final step of tyrosine and phenylalanine catabolism. A recombinant form of the fumarylacetoacetase family member TTHA0809 from Thermus thermophilus HB8 has been crystallized by the oil-microbatch method using sodium chloride as a precipitating agent. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 93.3, b = 73.4, c = 122.6 Å, β = 111.8°. The crystals are most likely to contain two dimers in the asymmetric unit, with a V{sub M} value of 3.32 Å{sup 3} Da{sup −1}. Diffraction data were collected at 2.2 Å resolution using synchrotron radiation at beamline BL26B1 of SPring-8, Japan.

  9. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DsrEFH from Allochromatium vinosum

    SciTech Connect

    Dahl, Christiane; Schulte, Andrea; Shin, Dong Hae

    2007-10-01

    DsrEFH from Allochromatium vinosum has been cloned, expressed, purified, and crystallized. A preliminary X-ray study of DsrEFH has been performed with a good quality crystal. In purple sulfur bacteria, the proteins encoded by dsr genes play an essential role in the oxidation of intracellular sulfur, which is an obligate intermediate during the oxidation of sulfide and thiosulfate. One such gene product, DsrEFH from Allochromatium vinosum, has been cloned, expressed, purified and crystallized. Synchrotron data were collected to 2.5 Å from a crystal of selenomethionine-substituted DsrEFH. The crystal belongs to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 56.6, b = 183.1, c = 107.8 Å, β = 99.6°. A full structure determination is under way in order to provide insight into the structure–function relationships of this protein.

  10. Overexpression, purification, crystallization and preliminary X-ray analysis of Rv2780 from Mycobacterium tuberculosis H37Rv

    SciTech Connect

    Tripathi, Sarvind Mani; Ramachandran, Ravishankar

    2008-05-01

    Rv2780, an alanine dehydrogenase from M. tuberculosis, has been crystallized in apo and NAD/pyruvate-bound forms. Preliminary crystallographic analysis shows that there is a hexamer and trimer in the asymmetric units of the apo and ternary complex forms, respectively. Rv2780, an alanine dehydrogenase from Mycobacterium tuberculosis (MtAlaDH), catalyzes the NAD-dependent interconversion of alanine and pyruvate. Alanine dehydrogenase is released into the culture medium in substantial amounts by virulent strains of mycobacteria and is not found in the vaccine strain of tuberculosis. Crystals of recombinant MtAlaDH were grown from 2 M ammonium sulfate solution at ∼12 mg ml{sup −1} protein concentration in two crystal forms which occur in the presence and absence of NAD/pyruvate, respectively. Diffraction data extending to 2.6 Å were collected at room temperature from both apo and ternary complex crystals. Crystals of the apoenzyme have unit-cell parameters a = 173.89, b = 127.07, c = 135.95 Å. They are rod-like in shape and belong to space group C2. They contain a hexamer in the asymmetric unit. Crystals of the ternary complex belong to space group P4{sub 3}2{sub 1}2 and have unit-cell parameters a = b = 88.99, c = 373.85 Å. There are three subunits in the asymmetric unit of the holoenzyme crystals.

  11. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of initiation factor 1 from Mycobacterium tuberculosis

    SciTech Connect

    Hatzopoulos, Georgios N.; Mueller-Dieckmann, Jochen

    2007-03-01

    Initiation factor 1 from M. tuberculosis was cloned, expressed, purified and crystallized. A complete data set has been collected to 1.47 Å resolution. Initiation factor 1 (IF-1; Rv3462c) from Mycobacterium tuberculosis, a component of the 30S initiation complex, was cloned and heterologously expressed in Escherichia coli. The protein was purified by affinity and size-exclusion chromatography and crystallized. A complete data set has been collected to high resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2, with two molecules per asymmetric unit which are related by translational symmetry.

  12. Purification, crystallization and preliminary X-ray diffraction study of human ribosomal protein L10 core domain

    SciTech Connect

    Nishimura, Mitsuhiro; Kaminishi, Tatsuya; Kawazoe, Masahito; Shirouzu, Mikako; Takemoto, Chie; Yokoyama, Shigeyuki; Tanaka, Akiko; Sugano, Sumio; Yoshida, Takuya; Ohkubo, Tadayasu; Kobayashi, Yuji

    2007-11-01

    A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution. Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to space group P3{sub 1}21 or P3{sub 2}21.

  13. Purification, crystallization and preliminary crystallographic analysis of the non-Pfam protein AF1514 from Archeoglobus fulgidus DSM 4304

    SciTech Connect

    Bahti, Pazilat; Chen, Shunmei; Li, Yang; Shaw, Neil; Zhang, Xuejun; Zhang, Min; Cheng, Chongyun; Song, Gaojie; Yin, Jie; Zhang, Hua; Che, Dongsheng; Abbas, Abdulla; Xu, Hao; Wang, Bi-Cheng; Liu, Zhi-Jie

    2008-02-01

    A non-Pfam protein, AF1514, from A. fulgidus has been crystallized. A 10.5 kDa non-Pfam hypothetical protein, AF1514, from the hyperthermophilic archaeon Archeoglobus fulgidus has been overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 2.09 Å resolution and a data set was collected at 100 K using Cu Kα radiation from a rotating-anode X-ray source. The crystals belong to space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 49.27, c = 106.61 Å. The calculated Matthews coefficient was 3.16 Å{sup 3} Da{sup −1}, suggesting the presence of one molecule in the asymmetric unit.

  14. Purification, crystallization and preliminary X-ray crystallographic analysis of rice bifunctional α-amylase/subtilisin inhibitor from Oryza sativa

    SciTech Connect

    Lin, Yi-Hung; Peng, Wen-Yan; Huang, Yen-Chieh; Guan, Hong-Hsiang; Hsieh, Ying-Cheng; Liu, Ming-Yih; Chang, Tschining; Chen, Chun-Jung

    2006-08-01

    The crystallization of rice α-amylase/subtilisin bifunctional inhibitor is reported. Rice bifunctional α-amylase/subtilisin inhibitor (RASI) can inhibit both α-amylase from larvae of the red flour beetle (Tribolium castaneum) and subtilisin from Bacillus subtilis. The synthesis of RASI is up-regulated during the late milky stage in developing seeds. The 8.9 kDa molecular-weight RASI from rice has been crystallized using the hanging-drop vapour-diffusion method. According to 1.81 Å resolution X-ray diffraction data from rice RASI crystals, the crystal belongs to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 79.99, b = 62.95, c = 66.70 Å. Preliminary analysis indicates two RASI molecules in an asymmetric unit with a solvent content of 44%.

  15. Expression, purification, crystallization and preliminary X-ray analysis of the receiver domain of Staphylococcus aureus LytR protein

    PubMed Central

    Shala, Agnesa; Patel, Kevin H.; Golemi-Kotra, Dasantila; Audette, Gerald F.

    2013-01-01

    The response-regulatory protein LytR belongs to a family of transcription factors involved in the regulation of important virulence factors in pathogenic bacteria. The protein consists of a receiver domain and an effector domain, which play an important role in controlled cell death and lysis. The LytR receiver domain (LytRN) has been overexpressed, purified and crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. The crystals grew as needles, with unit-cell parameters a = b = 84.82, c = 157.3 Å, α = β = 90, γ = 120°. LytRN crystallized in space group P6122 and the crystals diffracted to a maximum resolution of 2.34 Å. Based on the Matthews coefficient (V M = 5.44 Å3 Da−1), one molecule is estimated to be present in the asymmetric unit. PMID:24316844

  16. Purification, crystallization and preliminary X-ray diffraction studies of d-tagatose 3-epimerase from Pseudomonas cichorii

    SciTech Connect

    Yoshida, Hiromi; Yamada, Mitsugu; Nishitani, Takeyori; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2007-02-01

    Recombinant d-tagatose 3-epimerase from P. cichorii was purified and crystallized. Diffraction data were collected to 2.5 Å resolution. d-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of d-psicose has not been reported with epimerases other than P. cichorii D-TE and d-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 Å, β = 102.82°. Diffraction data were collected to 2.5 Å resolution. The asymmetric unit is expected to contain four molecules.

  17. Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast

    SciTech Connect

    Umehara, Takashi; Otta, Yumi; Tsuganezawa, Keiko; Matsumoto, Takehisa; Tanaka, Akiko; Horikoshi, Masami; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2005-11-01

    The histone chaperone cia1 from fission yeast has been overexpressed in E. coli, purified and crystallized using the vapour-diffusion method. In fission yeast, cia1{sup +} is an essential gene that encodes a histone chaperone, a homologue of human CIA (CCG1-interacting factor A) and budding yeast Asf1p (anti-silencing function-1), which both facilitate nucleosome assembly by interacting with the core histones H3/H4. The conserved domain (residues 1–161) of the cia1{sup +}-encoded protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The protein was crystallized in the monoclinic space group C2, with unit-cell parameters a = 79.16, b = 40.53, c = 69.79 Å, β = 115.93° and one molecule per asymmetric unit. The crystal diffracted to beyond 2.10 Å resolution using synchrotron radiation.

  18. Purification of molybdenum oxide, growth and characterization of medium size zinc molybdate crystals for the LUMINEU program

    NASA Astrophysics Data System (ADS)

    Shlegel, V. N.; Berge, L.; Boiko, R. S.; Chapellier, M.; Chernyak, D. M.; Coron, N.; Danevich, F. A.; Decourt, R.; Degoda, V. Ya.; Devoyon, L.; Drillien, A.; Dumoulin, L.; Enss, C.; Fleischmann, A.; Gastaldo, L.; Giuliani, A.; Gros, M.; Herve, S.; Ivanov, I. M.; Kobychev, V. V.; Kogut, Ya. P.; Koskas, F.; Loidl, M.; Magnier, P.; Makarov, E. P.; Mancuso, M.; de Marcillac, P.; Marnieros, S.; Marrache-Kikuchi, C.; Nasonov, S. G.; Navick, X. F.; Nones, C.; Olivieri, E.; Paul, B.; Penichot, Y.; Pessina, G.; Plantevin, O.; Poda, D. V.; Redon, T.; Rodrigues, M.; Strazzer, O.; Tenconi, M.; Torres, L.; Tretyak, V. I.; Vasiliev, Ya. V.; Velazquez, M.; Viraphong, O.; Zhdankov, V. N.

    2014-01-01

    The LUMINEU program aims at performing a pilot experiment on neutrinoless double beta decay of 100Mo using radiopure ZnMoO4 crystals operated as scintillating bolometers. Growth of high quality radiopure crystals is a complex task, since there are no commercially available molybdenum compounds with the required levels of purity and radioactive contamination. This paper discusses approaches to purify molybdenum and synthesize compound for high quality radiopure ZnMoO4 crystal growth. A combination of a double sublimation (with addition of zinc molybdate) with subsequent recrystallization in aqueous solutions (using zinc molybdate as a collector) was used. Zinc molybdate crystals up to 1.5 kg were grown by the low-thermal-gradient Czochralski technique, their optical, luminescent, diamagnetic, thermal and bolometric properties were tested.

  19. Purification, crystallization and preliminary X-ray crystallographic analysis of branched-chain aminotransferase from Deinococcus radiodurans

    SciTech Connect

    Chen, Chung-Der; Huang, Tien-Feng; Lin, Chih-Hao; Guan, Hong-Hsiang; Hsieh, Yin-Cheng; Lin, Yi-Hung; Huang, Yen-Chieh; Liu, Ming-Yih; Chang, Wen-Chang; Chen, Chun-Jung

    2007-06-01

    The crystallization of branched-chain aminotransferase from D. radiodurans is described. The branched-chain amino-acid aminotransferase (BCAT), which requires pyridoxal 5′-phosphate (PLP) as a cofactor, is a key enzyme in the biosynthetic pathway of the hydrophobic amino acids leucine, isoleucine and valine. DrBCAT from Deinococcus radiodurans, which has a molecular weight of 40.9 kDa, was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data to 2.50 Å resolution from a DrBCAT crystal, the crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 56.37, b = 90.70, c = 155.47 Å. Preliminary analysis indicates the presence of two DrBCAT molecules in the asymmetric unit, with a solvent content of 47.52%.

  20. Purification, crystallization and preliminary X-ray analysis of Enterococcus casseliflavus aminoglycoside-2′′-phosphotransferase-IVa

    PubMed Central

    Toth, Marta; Vakulenko, Sergei; Smith, Clyde A.

    2010-01-01

    The deactivation of aminoglycoside antibiotics by chemical modification is one of the major sources of bacterial resistance to this family of therapeutic compounds, which includes the clinically relevant drugs streptomycin, kanamycin and gentamicin. The aminoglycoside phosphotransferases (APHs) form one such family of enzymes responsible for this resistance. The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-IVa [APH(2′′)-IVa] from Enterococcus casseliflavus, has been cloned and the protein (comprising 306 amino-acid residues) has been expressed in Escherichia coli and purified. The enzyme was crystallized in three substrate-free forms. Two of the crystal forms belonged to the orthorhombic space group P212121 with similar unit-cell parameters, although one of the crystal forms had a unit-cell volume that was approximately 13% smaller than the other and a very low solvent content of around 38%. The third crystal form belonged to the monoclinic space group P21 and preliminary X-ray diffraction analysis was consistent with the presence of two molecules in the asymmetric unit. The orthorhombic crystal forms of apo APH(2′′)-IVa both diffracted to 2.2 Å resolution and the monoclinic crystal form diffracted to 2.4 Å resolution; synchrotron diffraction data were collected from these crystals at SSRL (Stanford, California, USA). Structure determination by molecular replacement using the structure of the related enzyme APH(2′′)-IIa is proceeding. PMID:20057078

  1. Purification, crystallization, and preliminary X-ray crystallographic analysis of the Group III chaperonin from Carboxydothermus hydrogenoformans.

    PubMed

    An, Young Jun; Rowland, Sara E; Robb, Frank T; Cha, Sun-Shin

    2016-06-01

    Chaperonins (CPNs) are megadalton sized ATP-dependent nanomachines that facilitate protein folding through complex cycles of complex allosteric articulation. They consist of two back-to-back stacked multisubunit rings. CPNs are usually classified into Group I and Group II. Here, we report the crystallization of both the AMPPNP (an ATP analogue) and ADP bound forms of a novel CPN, classified as belonging to a third Group, recently discovered in the extreme thermophile Carboxydothermus hydrogenoformans. Crystals of the two forms were grown by the vapor batch crystallization method at 295 K. Crystals of the Ch-CPN/AMPPNP complex diffracted to 3.0 Å resolution and belonged to the space group P422, with unit-cell parameters a = b = 186.166, c = 160.742 Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 60.02%. Crystals of the Ch-CPN/ADP complex diffracted to 4.0 Å resolution and belonged to the space group P4212, with unit-cell parameters a = b = 209.780, c = 169.813Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 70.19%.

  2. Purification, crystallization and preliminary X-ray crystallographic analysis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1

    PubMed Central

    Rohman, Ali; van Oosterwijk, Niels; Dijkstra, Bauke W.

    2012-01-01

    3-Ketosteroid Δ1-dehydrogenase plays a crucial role in the early steps of steroid degradation by introducing a double bond between the C1 and C2 atoms of the A-ring of its 3-ketosteroid substrates. The 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1, a 56 kDa flavoprotein, was crystallized using the sitting-drop vapour-diffusion method at room temperature. The crystals grew in various buffers over a wide pH range (from pH 5.5 to 10.5), but the best crystallization condition consisted of 2%(v/v) PEG 400, 0.1 M HEPES pH 7.5, 2.0 M ammonium sulfate. A native crystal diffracted X-rays to 2.0 Å resolution. It belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 107.4, b = 131.6, c = 363.2 Å, and contained eight molecules in the asymmetric unit. The initial structure of the enzyme was solved using multi-wavelength anomalous dispersion (MAD) data collected from a Pt-derivatized crystal. PMID:22691786

  3. Purification, crystallization and preliminary crystallographic analysis of DR0248, an MNT-HEPN fused protein from Deinococcus radiodurans.

    PubMed

    Pesce, Gaelle; Pellegrino, Simone; McSweeney, Sean; Goncalves, AnaMaria; de Sanctis, Daniele

    2015-01-01

    DR0248 is a protein identified in the Deinococcus radiodurans (DR) genome that is predicted to encompass two domains: an N-terminal minimal nucleotidyl transferase domain (MNT) and a C-terminal higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN). These two domains, usually encoded in two ORFs, have been suggested to play the role of a toxin-antitoxin (TA) system in prokaryotes. Recombinant DR0248 was overexpressed and purified from Escherichia coli and diffraction-quality crystals were obtained in the presence of the detergent molecules dodecyldimethylamine oxide (DDAO) and octaethylene glycol monododecyl ether (C12E8), which were used as crystallization additives. Crystals grown with DDAO diffracted to a resolution of 2.24 Å and belonged to space group C222(1), with unit-cell parameters a=98.4, b=129.9, c=59.2 Å. Crystals grown with C12E8 diffracted to a resolution of 1.83 Å and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=51.6, b=87.2, c=108.2 Å. The structure was solved by multiwavelength anomalous dispersion from zinc bound to the protein using a single crystal obtained in the presence of DDAO.

  4. Purification, crystallization and preliminary crystallographic analysis of the vacuole-type ATPase subunit E from Pyrococcus horikoshii OT3

    SciTech Connect

    Lokanath, Neratur K.; Ukita, Yoko; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-01-01

    The E subunit of vacuole-type ATPase from P. horikoshii OT3 was overexpressed, purified and crystallized. The native crystals diffracted X-rays to 1.85 Å resolution. The vacuole-type ATPases in eukaryotic cells translocate protons across various biological membranes including the vacuolar membrane by consuming ATP molecules. The E subunit of the multisubunit complex V-ATPase from Pyrococcus horikoshii OT3, which has a molecular weight of 22.88 kDa, has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. A data set to 1.85 Å resolution with 98.8% completeness and an R{sub merge} of 6.5% was collected from a single flash-cooled crystal using synchrotron radiation. The crystal belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 Å, and is most likely to contain one molecule per asymmetric unit.

  5. Protein purification, crystallization and preliminary X-ray diffraction analysis of L-arabinose isomerase from Lactobacillus fermentum CGMCC2921.

    PubMed

    Xu, Zheng; Li, Sha; Liang, Jinfeng; Feng, Xiaohai; Xu, Hong

    2015-01-01

    L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose, as well as that of D-galactose to D-tagatose. A thermophilic AI derived from Lactobacillus fermentum CGMCC2921 (LFAI) was overexpressed in Escherichia coli BL21 (DE3). This enzyme was purified to over 95% purity by nickel affinity, Mono-Q ion-exchange and size-exclusion chromatography. The LFAI protein was crystallized from either 0.1 M bis-tris pH 6.5, 23% PEG 3350, 0.3 M NaCl (form 1 crystals) or 0.1 M bis-tris pH 6.0, 25% PEG monomethyl ether 5000 (form 2 crystals). Diffraction data from form 1 LFAI crystals were collected to 2.80 Å resolution using synchrotron radiation. The form 1 crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=85.11, b=184.57, c=186.26 Å, α=β=γ=90°. The asymmetric unit contained six LFAI subunits, corresponding to a calculated Matthews coefficient of 2.29 Å3 Da(-1) and a solvent content of 46.22%.

  6. PredPPCrys: Accurate Prediction of Sequence Cloning, Protein Production, Purification and Crystallization Propensity from Protein Sequences Using Multi-Step Heterogeneous Feature Fusion and Selection

    PubMed Central

    Wang, Huilin; Wang, Mingjun; Tan, Hao; Li, Yuan; Zhang, Ziding; Song, Jiangning

    2014-01-01

    X-ray crystallography is the primary approach to solve the three-dimensional structure of a protein. However, a major bottleneck of this method is the failure of multi-step experimental procedures to yield diffraction-quality crystals, including sequence cloning, protein material production, purification, crystallization and ultimately, structural determination. Accordingly, prediction of the propensity of a protein to successfully undergo these experimental procedures based on the protein sequence may help narrow down laborious experimental efforts and facilitate target selection. A number of bioinformatics methods based on protein sequence information have been developed for this purpose. However, our knowledge on the important determinants of propensity for a protein sequence to produce high diffraction-quality crystals remains largely incomplete. In practice, most of the existing methods display poorer performance when evaluated on larger and updated datasets. To address this problem, we constructed an up-to-date dataset as the benchmark, and subsequently developed a new approach termed ‘PredPPCrys’ using the support vector machine (SVM). Using a comprehensive set of multifaceted sequence-derived features in combination with a novel multi-step feature selection strategy, we identified and characterized the relative importance and contribution of each feature type to the prediction performance of five individual experimental steps required for successful crystallization. The resulting optimal candidate features were used as inputs to build the first-level SVM predictor (PredPPCrys I). Next, prediction outputs of PredPPCrys I were used as the input to build second-level SVM classifiers (PredPPCrys II), which led to significantly enhanced prediction performance. Benchmarking experiments indicated that our PredPPCrys method outperforms most existing procedures on both up-to-date and previous datasets. In addition, the predicted crystallization targets of

  7. Expression, purification, crystallization and preliminary X-ray characterization of two crystal forms of stationary-phase survival E protein from Campylobacter jejuni

    SciTech Connect

    Gonçalves, A. M. D.; Rêgo, A. T.; Thomaz, M.; Enguita, F. J.; Carrondo, M. A.

    2008-03-01

    Survival E (SurE) protein from Campylobacter jejuni, a Gram-negative mesophile, has been overexpressed in Escherichia coli as a soluble protein, successfully purified and crystallized in two distinct crystal forms. Survival E (SurE) protein from Campylobacter jejuni, a Gram-negative mesophile, has been overexpressed in Escherichia coli as a soluble protein, successfully purified and crystallized in two distinct crystal forms. The first form belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with a tetramer in the asymmetric unit and unit-cell parameters a = 80.5, b = 119.0, c = 135.3 Å. The second form belongs to space group C2, with unit-cell parameters a = 121.4, b = 47.1, c = 97.8 Å, and contains a dimer in the asymmetric unit. Diffraction data have been collected from these crystal forms to 2.5 and 2.95 Å resolution, respectively.

  8. Purification, crystallization and preliminary X-ray diffraction analysis of Cif, a virulence factor secreted by Pseudomonas aeruginosa.

    PubMed

    Bahl, Christopher D; MacEachran, Daniel P; O'Toole, George A; Madden, Dean R

    2010-01-01

    The opportunistic pathogen Pseudomonas aeruginosa secretes a protein that triggers the accelerated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. This protein, which is known as the CFTR inhibitory factor (Cif), acts as a virulence factor and may facilitate airway colonization by P. aeruginosa. Based on sequence similarity Cif appears to be an epoxide hydrolase (EH), but it lacks several of the conserved features found in the active sites of canonical members of the EH family. Here, the crystallization of purified recombinant Cif by vapor diffusion is reported. The crystals formed in space group C2, with unit-cell parameters a = 167.4, b = 83.6, c = 88.3 A, beta = 100.6 degrees . The crystals diffracted to 2.39 A resolution on a rotating-anode source. Based on the calculated Matthews coefficient (2.2 A(3) Da(-1)), it appears that the asymmetric unit contains four molecules. PMID:20057063

  9. Purification, crystallization and preliminary X-ray analysis of an aminoacylhistidine dipeptidase (PepD) from Vibrio alginolyticus.

    PubMed

    Chang, Chin Yuan; Hsieh, Yin Cheng; Wang, Ting Yi; Chen, Chun Jung; Wu, Tung Kung

    2009-03-01

    The aminoacylhistidine dipeptidase (PepD) protein encoded by Vibrio alginolyticus pepD was successfully overexpressed and characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The purified enzyme contained two zinc ions per monomer. The recombinant dipeptidase enzyme, which was identified as a homodimer in solution, exhibited broad substrate specificity for Xaa-His dipeptides, with highest activity towards the His-His dipeptide. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Preliminary crystallographic analysis showed that the crystal belonged to space group P6(1) or P6(5), with unit-cell parameters a = b = 80.42, c = 303.11 A. The crystal contained two molecules per asymmetric unit and the predicted solvent content was 53.4%.

  10. Purification, crystallization and preliminary X-ray crystallographic analysis of mammalian MSS4–Rab8 GTPase protein complex

    SciTech Connect

    Itzen, Aymelt; Bleimling, Nathalie; Ignatev, Alexander; Pylypenko, Olena; Rak, Alexey

    2006-02-01

    The MSS4 (mammalian suppressor of Sec4) protein in complex with nucleotide-free Rab8 GTPase has been purified and crystallized in a form suitable for structure analysis and a complete data set has been collected to 2 Å resolution. Rab GTPases function as ubiquitous key regulators of membrane-vesicle transport in eukaryotic cells. MSS4 is an evolutionarily conserved protein that binds to exocytotic Rabs and facilitates nucleotide release. The MSS4 protein in complex with nucleotide-free Rab8 GTPase has been purified and crystallized in a form suitable for structure analysis. The crystals belonged to space group P1, with unit-cell parameters a = 40.92, b = 49.85, c = 83.48 Å, α = 102.88, β = 97.46, γ = 90.12°. A complete data set has been collected to 2 Å resolution.

  11. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

    SciTech Connect

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Kuroishi, Chizu; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2007-02-01

    DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K{sub 2}) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit.

  12. Purification, crystallization and preliminary X-ray diffraction analysis of the seryl-tRNA synthetase from Candida albicans.

    PubMed

    Rocha, Rita; Barbosa Pereira, Pedro José; Santos, Manuel A S; Macedo-Ribeiro, Sandra

    2011-01-01

    The seryl-tRNA synthetase (SerRS) from Candida albicans exists naturally as two isoforms resulting from ambiguity in the natural genetic code. Both enzymes were crystallized by the sitting-drop vapour-diffusion method using 3.2-3.4 M ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6(1)22 and contained one monomer per asymmetric unit, despite the synthetase existing as a homodimer (with a molecular weight of ∼116 kDa) in solution. Diffraction data were collected to 2.0 Å resolution at a synchrotron source and the crystal structures of unliganded SerRS and of its complexes with ATP and with a seryl-adenylate analogue were solved by molecular replacement. The structure of C. albicans SerRS represents the first reported structure of a eukaryotic cytoplasmic SerRS. PMID:21206050

  13. Purification, crystallization and preliminary crystallographic analysis of Est25: a ketoprofen-specific hormone-sensitive lipase

    SciTech Connect

    Kim, SeungBum; Joo, Sangbum; Yoon, Hyun C.; Ryu, Yeonwoo; Kim, Kyeong Kyu; Kim, T. Doohun

    2007-07-01

    Est25, a ketoprofen-specific hormone-sensitive lipase from a metagenomic library, was crystallized and diffraction data were collected to 1.49 Å resolution. Ketoprofen, a nonsteroidal anti-inflammatory drug, inhibits the synthesis of prostaglandin. A novel hydrolase (Est25) with high ketoprofen specificity has previously been identified using a metagenomic library from environmental samples. Recombinant Est25 protein with a histidine tag at the N-terminus was expressed in Escherichia coli and purified in a homogenous form. Est25 was crystallized from 2.4 M sodium malonate pH 7.0 and X-ray diffraction data were collected to 1.49 Å using synchrotron radiation. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 197.8, b = 95.2, c = 99.4 Å, β = 97.1°.

  14. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the glutamate-1-semialdehyde aminotransferase from Bacillus subtilis

    SciTech Connect

    Lv, Xinhuai; Fan, Jun; Ge, Honghua; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun Niu, Liwen

    2006-05-01

    Crystals of glutamate-1-semialdehyde aminotransferase (GSAT) from B. subtilis were obtained and diffraction data were collected to 2.0 Å resolution. 5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole-biosynthesis pathway. Plants, algae and many other bacteria synthesize ALA from glutamate by a C5 pathway in which the carbon skeleton of glutamate is converted into ALA by a series of enzymes. Glutamate-1-semialdehyde aminotransferase (GSAT) is the last enzyme in this pathway. The gene that codes for GSAT was amplified from the cDNA library of Bacillus subtilis and overexpressed in Escherichia coli strain BL21(DE3). The protein was purified and crystallized. Well diffracting single crystals were obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray diffraction studies yielded excellent diffraction data to a resolution of 2.0 Å.

  15. Purification, crystallization and preliminary X-ray diffraction studies of disintegrin (schistatin) from saw-scaled viper (Echis carinatus).

    PubMed

    Tomar, S; Yadav, S; Chandra, V; Kumar, P; Singh, T P

    2001-11-01

    This is the first report of crystallographic data on a disintegrin molecule from any source. The heterodimeric disintegrin with a molecular weight of 14 kDa from Echis carinatus venom is a potent antagonist of alpha4 integrins. The intact disintegrin, containing two subunits A and B, was isolated and purified using affinity and ion-exchange columns. It has been crystallized using 1.6 M ammonium sulfate as a precipitating agent. The crystals grew to dimensions of 0.25 x 0.20 x 0.20 mm and diffracted to 2.5 A resolution. The crystals belong to space group I4(1)22, with unit-cell parameters a = b = 91.7, c = 55.1 A. Assuming a molecular weight of 14 kDa, a V(M) of 2.1 A(3) Da(-1) is obtained for one molecule of disintegrin in the asymmetric unit. PMID:11679739

  16. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the DDX3 RNA helicase domain

    SciTech Connect

    Rodamilans, Bernardo; Montoya, Guillermo

    2007-04-01

    Crystals of DDX3 RNA helicase domain have been obtained in a monoclinic form that diffract to 2.2 Å resolution using synchrotron radiation at the ID14-1 ESRF beamline. DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD-box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp-Glu-Ala-Asp motif that defines the family. DDX3 has two distinct domains: an ATP-binding domain in the central region of the protein and a helicase domain in the carboxy-terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that were suitable for X-ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 mM spermine tetrahydrochloride and a protein solution containing 10 mM HEPES, 500 mM ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 Å, α = γ = 90, β = 101.02°, and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS)

  17. Purification and crystallization of the entire recombinant subunit E of the energy producer A(1)A(o) ATP synthase.

    PubMed

    Balakrishna, Asha Manikkoth; Hunke, Cornelia; Grüber, Gerhard

    2010-03-01

    A(1)A(o) ATP synthases are the major energy producers in archaea. Subunit E of the stator domain of the ATP synthase from Pyrococcus horikoshii OT3 was cloned, expressed and purified to homogeneity. The monodispersed protein was crystallized by vapour diffusion. A complete diffraction data set was collected to 3.3 A resolution with 99.4% completeness using a synchrotron-radiation source. The crystals belonged to space group I4, with unit-cell parameters a = 112.51, b = 112.51, c = 96.25 A, and contained three molecules in the asymmetric unit.

  18. Expression, purification, crystallization and preliminary diffraction studies of the tRNA pseudouridine synthase TruD from Escherichia coli.

    PubMed

    Ericsson, Ulrika B; Andersson, Martin E; Engvall, Benita; Nordlund, Pär; Hallberg, B Martin

    2004-04-01

    Pseudouridine, the 5-ribosyl isomer of uridine, is the most common modification of structural RNA. The recently identified pseudouridine synthase TruD belongs to a widespread class of pseudouridine synthases without significant sequence homology to previously known families. TruD from Escherichia coli was overexpressed, purified and crystallized. The crystals diffract to a minimum Bragg spacing of 2.4 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 63.4, b = 108.6, c = 111.7 A.

  19. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of ScpB (Rv1710) from Mycobacterium tuberculosis

    SciTech Connect

    Kwon, Soo-Young; Kang, Beom Sik; Kim, Myung Hee; Kim, Kyung Jin

    2007-12-01

    ScpB from M. tuberculosis was crystallized using the sitting-drop vapour-diffusion method in the presence of 2 M NaCl and 10% PEG 6000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å at a synchrotron beamline. Structural maintenance of chromosome (SMC) proteins play diverse roles in cellular DNA reassembly by directly interacting with DNA. They require non-SMC proteins for their proper function; these include the conserved segregation and condensation proteins (Scps) in prokaryotes. ScpB from Mycobacterium tuberculosis was crystallized using the sitting-drop vapour-diffusion method in the presence of 2 M NaCl and 10% PEG 6000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å at a synchrotron beamline. The crystal belongs to the hexagonal space group R32, with unit-cell parameters a = b = 136.69, c = 78.55 Å, γ = 120°. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V{sub M}) is 2.95 Å{sup 3} Da{sup −1}. The structure was solved by the single anomalous dispersion method and structure refinement is in progress.

  20. The purification, crystallization and preliminary structural characterization of PhzM, a phenazine-modifying methyltransferase from Pseudomonas aeruginosa

    SciTech Connect

    Gohain, Neelakshi; Thomashow, Linda S.; Mavrodi, Dmitri V.; Blankenfeldt, Wulf

    2006-09-01

    PhzM, an S-adenosylmethionine-dependent methyltransferase enzyme that catalyzes a reaction involved in the biosynthesis of pyocyanin in P. aeruginosa, was cloned, overexpressed and crystallized. Data collection from native and selenomethionine-labelled crystals is reported. Pyocyanin, phenazine-1-carboxylic acid and more than 70 related compounds collectively known as phenazines are produced by various species of Pseudomonas, including the fluorescent pseudomonad P. aeruginosa, a Gram-negative opportunistic pathogen in humans and animals. P. aeruginosa synthesizes a characteristic blue water-soluble compound called pyocyanin (1-hydroxy-5-methyl-phenazine). Two enzymes designated PhzM and PhzS are involved in the terminal steps of its synthesis and very little is known about these enzymes. In this study, PhzM, a dimeric S-adenosylmethionine-dependent methyltransferase, was purified and crystallized from PEG 3350/sodium cacodylate/sodium citrate pH 6.5. The crystals belong to space group P1, with unit-cell parameters a = 46.1, b = 61.8, c = 69.6 Å, α = 96.3, β = 106.6, γ = 106.9°. They contain one dimer in the asymmetric unit and diffract to a resolution of 1.8 Å. Anomalous data to 2.3 Å resolution have been collected from seleno-l-methionine-labelled PhzM.

  1. Cloning, purification, crystallization and preliminary crystallographic analysis of a penicillin-binding protein homologue from Pyrococcus abyssi

    SciTech Connect

    Delfosse, Vanessa; Hugonnet, Jean-Emmanuel; Sougakoff, Wladimir; Mayer, Claudine

    2005-11-01

    The crystallization of a hypothetical penicillin-binding protein from the archaeon P. abyssi in space group C2 by hanging-drop vapour diffusion is reported. The genome of the hyperthermophilic archaeon Pyrococcus abyssi contains a gene (pab0087) encoding a penicillin-binding protein (PBP) homologue. This sequence consists of 447 residues and shows significant sequence similarity to low-molecular-weight PBPs and class C β-lactamases. The Pab0087 protein was overexpressed, purified and crystallized. Diffraction data from two different crystal forms were collected to 2.7 and 2.0 Å resolution. Both crystals belong to space group C2, with unit-cell parameters a = 160.59, b = 135.74, c = 113.02 Å, β = 117.36° and a = 166.97, b = 131.25, c = 189.39 Å, β = 113.81°, respectively. The asymmetric unit contains four and eight molecules, respectively, with fourfold non-crystallographic symmetry.

  2. Purification, crystallization and preliminary crystallographic analysis of KatB, a manganese catalase from Anabaena PCC 7120.

    PubMed

    Bihani, Subhash Chandra; Chakravarty, Dhiman; Ballal, Anand

    2013-11-01

    Catalases are enzymes that play an important role in the detoxification of hydrogen peroxide (H2O2) in aerobic organisms. Among catalases, haem-containing catalases are ubiquitously distributed and their enzymatic mechanism is very well understood. On the other hand, manganese catalases that contain a bimanganese core in the active site have been less well characterized and their mode of action is not fully understood. The genome of Anabaena PCC 7120 does not show the presence of a haem catalase-like gene; instead, two ORFs encoding manganese catalases (Mn-catalases) are present. Here, the crystallization and preliminary X-ray crystallographic analysis of KatB, one of the two Mn-catalases from Anabaena, are reported. KatB was crystallized using the hanging-drop vapour-diffusion method with PEG 400 as a precipitant and calcium acetate as an additive. Diffraction data were collected in-house on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 2.2 Å resolution at 100 K. The tetragonal crystal belonged to space group P4(1)2(1)2 (or enantiomer), with unit-cell parameters a = b = 101.87, c = 138.86 Å. Preliminary X-ray diffraction analysis using the Matthews coefficient and self-rotation function suggests the presence of a trimer in the asymmetric unit.

  3. Purification, crystallization and preliminary X-ray diffraction analysis of NADP-dependent glutamate dehydrogenase from Aspergillus niger

    PubMed Central

    Prakash, Prem; Walvekar, Adhish S.; Punekar, Narayan S.; Bhaumik, Prasenjit

    2014-01-01

    Glutamate dehydrogenase (GDH) catalyzes the NAD-dependent or NADP-dependent oxidative deamination of l-glutamate to 2-oxoglutarate and ammonia. This important reversible reaction establishes the link between carbon and nitrogen metabolism. In this study, Aspergillus niger NADP-GDH (AnGDH) has been overexpressed and purified. Purified AnGDH, with a high specific activity of 631.1 units per milligram of protein, was crystallized and the crystal diffracted to 2.9 Å resolution using a home X-ray source. Preliminary analysis of the X-ray diffraction data showed that the crystal belonged to space group R32, with unit-cell parameters a = b = 173.8, c = 241.5 Å, α = β = 90, γ = 120°. The crystals exhibited an unusually high solvent content (83.0%) and had only one molecule in the asymmetric unit. Initial phases were obtained by molecular replacement, and model building and structure refinement of AnGDH are in progress. PMID:25372818

  4. Expression, purification and crystallization of a birnavirus-encoded protease, VP4, from blotched snakehead virus (BSNV).

    PubMed

    Lee, Jaeyong; Feldman, Anat R; Delmas, Bernard; Paetzel, Mark

    2006-04-01

    Blotched snakehead virus (BSNV) is a member of the Birnaviridae family that requires a virally encoded protease known as VP4 in order to process its polyprotein into viral capsid protein precursors (pVP2 and VP3). VP4 belongs to a family of serine proteases that utilize a serine/lysine catalytic dyad mechanism. A mutant construct of VP4 with a short C-terminal truncation was overexpressed in Escherichia coli and purified to homogeneity for crystallization. Using the sitting-drop vapour-diffusion method at room temperature, protein crystals with two distinct morphologies were observed. Cubic crystals grown in PEG 2000 MME and magnesium acetate at pH 8.5 belong to space group I23, with unit-cell parameters a = b = c = 143.8 angstroms. Trigonal crystals grown in ammonium sulfate and glycerol at pH 8.5 belong to space group P321/P312, with unit-cell parameters a = b = 158.2, c = 126.4 angstroms.

  5. The VapBC1 toxin-antitoxin complex from Mycobacterium tuberculosis: purification, crystallization and X-ray diffraction analysis.

    PubMed

    Lu, Zuokun; Wang, Han; Zhang, Aili; Tan, Yusheng

    2016-06-01

    Mycobacterium tuberculosis, a major human pathogen, encodes at least 88 toxin-antitoxin (TA) systems. Remarkably, more than half of these modules belong to the VapBC family. Under normal growth conditions, the toxicity of the toxin VapC is neutralized by the protein antitoxin VapB. When bacteria face an unfavourable environment, the antitoxin is degraded and the free toxin VapC targets important cellular processes in order to inhibit cell growth. TA systems function in many biological processes, such as in the stringent response, in biofilm formation and in drug tolerance. To explore the structure of the VapBC1 complex, the toxin VapC1 and the antitoxin VapB1 were separately cloned, co-expressed and crystallized. The best crystal was obtained using a crystallization solution consisting of optimized solution with commercial sparse-matrix screen solutions as additives. The crystal diffracted to a resolution of 2.7 Å and belonged to space group P21, with unit-cell parameters a = 59.3, b = 106.7, c = 250.0 Å, β = 93.75°. PMID:27303903

  6. Purification, crystallization and preliminary crystallographic analysis of a GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus

    SciTech Connect

    Wu, Hao; Sun, Lei; Brouns, Stan J. J.; Fu, Sheng; Akerboom, Jasper; Li, Xuemei; Oost, John van der

    2007-03-01

    A GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus has been crystallized. Combined with biochemical analyses, it is expected that the structure of this protein will give insight in the function of a relatively unknown subfamily of the GTPase superfamily. A predicted GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus, termed SsGBP, has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion technique in the presence of 0.05 M cadmium sulfate and 0.8 M sodium acetate pH 7.5. A single-wavelength anomalous dispersion data set was collected to a maximum resolution of 2.0 Å using a single cadmium-incorporated crystal. The crystal form belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with approximate unit-cell parameters a = 65.0, b = 72.6, c = 95.9 Å and with a monomer in the asymmetric unit.

  7. Purification, crystallization and preliminary X-ray analysis of a Nup107–Nup133 heterodimeric nucleoporin complex

    PubMed Central

    Boehmer, Thomas; Schwartz, Thomas U.

    2007-01-01

    The nuclear pore complex (NPC), the sole gateway of traffic between the nucleus and the cytoplasm, is built up from multiple copies of about 30 proteins collectively termed nucleoporins (nups). Nups are organized into distinct subcomplexes. Nup107 and Nup133 are members of the essential Nup107–160 subcomplex, a component of the central NPC architecture. A dimeric complex of the C-­terminal domains of human Nup107 and Nup133 was expressed from a bicistronic vector in Escherichia coli, purified and crystallized in two different crystal forms. Crystals grown in the presence of 18–22% PEG 3350 belong to space group P212121 and diffracted to 2.9 Å. Native and seleno-l-methionine-derivative crystals grown in the presence of 1.1 M sodium malonate belong to space group C2 and diffracted to 2.55 and 2.9 Å, respectively. Structure determination of this complex will give the first insights into the protein–protein interactions within a core module of the NPC. PMID:17768364

  8. Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

    SciTech Connect

    Troffer-Charlier, Nathalie; Cura, Vincent; Hassenboehler, Pierre; Moras, Dino; Cavarelli, Jean

    2007-04-01

    Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering. Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM1{sub 28–507} and two structural states of CARM1{sub 140–480} were expressed, purified and crystallized. Crystals of CARM1{sub 28–507} belong to space group P6{sub 2}22, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM1{sub 28–507} was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1{sub 140–480} belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1{sub 140–480} in complex with S-adenosyl-l-homocysteine belong to space P2{sub 1}2{sub 1}2, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1{sub 140–480} were solved by molecular-replacement techniques from the structure of CARM1{sub 28–507}.

  9. Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB

    SciTech Connect

    Garnett, James A.; Diallo, Mamou; Matthews, Steve J.

    2015-05-20

    In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher pathway that plays a major role in both early biofilm formation and host-cell adhesion. Initial attempts at crystallizing the chaperone EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. This is the first time that this refolding strategy has been used to purify CU chaperones. Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it

  10. Purification, crystallization and preliminary crystallographic studies of a Kunitz-type proteinase inhibitor from tamarind (Tamarindus indica) seeds.

    PubMed

    Patil, Dipak N; Chaudhry, Anshul; Sharma, Ashwani K; Tomar, Shailly; Kumar, Pravindra

    2009-07-01

    A Kunitz-type proteinase inhibitor has been purified from tamarind (Tamarindus indica) seeds. SDS-PAGE analysis of a purified sample showed a homogeneous band corresponding to a molecular weight of 21 kDa. The protein was identified as a Kunitz-type proteinase inhibitor based on N-terminal amino-acid sequence analysis. It was crystallized by the vapour-diffusion method using PEG 6000. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 37.2, b = 77.1, c = 129.1 A. Diffraction data were collected to a resolution of 2.7 A. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%.

  11. Purification and crystallization of Bacillus subtilis NrnA, a novel enzyme involved in nanoRNA degradation

    SciTech Connect

    Nelersa, Claudiu M.; Schmier, Brad J.; Malhotra, Arun

    2012-05-08

    The final step in RNA degradation is the hydrolysis of RNA fragments five nucleotides or less in length (nanoRNA) to mononucleotides. In Escherichia coli this step is carried out by oligoribonuclease (Orn), a DEDD-family exoribonuclease that is conserved throughout eukaryotes. However, many bacteria lack Orn homologs, and an unrelated DHH-family phosphoesterase, NrnA, has recently been identified as one of the enzymes responsible for nanoRNA degradation in Bacillus subtilis. To understand its mechanism of action, B. subtilis NrnA was purified and crystallized at room temperature using the hanging-drop vapor-diffusion method with PEG 4000, PEG 3350 or PEG MME 2000 as precipitant. The crystals belonged to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 50.62, b = 121.3, c = 123.4 {angstrom}, {alpha} = 90, {beta} = 91.31, {gamma} = 90{sup o}.

  12. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the extracellular olfactomedin domain of gliomedin

    PubMed Central

    Han, Huijong; Kursula, Petri

    2014-01-01

    Gliomedin (GLDN) is one of the essential proteins in the development of the nodes of Ranvier in the vertebrate peripheral nervous system. An olfactomedin (OLF) domain is located at the GLDN extracellular C-terminus and is involved in the accumulation of neuronal plasma membrane voltage-gated sodium channels in the nodes by interacting with neurofascin and NrCAM. No structures of OLF domains have previously been reported. Here, the crystallization of the rat GLDN OLF domain, which was expressed in an insect-cell system, is reported. The crystal diffracted to 1.55 Å resolution and belonged to space group P21, with unit-cell parameters a = 37.5, b = 141.7, c = 46.0 Å, β = 110.6°, and had two molecules in the asymmetric unit. PMID:25372825

  13. Purification, crystallization and preliminary X-ray crystallographic analysis of rice Bowman–Birk inhibitor from Oryza sativa

    PubMed Central

    Lin, Yi-Hung; Li, Hsin-Tai; Huang, Yen-Chieh; Hsieh, Ying-Cheng; Guan, Hong-Hsiang; Liu, Ming-Yih; Chang, Tschining; Wang, Andrew H.-J.; Chen, Chun-Jung

    2006-01-01

    Bowman–Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 Å, the unit cell belongs to space group P212121, with unit-cell parameters a = 74.37, b = 96.69, c = 100.36 Å. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%. PMID:16754971

  14. Expression, purification, crystallization and preliminary X-ray analysis of glucose-1-phosphate uridylyltransferase (GalU) from Erwinia amylovora

    PubMed Central

    Toccafondi, Mirco; Cianci, Michele; Benini, Stefano

    2014-01-01

    Glucose-1-phosphate uridylyltransferase from Erwinia amylovora CFPB1430 was expressed as a His-tag fusion protein in Escherichia coli. After tag removal, the purified protein was crystallized from 100 mM Tris pH 8.5, 2 M ammonium sulfate, 5% ethylene glycol. Diffraction data sets were collected to a maximum resolution of 2.46 Å using synchrotron radiation. The crystals belonged to the hexagonal space group P62, with unit-cell parameters a = 80.67, b = 80.67, c = 169.18. The structure was solved by molecular replacement using the structure of the E. coli enzyme as a search model. PMID:25195902

  15. Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB

    PubMed Central

    Garnett, James A.; Diallo, Mamou; Matthews, Steve J.

    2015-01-01

    Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals. PMID:26057794

  16. Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB.

    PubMed

    Garnett, James A; Diallo, Mamou; Matthews, Steve J

    2015-06-01

    Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone-usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.

  17. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of β-ketothiolase B from Ralstonia eutropha H16

    PubMed Central

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Chang, Jeong Ho; Kim, Kyung-Jin

    2014-01-01

    Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. β-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M bis-tris pH 6.5, 0.2 M lithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space group C2221, with unit-cell parameters a = 106.95, b = 107.24, c = 144.14 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (V M) is 2.54 Å3 Da−1, which corresponds to a solvent content of approximately 51.5%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress. PMID:24598917

  18. Purification, crystallization and preliminary X-ray diffraction analysis of pathogen-inducible oxygenase (PIOX) from Oryza sativa

    SciTech Connect

    Lloyd, Tracy; Krol, Adam; Campanaro, Danielle; Malkowski, Michael

    2006-04-01

    The heme-containing membrane-associated fatty-acid α-dioxygenase pathogen-inducible oxygenase (PIOX) from O. sativa has been crystallized and a data set collected to 3.0 Å using a rotating-anode generator and R-AXIS IV detector. Pathogen-inducible oxygenase (PIOX) is a heme-containing membrane-associated protein found in monocotyledon and dicotyledon plants that utilizes molecular oxygen to convert polyunsaturated fatty acids into their corresponding 2R-hydroperoxides. PIOX is a member of a larger family of fatty-acid α-dioxygenases that includes the mammalian cyclooxygenase enzymes cyclooxygenase 1 and 2 (COX-1 and COX-2). Single crystals of PIOX from rice (Oryza sativa) have been grown from MPD using recombinant protein expressed in Escherichia coli and subsequently extracted utilizing decyl maltoside as the solubilizing detergent. Crystals diffract to 3.0 Å resolution using a rotating-anode generator and R-AXIS IV detector, and belong to space group P1. Based on the Matthews coefficient and self-rotation function analyses, there are presumed to be four molecules in the asymmetric unit related by noncrystallographic 222 symmetry.

  19. Purification, Crystallization And Preliminary X-Ray Analysis of Aminoglycoside-2 ''-Phosphotransferase-Ic [APH(2 '')-Ic] From Enterococcus Gallinarum

    SciTech Connect

    Byrnes, L.J.; Badarau, A.; Vakulenko, S.B.; Smith, C.A.; /SLAC, SSRL

    2009-04-30

    Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2{double_prime}-phosphotransferase-Ic [APH(2{double_prime})-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2{double_prime})-Ic variants were crystallized in the presence of 14-20%(w/v) PEG 4000, 0.25 M MgCl{sub 2}, 0.1 M Tris-HCl pH 8.5 and 1 mM Mg{sub 2}GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 {angstrom}, {beta} = 108.8{sup o}. X-ray diffraction data were collected to approximately 2.15 {angstrom} resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.

  20. Purification, Crystallization and Preliminary X-ray Diffraction Analysis of Cif, a Virulence Factor Secreted by Pseudomonas aeruginosa

    SciTech Connect

    Bahl, C.; MacEachran, D; O' Toole, G; Madden, D

    2010-01-01

    The opportunistic pathogen Pseudomonas aeruginosa secretes a protein that triggers the accelerated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. This protein, which is known as the CFTR inhibitory factor (Cif), acts as a virulence factor and may facilitate airway colonization by P. aeruginosa. Based on sequence similarity Cif appears to be an epoxide hydrolase (EH), but it lacks several of the conserved features found in the active sites of canonical members of the EH family. Here, the crystallization of purified recombinant Cif by vapor diffusion is reported. The crystals formed in space group C2, with unit-cell parameters a = 167.4, b = 83.6, c = 88.3 {angstrom}, {beta} = 100.6{sup o}. The crystals diffracted to 2.39 {angstrom} resolution on a rotating-anode source. Based on the calculated Matthews coefficient (2.2 {angstrom}{sup 3} Da{sup -1}), it appears that the asymmetric unit contains four molecules.

  1. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of D-lactate dehydrogenase from Lactobacillus jensenii.

    PubMed

    Kim, Sangwoo; Kim, Yong Hwan; Kim, Kyung-Jin

    2014-08-01

    The thermostable D-lactate dehydrogenase from Lactobacillus jensenii (LjD-LDH) is a key enzyme for the production of the D-form of lactic acid from pyruvate concomitant with the oxidation of NADH to NAD(+). The polymers of lactic acid are used as biodegradable bioplastics. The LjD-LDH protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 28%(w/v) polyethylene glycol 400, 100 mM Tris-HCl pH 9, 200 mM magnesium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The crystal belonged to space group P3121, with unit-cell parameters a = b = 90.5, c = 157.8 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.58 Å(3) Da(-1), which corresponds to a solvent content of approximately 52.3%. The structure was solved by single-wavelength anomalous dispersion using a selenomethionine derivative. PMID:25084378

  2. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of β-ketothiolase B from Ralstonia eutropha H16.

    PubMed

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Chang, Jeong Ho; Kim, Kyung-Jin

    2014-03-01

    Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. β-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M bis-tris pH 6.5, 0.2 M lithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space group C2221, with unit-cell parameters a = 106.95, b = 107.24, c = 144.14 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.54 Å(3) Da(-1), which corresponds to a solvent content of approximately 51.5%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress. PMID:24598917

  3. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of PhaA from Ralstonia eutropha.

    PubMed

    Kim, Eun-Jung; Kim, Kyung-Jin

    2014-11-01

    Polyhydroxybutyrate (PHB) is a biopolymer that is in the spotlight because of its broad applications in bioplastics, fine chemicals, implant biomaterials and biofuels. PhaA from Ralstonia eutropha (RePhaA) is the first enzyme in the PHB biosynthetic pathway and catalyzes the condensation reaction of two acetyl-CoA molecules to give acetoacetyl-CoA. RePhaA was crystallized using the hanging-drop vapour-diffusion method in the presence of 20% polyethylene glycol monomethyl ether 2K, 0.1 M Tris-HCl pH 8.5 and 0.2 M trimethylamine N-oxide dihydrate at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.96 Å on a synchrotron beamline. The crystal belonged to space group P2₁, with unit-cell parameters a=68.38, b=105.47, c=106.91 Å, α=γ=90, β=106.18°. With four subunits per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.3 Å3 Da(-1), which corresponds to a solvent content of approximately 46.2%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress. PMID:25372833

  4. Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme from Gloydius saxatilis venom

    SciTech Connect

    Wei, Wenqing; Zhao, Wei; Wang, Xiaoping; Teng, Maikun Niu, Liwen

    2007-08-01

    The thrombin-like enzyme saxthrombin has been purified from G. saxatilis snake venom. Crystallization conditions were found and a data set was obtained to 1.43 Å. The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatography procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS–PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 Å. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 Å, β = 96.72°, and the Matthews coefficient (V{sub M}) was calculated to be 2.13 Å{sup 3} Da{sup −1} with one molecule in the asymmetric unit.

  5. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of PhaA from Ralstonia eutropha

    PubMed Central

    Kim, Eun-Jung; Kim, Kyung-Jin

    2014-01-01

    Polyhydroxybutyrate (PHB) is a biopolymer that is in the spotlight because of its broad applications in bioplastics, fine chemicals, implant biomaterials and biofuels. PhaA from Ralstonia eutropha (RePhaA) is the first enzyme in the PHB biosynthetic pathway and catalyzes the condensation reaction of two acetyl-CoA molecules to give acetoacetyl-CoA. RePhaA was crystallized using the hanging-drop vapour-diffusion method in the presence of 20% polyethylene glycol monomethyl ether 2K, 0.1 M Tris–HCl pH 8.5 and 0.2 M trimethylamine N-oxide dihydrate at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.96 Å on a synchrotron beamline. The crystal belonged to space group P21, with unit-cell parameters a = 68.38, b = 105.47, c = 106.91 Å, α = γ = 90, β = 106.18°. With four subunits per asymmetric unit, the crystal volume per unit protein weight (V M) is 2.3 Å3 Da−1, which corresponds to a solvent content of approximately 46.2%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress. PMID:25372833

  6. Cloning, expression, purification, crystallization and preliminary X-ray characterization of allantoinase from Bacillus licheniformis ATCC 14580

    PubMed Central

    Conejero-Muriel, Mayte; Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Gavira, Jose A.

    2014-01-01

    Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N1—C2 amide bond of the five-membered hydantoin ring. Allantoinase from Bacillus licheniformis (AllBali) presents an inverted enantioselectivity towards allantoin (R-enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work, B. licheniformis ATCC 14580 allantoinase (AllBali) containing a C-terminal His6 tag was overproduced in Escherichia coli and purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 M potassium thiocyanate, 20%(w/v) polyethylene glycol 3350 as a crystallization solution. X-ray diffraction data were collected to a resolution of 3.5 Å with an R merge of 29.2% from a crystal belonging to space group P1211, with unit-cell parameters a = 54.93, b = 164.74, c = 106.89 Å, β = 98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient (V M = 2.34 Å3 Da−1). PMID:25372819

  7. Expression, purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of metabotropic glutamate receptor 7

    SciTech Connect

    Muto, Takanori; Tsuchiya, Daisuke; Morikawa, Kosuke Jingami, Hisato

    2007-07-01

    The ligand-binding domain of metabotropic glutamate receptor 7 has been overexpressed, purified, and crystallized by the hanging-drop vapour-diffusion method. A complete data set has been collected to 3.30 Å. Glutamate is the major excitatory neurotransmitter and its metabotropic glutamate receptor (mGluR) plays an important role in the central nervous system. The ligand-binding domain (LBD) of mGluR subtype 7 (mGluR7) was produced using the baculovirus expression system and purified from the culture medium. The purified protein was characterized by gel-filtration chromatography, SDS–PAGE and a ligand-binding assay. Crystals of mGluR7 LBD were grown at 293 K by the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 3.30 Å resolution using synchrotron radiation and belong to the trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 92.4, c = 114.3 Å. Assuming the presence of one protomer per crystallographic asymmetric unit, the Matthews coefficient V{sub M} was calculated to be 2.5 Å{sup 3} Da{sup −1} and the solvent content was 51%.

  8. Expression, purification, crystallization and preliminary diffraction studies of the mammalian DAG kinase homologue YegS from Escherichia coli

    SciTech Connect

    Bakali H, M. Amin; Nordlund, Pär; Hallberg, B. Martin

    2006-03-01

    The overexpression, crystallization and preliminary diffraction analysis of E. coli YegS are reported. yegS is a gene encoding a 32 kDa cytosolic protein with unknown function but with strong sequence homology to a family of structurally uncharacterized eukaryotic non-protein kinases: diacylglycerol kinases, sphingosine kinases and ceramide kinases. Here, the overexpression, crystallization and preliminary diffraction analysis of Escherichia coli YegS are reported. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 42.4, b = 166.1, c = 48.5 Å, β = 96.97°. The presence of a dimer in the asymmetric unit was estimated to give a Matthews coefficient (V{sub M}) of 2.5 Å{sup 3} Da{sup −1} and a solvent content of 50.8%(v/v). Single-wavelength diffraction data were collected to a resolution of 1.9 Å using synchrotron radiation.

  9. Purification, crystallization and preliminary X-ray analysis of cytochrome P450 219A1 from Novosphingobium aromaticivorans DSM 12444

    PubMed Central

    Hong, Chuan; Bell, Stephen G.; Yang, Wen; Wang, Hui; Hao, Yiming; Li, Xin; Zhou, Weihong; Bartlam, Mark; Wong, Luet-Lok

    2009-01-01

    Cytochrome P450 enzymes catalyze a variety of reactions and are widely distributed in living organisms. In recent studies, the first members of five new families of cytochrome P450 enzymes have been identified, including cyto­chrome P450 219A1 (CYP219A1) from Novosphingobium aromaticivorans DSM 12444. It has also been reported that isolongifolen-9-one (C15H22O), a sesqui­terpenoid ketone derivative, is a potential substrate for CYP219A1, inducing a ≥95% shift of the haem spin state to high spin upon binding. The CYP219A1 protein has been crystallized and single crystals have been studied by X-ray crystallography. Diffraction data were collected to 2.4 Å resolution. The crystals belonged to space group P6, with unit-cell parameters a = 93.1, b = 93.1, c = 98.0 Å. Preliminary X-ray diffraction data analysis revealed that the asymmetric unit contained one protein molecule. PMID:19342781

  10. Systems Harmonization and Convergence - the GIGAS Approach

    NASA Astrophysics Data System (ADS)

    Marchetti, P. G.; Biancalana, A.; Coene, Y.; Uslander, T.

    2009-04-01

    0.1 Background The GIGAS1 Support Action promotes the coherent and interoperable development of the GMES, INSPIRE and GEOSS initiatives through their concerted adoption of standards, protocols, and open architectures. 0.2 Preparing for Coordinated Data Access The GMES Coordinated Data Access System is under design and implementation2. This objective has motivated the definition of the interoperability standards between the contributing missions. The following elements have been addressed with associated papers submitted to OGC: The EO Product Metadata has been based on the OGC Geographic Markup Language, addressing sensor characteristics for optical, radar and atmospheric products. Collection and service discovery: an ISO extension package for CSW ebRim has been proposed. Catalogue Service (CSW): an Earth Observation extension package of the CSW ebRim has been proposed. Feasibility Analysis and Order: an Order interface control document and an Earth Observation profile of the Sensor Planning Service have been proposed. Online Data Access: an Earth Observation profile of the Web Map Services (WMS) for visualization and evaluation purposes has been proposed. Identity (user) management: the objective in the long term is to allow for a single sign-on to the Coordinated Data Access system by users registered in the various Earth Observation ground segments by providing a federated identity across participating ground segments, exploiting OASIS standards. 0.3 The GIGAS proposed harmonization approach The approach proposed by GIGAS is based on three elements: Technology watch Comparative analysis Shaping of initiatives and standards This paper concentrates on the methodology for technology watch and comparative analysis. The complexity of the GIGAS scenario involving huge systems (i.e. GEOSS, INSPIRE, GMES etc.) entails the interaction with different heterogeneous partners, each with a specific competence, expertise and know-how. 0.3.1 Technology watch The methodology

  11. Purification, crystallization and X-ray crystallographic studies of a Bacillus cereus MepR-like transcription factor, BC0657.

    PubMed

    Cho, Min Uk; Kim, Meong Il; Hong, Minsun

    2015-06-01

    Transcription factors of the MarR family respond to internal and external changes and regulate a variety of biological functions through ligand association with microorganisms. MepR belongs to the MarR family, and its mutations are associated with the development of multidrug resistance in Staphylococcus aureus, which has caused a growing health problem. In this study, a Bacillus cereus MepR-like transcription regulator, BC0657, was crystallized. The BC0657 crystals diffracted to 2.05 Å resolution and belonged to either space group P6(2)22 or P6(4)22, with unit-cell parameters a = 110.57, b = 110.57, c = 67.29 Å. There was one molecule per asymmetric unit. Future comparative structural studies on BC0657 would extend knowledge of ligand-induced transcriptional regulatory mechanisms in the MarR family and would make a significant contribution to the design of antibiotic drugs against multidrug-resistant bacteria.

  12. Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease from Pseudomonas aeruginosa

    PubMed Central

    Nji, Emmanuel; Li, Dianfan; Doyle, Declan A.; Caffrey, Martin

    2014-01-01

    The prokaryotic lysine-specific permease (LysP) belongs to the amino acid–polyamine–organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of l-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP from Pseudomonas aeruginosa was recombinantly expressed in Escherichia coli and purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of l-lysine and the l-lysine analogue l-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space group P21, with unit-cell parameters a = 169.53, b = 169.53, c = 290.13 Å, γ = 120°. PMID:25286940

  13. Purification, crystallization and preliminary X-ray studies of MbtN (Rv1346) from Mycobacterium tuberculosis.

    PubMed

    Chai, Ai Fen; Johnston, Jodie M; Bunker, Richard D; Bulloch, Esther M M; Evans, Genevieve L; Lott, J Shaun; Baker, Edward N

    2013-12-01

    In Mycobacterium tuberculosis, the protein MbtN (Rv1346) catalyzes the formation of a double bond in the fatty-acyl moiety of the siderophore mycobactin, which is used by this organism to acquire essential iron. MbtN is homologous to acyl-CoA dehydrogenases, whose general role is to catalyze the α,β-dehydrogenation of fatty-acyl-CoA conjugates. Mycobactins, however, contain a long unsaturated fatty-acid chain with an unusual cis double bond conjugated to the carbonyl group of the mycobactin core. To characterize the role of MbtN in the dehydrogenation of this fatty-acyl moiety, the enzyme has been expressed, purified and crystallized. The crystals diffracted to 2.3 Å resolution at a synchrotron source and were found to belong to the hexagonal space group H32, with unit-cell parameters a = b = 139.10, c = 253.09 Å, α = β = 90, γ = 120°. PMID:24316828

  14. Purification, crystallization and preliminary X-ray studies of MbtN (Rv1346) from Mycobacterium tuberculosis

    PubMed Central

    Chai, Ai-Fen; Johnston, Jodie M.; Bunker, Richard D.; Bulloch, Esther M. M.; Evans, Genevieve L.; Lott, J. Shaun; Baker, Edward N.

    2013-01-01

    In Mycobacterium tuberculosis, the protein MbtN (Rv1346) catalyzes the formation of a double bond in the fatty-acyl moiety of the siderophore mycobactin, which is used by this organism to acquire essential iron. MbtN is homologous to acyl-CoA dehydrogenases, whose general role is to catalyze the α,β-dehydrogenation of fatty-acyl-CoA conjugates. Mycobactins, however, contain a long unsaturated fatty-acid chain with an unusual cis double bond conjugated to the carbonyl group of the mycobactin core. To characterize the role of MbtN in the dehydrogenation of this fatty-acyl moiety, the enzyme has been expressed, purified and crystallized. The crystals diffracted to 2.3 Å resolution at a synchrotron source and were found to belong to the hexagonal space group H32, with unit-cell parameters a = b = 139.10, c = 253.09 Å, α = β = 90, γ = 120°. PMID:24316828

  15. High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

    PubMed Central

    Truan, Daphné; Vasil, Adriana; Stonehouse, Martin; Vasil, Michael L.; Pohl, Ehmke

    2013-01-01

    The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secreted as a fully folded heterodimer (PlcHR2) of approximately 95 kDa, by the twin arginine translocase (TAT) via the cytoplasmic membrane and through the outer membrane, by the Xcp (TypeII) secretory system. PlcHR2 has been shown to be an important virulence factor in model P. aeruginosa infections and is selectively cytotoxic, at picomolar concentrations to mammalian endothelial cells. Here we report how the various challenges starting from protein overexpression in the native organism P. aeruginosa, the use of detergents in the crystallization and data collection using the most advanced μ-focus synchrotron beam lines were overcome. Native diffraction data of this heterodimeric protein complex were collected up to a resolution of 4 Å, whereas needle-shaped crystals of l-selenomethionine substituted PlcHR2 with a maximum diameter of 10 micron were used to collect data sets with a maximum resolution of 2.75 Å. PMID:23201280

  16. Improved expression, purification and crystallization of a putative N-acetyl-γ-glutamyl-phosphate reductase from rice (Oryza sativa)

    SciTech Connect

    Miura-Ohnuma, Jun; Nonaka, Tsuyoshi; Katoh, Shizue; Murata, Katsuyoshi; Kita, Akiko; Miki, Kunio

    2005-12-01

    Crystals of OsAGPR were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 Å resolution. They belong to the hexagonal space group P6{sub 1}, with unit-cell parameters a = 86.11, c = 316.3 Å. N-Acetyl-γ-glutamyl-phosphate reductase (AGPR) catalyzes the third step in an eight-step arginine-biosynthetic pathway that starts with glutamate. This enzyme converts N-acetyl-γ-glutamyl phosphate to N-acetylglutamate-γ-semialdehyde by an NADPH-dependent reductive dephosphorylation. AGPR from Oryza sativa (OsAGPR) was expressed in Escherichia coli at 291 K as a soluble fusion protein with an upstream thioredoxin-hexahistidine [Trx-(His){sub 6}] extension. OsAGPR(Ala50–Pro366) was purified and crystals were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 Å resolution. They belong to the hexagonal space group P6{sub 1}, with unit-cell parameters a = 86.11, c = 316.3 Å.

  17. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of propionate kinase (TdcD) from Salmonella typhimurium

    SciTech Connect

    Simanshu, Dhirendra K.; Murthy, M. R. N.

    2005-01-01

    Propionate kinase (TdcD) from S. typhimurium has been expressed, purified and crystallized. A diffraction data set has been collected to 2.2 Å resolution. In the cell, propionate is mainly formed during β-oxidation of odd-numbered carbon-chain fatty acids, fermentation of carbohydrates and degradation of the amino acids threonine, valine, isoleucine and methionine. Recently, it has been shown that l-threonine is non-oxidatively cleaved to propionate via 2-ketobutyrate. The last step in this process, conversion of propionyl phosphate and ADP to propionate and ATP, is catalysed by propionate kinase (EC 2.7.1.–). Here, the cloning of propionate kinase (molecular weight 44 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli are reported. Purified propionate kinase was found to cocrystallize with ADP in the hanging-drop vapour-diffusion and microbatch methods. Crystals belong to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 111.47, c = 66.52 Å. A complete data set to 2.2 Å resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.

  18. Purification, crystallization and preliminary crystallographic studies of haemoglobin from mongoose (Helogale parvula) in two different crystal forms induced by pH variation.

    PubMed

    Mohamed Abubakkar, M; Saraboji, K; Ponnuswamy, M N

    2013-02-01

    Haemoglobin (Hb) is a respiratory pigment; it is a tetrameric protein that ferries oxygen from the lungs to tissues and transports carbon dioxide on the return journey. The oxygen affinity of haemoglobin is regulated by the concentration of oxygen surrounding it and several efforts have revealed the shapes of Hb in different states and with different functions. However, study of the molecular basis of Hbs from low-oxygen-affinity species is critically needed in order to increase the understanding of the mechanism behind oxygen adaptation. The present study reports the preliminary crystallographic study of low-oxygen-affinity haemoglobin from mongoose, a burrowing mammal. Haemoglobin from mongoose was purified by anion-exchange chromatography, crystallized using the hanging-drop vapour-diffusion method and diffraction data sets were collected from monoclinic (2.3 Å resolution) and orthorhombic (2.9 Å resolution) crystal forms obtained by pH variation. The monoclinic and orthorhombic asymmetric units contained half and a whole biological molecule, respectively.

  19. Purification, crystallization and preliminary crystallographic studies of haemoglobin from mongoose (Helogale parvula) in two different crystal forms induced by pH variation.

    PubMed

    Mohamed Abubakkar, M; Saraboji, K; Ponnuswamy, M N

    2013-02-01

    Haemoglobin (Hb) is a respiratory pigment; it is a tetrameric protein that ferries oxygen from the lungs to tissues and transports carbon dioxide on the return journey. The oxygen affinity of haemoglobin is regulated by the concentration of oxygen surrounding it and several efforts have revealed the shapes of Hb in different states and with different functions. However, study of the molecular basis of Hbs from low-oxygen-affinity species is critically needed in order to increase the understanding of the mechanism behind oxygen adaptation. The present study reports the preliminary crystallographic study of low-oxygen-affinity haemoglobin from mongoose, a burrowing mammal. Haemoglobin from mongoose was purified by anion-exchange chromatography, crystallized using the hanging-drop vapour-diffusion method and diffraction data sets were collected from monoclinic (2.3 Å resolution) and orthorhombic (2.9 Å resolution) crystal forms obtained by pH variation. The monoclinic and orthorhombic asymmetric units contained half and a whole biological molecule, respectively. PMID:23385751

  20. Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the C-terminal domain of Par-4 (PAWR)

    PubMed Central

    Tiruttani Subhramanyam, Udaya Kumar; Kubicek, Jan; Eidhoff, Ulf B.; Labahn, Joerg

    2014-01-01

    Prostate apoptosis response-4 protein is an intrinsically disordered pro-apoptotic protein with tumour suppressor function. Par-4 is known for its selective induction of apoptosis in cancer cells only and its ability to interact with various apoptotic proteins via its C-terminus. Par-4, with its unique function and various interacting partners, has gained importance as a potential target for cancer therapy. The C-terminus of the rat homologue of Par-4 was crystallized and a 3.7 Å resolution X-ray diffraction data set was collected. Preliminary data analysis shows the space group to be P41212. The unit-cell parameters are a = b = 115.351, c = 123.663 Å, α = β = γ = 90°. PMID:25195896

  1. Cloning, expression, purification, crystallization and X-ray analysis of inositol monophosphatase from Mus musculus and Homo sapiens

    PubMed Central

    Singh, Nisha; Halliday, Amy C.; Knight, Matthew; Lack, Nathan A.; Lowe, Edward; Churchill, Grant C.

    2012-01-01

    Inositol monophosphatase (IMPase) catalyses the hydrolysis of inositol monophosphate to inositol and is crucial in the phosphatidylinositol (PI) signalling pathway. Lithium, which is the drug of choice for bipolar disorder, inhibits IMPase at therapeutically relevant plasma concentrations. Both mouse IMPase 1 (MmIMPase 1) and human IMPase 1 (HsIMPase 1) were cloned into pRSET5a, expressed in Escherichia coli, purified and crystallized using the sitting-drop method. The structures were solved at resolutions of 2.4 and 1.7 Å, respectively. Comparison of MmIMPase 1 and HsIMPase 1 revealed a core r.m.s. deviation of 0.516 Å. PMID:23027737

  2. Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.

    PubMed Central

    Malinowski, J. J.; Grasberger, B. L.; Trakshel, G.; Huston, E. E.; Helaszek, C. T.; Smallwood, A. M.; Ator, M. A.; Banks, T. M.; Brake, P. G.; Ciccarelli, R. B.

    1995-01-01

    Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease. PMID:8535252

  3. GigaDB: promoting data dissemination and reproducibility

    PubMed Central

    Sneddon, Tam P.; Si Zhe, Xiao; Edmunds, Scott C.; Li, Peter; Goodman, Laurie; Hunter, Christopher I.

    2014-01-01

    Often papers are published where the underlying data supporting the research are not made available because of the limitations of making such large data sets publicly and permanently accessible. Even if the raw data are deposited in public archives, the essential analysis intermediaries, scripts or software are frequently not made available, meaning the science is not reproducible. The GigaScience journal is attempting to address this issue with the associated data storage and dissemination portal, the GigaScience database (GigaDB). Here we present the current version of GigaDB and reveal plans for the next generation of improvements. However, most importantly, we are soliciting responses from you, the users, to ensure that future developments are focused on the data storage and dissemination issues that still need resolving. Database URL: http://www.gigadb.org PMID:24622612

  4. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  5. Water purification using organic salts

    DOEpatents

    Currier, Robert P.

    2004-11-23

    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.

  6. An electron microscopy study of the microstructure and microarchitecture of the Strombus gigas shell

    SciTech Connect

    Rieke, P.C.; Laraia, V.J. ); Heuer, A.H. ); Aindow, M. )

    1989-11-01

    A scanning and transmission electron microscopy study is presented of the microstructure of the Strombus gigas shell. The hierarchical nature of this crossed-lamellar structure and the defect content of the mineral component are described. The mineral component consists of small single crystal grains of aragonite, the metastable orthorhombic polymorph of CaCO{sub 3}. The habit and morphology of the grains discussed here have not been determined previously. The observed habit and defect structure suggest that the organic matrix exerts a high degree of control over the crystal growth of the mineral phase and is responsible for the long range order in the microarhitecture. Electron beam heating of the mineral component leads to certain phase changes and these are discussed. 15 refs., 6 figs.

  7. Hamiltonian purification

    SciTech Connect

    Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo; Pascazio, Saverio; Nakazato, Hiromichi; Yuasa, Kazuya; Giovannetti, Vittorio

    2015-12-15

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians (h{sub 1}, …, h{sub m}) operating on a d-dimensional quantum system ℋ{sub d}, the problem consists in identifying a set of commuting Hamiltonians (H{sub 1}, …, H{sub m}) operating on a larger d{sub E}-dimensional system ℋ{sub d{sub E}} which embeds ℋ{sub d} as a proper subspace, such that h{sub j} = PH{sub j}P with P being the projection which allows one to recover ℋ{sub d} from ℋ{sub d{sub E}}. The notions of spanning-set purification and generator purification of an algebra are also introduced and optimal solutions for u(d) are provided.

  8. Hamiltonian purification

    NASA Astrophysics Data System (ADS)

    Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo; Nakazato, Hiromichi; Pascazio, Saverio; Yuasa, Kazuya; Giovannetti, Vittorio

    2015-12-01

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians {h1, …, hm} operating on a d-dimensional quantum system ℋd, the problem consists in identifying a set of commuting Hamiltonians {H1, …, Hm} operating on a larger dE-dimensional system ℋdE which embeds ℋd as a proper subspace, such that hj = PHjP with P being the projection which allows one to recover ℋd from ℋdE. The notions of spanning-set purification and generator purification of an algebra are also introduced and optimal solutions for 𝔲(d) are provided.

  9. Structural basis for the fracture toughness of the shell of the conch Strombus gigas

    NASA Astrophysics Data System (ADS)

    Kamat, S.; Su, X.; Ballarini, R.; Heuer, A. H.

    2000-06-01

    Natural composite materials are renowned for their mechanical strength and toughness: despite being highly mineralized, with the organic component constituting not more than a few per cent of the composite material, the fracture toughness exceeds that of single crystals of the pure mineral by two to three orders of magnitude. The judicious placement of the organic matrix, relative to the mineral phase, and the hierarchical structural architecture extending over several distinct length scales both play crucial roles in the mechanical response of natural composites to external loads. Here we use transmission electron microscopy studies and beam bending experiments to show that the resistance of the shell of the conch Strombus gigas to catastrophic fracture can be understood quantitatively by invoking two energy-dissipating mechanisms: multiple microcracking in the outer layers at low mechanical loads, and crack bridging in the shell's tougher middle layers at higher loads. Both mechanisms are intimately associated with the so-called crossed lamellar microarchitecture of the shell, which provides for `channel' cracking in the outer layers and uncracked structural features that bridge crack surfaces, thereby significantly increasing the work of fracture, and hence the toughness, of the material. Despite a high mineral content of about 99% (by volume) of aragonite, the shell of Strombus gigas can thus be considered a `ceramic plywood', and can guide the biomimetic design of tough, lightweight structures.

  10. Structural basis for the fracture toughness of the shell of the conch Strombus gigas.

    PubMed

    Kamat, S; Su, X; Ballarini, R; Heuer, A H

    2000-06-29

    Natural composite materials are renowned for their mechanical strength and toughness: despite being highly mineralized, with the organic component constituting not more than a few per cent of the composite material, the fracture toughness exceeds that of single crystals of the pure mineral by two to three orders of magnitude. The judicious placement of the organic matrix, relative to the mineral phase, and the hierarchical structural architecture extending over several distinct length scales both play crucial roles in the mechanical response of natural composites to external loads. Here we use transmission electron microscopy studies and beam bending experiments to show that the resistance of the shell of the conch Strombus gigas to catastrophic fracture can be understood quantitatively by invoking two energy-dissipating mechanisms: multiple microcracking in the outer layers at low mechanical loads, and crack bridging in the shell's tougher middle layers at higher loads. Both mechanisms are intimately associated with the so-called crossed lamellar microarchitecture of the shell, which provides for 'channel' cracking in the outer layers and uncracked structural features that bridge crack surfaces, thereby significantly increasing the work of fracture, and hence the toughness, of the material. Despite a high mineral content of about 99% (by volume) of aragonite, the shell of Strombus gigas can thus be considered a 'ceramic plywood' and can guide the biomimetic design of tough, lightweight structures.

  11. Structural basis for the fracture toughness of the shell of the conch Strombus gigas.

    PubMed

    Kamat, S; Su, X; Ballarini, R; Heuer, A H

    2000-06-29

    Natural composite materials are renowned for their mechanical strength and toughness: despite being highly mineralized, with the organic component constituting not more than a few per cent of the composite material, the fracture toughness exceeds that of single crystals of the pure mineral by two to three orders of magnitude. The judicious placement of the organic matrix, relative to the mineral phase, and the hierarchical structural architecture extending over several distinct length scales both play crucial roles in the mechanical response of natural composites to external loads. Here we use transmission electron microscopy studies and beam bending experiments to show that the resistance of the shell of the conch Strombus gigas to catastrophic fracture can be understood quantitatively by invoking two energy-dissipating mechanisms: multiple microcracking in the outer layers at low mechanical loads, and crack bridging in the shell's tougher middle layers at higher loads. Both mechanisms are intimately associated with the so-called crossed lamellar microarchitecture of the shell, which provides for 'channel' cracking in the outer layers and uncracked structural features that bridge crack surfaces, thereby significantly increasing the work of fracture, and hence the toughness, of the material. Despite a high mineral content of about 99% (by volume) of aragonite, the shell of Strombus gigas can thus be considered a 'ceramic plywood' and can guide the biomimetic design of tough, lightweight structures. PMID:10890440

  12. Population structure of the giant garter snake, Thamnophis gigas

    USGS Publications Warehouse

    Paquin, M.M.; Wylie, G.D.; Routman, E.J.

    2006-01-01

    The giant garter snake, Thamnophis gigas, is a threatened species endemic to California's Central Valley. We tested the hypothesis that current watershed boundaries have caused genetic differentiation among populations of T. gigas. We sampled 14 populations throughout the current geographic range of T. gigas and amplified 859 bp from the mitochondrial gene ND4 and one nuclear microsatellite locus. DNA sequence variation from the mitochondrial gene indicates there is some genetic structuring of the populations, with high F ST values and unique haplotypes occurring at high frequency in several populations. We found that clustering populations by watershed boundary results in significant between-region genetic variance for mtDNA. However, analysis of allele frequencies at the microsatellite locus NSU3 reveals very low F ST values and little between-region variation in allele frequencies. The discordance found between mitochondrial and microsatellite data may be explained by aspects of molecular evolution and/or T. gigas life history characteristics. Differences in effective population size between mitochondrial and nuclear DNA, or male-biased gene flow, result in a lower migration rate of mitochondrial haplotypes relative to nuclear alleles. However, we cannot exclude homoplasy as one explanation for homogeneity found for the single microsatellite locus. The mitochondrial nucleotide sequence data supports conservation practices that identify separate management units for T. gigas. ?? Springer 2006.

  13. Preparation and antioxidant activities of oligosaccharides from Crassostrea gigas.

    PubMed

    Wu, Shengjun; Huang, Xiaolian

    2017-02-01

    Oligosaccharides were prepared from Crassostrea gigas by hydrolysis of polysaccharide in C. gigas with peroxide oxygen (H2O2). The hydrolysates were cleared of protein, filtered, ultrafiltered and precipitated with absolute ethanol to give C. gigas oligosaccharides (CGOs). Factors affecting CGO yields, i.e., reaction time, temperature, and H2O2 concentration, were optimised as follows: 2.96h reaction time, 84.71°C reaction temperature, and 2.46% H2O2 concentration. Under these conditions, the maximum yield of CGOs reached 10.61%. The CGOs were then partially characterised by Fourier transform infrared spectroscopy, UV spectroscopy, monosaccharide composition, and antioxidant activities. Results indicate that CGOs possessed strong hydroxyl radical activity, 2,2-diphenyl-β-picrylhydrazyl-radical-scavenging activity and reducing capacity at a concentration of 100μg/mL. PMID:27596415

  14. Polonium purification

    SciTech Connect

    Baker, J.D.

    1996-09-01

    Three processes for the purification of {sup 210}Po from irradiated bismuth targets are described. Safety equipment includes shielded hotcells for the initial separation from other activation products, gloveboxes for handling the volatile and highly toxic materials, and provisions for ventilation. All chemical separations must be performed under vacuum or in inerted systems. Two of the processes require large amounts of electricity; the third requires vessels made from exotic materials.

  15. Pool Purification

    NASA Technical Reports Server (NTRS)

    1988-01-01

    Caribbean Clear, Inc. used NASA's silver ion technology as a basis for its automatic pool purifier. System offers alternative approach to conventional purification chemicals. Caribbean Clear's principal markets are swimming pool owners who want to eliminate chlorine and bromine. Purifiers in Caribbean Clear System are same silver ions used in Apollo System to kill bacteria, plus copper ions to kill algae. They produce spa or pool water that exceeds EPA Standards for drinking water.

  16. Semiconductor grade, solar silicon purification project

    NASA Technical Reports Server (NTRS)

    Ingle, W. M.; Rosler, R. R.; Thompson, S. W.; Chaney, R. E.

    1979-01-01

    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.

  17. Heritability of shell pigmentation in the Pacific oyster, Crassostrea gigas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Pacific oyster (Crassostrea gigas) is a species of considerable economic importance, with among the highest global production of any cultured aquatic animal species. In the interest of increasing the value of Pacific oysters sold as “singles” for the half-shell market, we explored the feasibili...

  18. Purification, crystallization and preliminary X-ray crystallographic analysis of the flagellar accessory protein FlaH from the methanogenic archaeon Methanocaldococcus jannaschii

    PubMed Central

    Meshcheryakov, Vladimir A.; Yoon, Young-Ho; Matsunami, Hideyuki; Wolf, Matthias

    2014-01-01

    The flagellar accessory protein FlaH is thought to be one of the essential components of an archaeal motility system. However, to date biochemical and structural information about this protein has been limited. Here, the crystallization of FlaH from the hyperthermophilic archaeon Methanocaldococcus jannaschii is reported. Protein crystals were obtained by the vapour-diffusion method. These crystals belonged to space group P3121, with unit-cell parameters a = b = 131.42, c = 89.35 Å. The initial solution of the FlaH structure has been determined by multiple-wavelength anomalous dispersion phasing using a selenomethionine-derivatized crystal. PMID:25372827

  19. PURIFICATION PROCESS

    DOEpatents

    Wibbles, H.L.; Miller, E.I.

    1958-01-14

    This patent deals with the separation of uranium from molybdenum compounds, and in particular with their separation from ether solutions containing the molybdenum in the form of acids, such as silicomolybdic and phosphomolybdic acids. After the nitric acid leach of pitchblende, the molybdenum values present in the ore are found in the leach solution in the form of complex acids. The uranium bearing solution may be purified of this molybdenum content by comtacting it with activated charcoal. The purification is improved when the acidity of the solution is low ad agitation is also beneficial. The molybdenum may subsequently be recovered from the charcosl ad the charcoal reused.

  20. Water Purification

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Silver ionization water purification technology was originally developed for Apollo spacecraft. It was later used to cleanse swimming pools and has now been applied to industrial cooling towers and process coolers. Sensible Technologies, Inc. has added two other technologies to the system, which occupies only six square feet. It is manufactured in three capacities, and larger models are custom built on request. The system eliminates scale, corrosion, algae, bacteria and debris, and because of the NASA technology, viruses and waterborne bacteria are also destroyed. Applications include a General Motors cooling tower, amusement parks, ice manufacture and a closed-loop process cooling system.

  1. Cloning, purification, crystallization and preliminary X-ray diffraction of the OleC protein from Stenotrophomonas maltophilia involved in head-to-head hydrocarbon biosynthesis

    SciTech Connect

    Frias, JA; Goblirsch, BR; Wackett, LP; Wilmot, CM

    2010-08-28

    OleC, a biosynthetic enzyme involved in microbial hydrocarbon biosynthesis, has been crystallized. Synchrotron X-ray diffraction data have been collected to 3.4 A resolution. The crystals belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 98.8, c = 141.0 A.

  2. The expression, purification, crystallization and preliminary X-ray analysis of a subcomplex of the peripheral stalk of ATP synthase from bovine mitochondria

    SciTech Connect

    Silvester, Jocelyn A.; Kane Dickson, Veronica; Runswick, Michael J.; Leslie, Andrew G. W.; Walker, John E.

    2006-06-01

    A recombinant subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been crystallized and a native data set has been collected to 2.8 Å resolution. A subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been expressed to high levels in a soluble form in Escherichia coli. The subcomplex consists of residues 79–184 of subunit b, residues 1–124 of subunit d and the entire F{sub 6} subunit (76 residues). It has been purified and crystallized by vapour diffusion. The morphology and diffraction properties of the crystals of the subcomplex were improved by the presence of thioxane or 4-methylpyridine in the crystallization liquor. With a synchrotron-radiation source, these crystals diffracted to 2.8 Å resolution. They belong to the monoclinic space group P2{sub 1}.

  3. Expression, purification, crystallization and preliminary crystallographic analysis of hepatitis B virus core protein dimerized via a peptide linker containing an EGFP insertion.

    PubMed

    Kikuchi, Masaki; Iwabuchi, Shinichiro; Kikkou, Tatsuhiko; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi; Kawata, Masaaki; Sato, Chikara; Matsumoto, Osamu

    2013-08-01

    Virus-like particles (VLPs) have many potentially useful applications. The core proteins of human hepatitis B virus self-assemble into icosahedral VLPs. As previously reported, core protein dimers (CPDs), produced by connecting two core proteins via a peptide linker, can also assemble into VLPs. CPDs in which heterologous proteins were connected to the C-terminus (CPD1) were found to rearrange into symmetrical octahedra during crystallization. In this study, a heterologous protein was inserted into the peptide linker of the CPD (CPD2). CPD2 was expressed in Escherichia coli, assembled into VLPs, purified and crystallized. A single crystal diffracted to 2.8 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 218.6 Å. Single-crystal analysis showed that CPD1 and CPD2 rearranged into the same octahedral organization in a crystallization solution.

  4. SnRK2.6/OST1 from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis of K50N and D160A mutants

    PubMed Central

    Yunta, Cristina; Martinez-Ripoll, Martin; Albert, Armando

    2011-01-01

    The SnRK2.6 (SNF1-related kinase 2.6) gene from Arabidopsis thaliana encodes the serine/threonine protein kinase SnRK2.6/OST1 (OPEN STOMATA 1). It plays a central role in the drought-tolerance mechanism. OST1 is in fact the main positive effector in the hydric stress response. The SnRK2.6 gene was cloned into the pGEX4T1 plasmid, mutated and expressed in Escherichia coli, allowing purification to homogeneity in two chromatographic steps. Various OST1 mutants yielded crystals using vapour-diffusion techniques, but only one mutant showed a good diffraction pattern. Its crystals diffracted to 2.8 Å resolution and belonged to space group P2221, with unit-cell parameters a = 77.7, b = 99.4, c = 108.4 Å. A promising molecular-replacement solution was found using the structure of the kinase domain of the yeast AMP-activated protein kinase SNF1 (PDB entry 3hyh) as the search model. PMID:21393844

  5. Purification, crystallization and initial X-ray analysis of the C1 subunit of the astaxanthin protein, V600, of the chondrophore Velella velella.

    PubMed

    Chayen, N E; Boggon, T J; Raftery, J; Helliwell, J R; Zagalsky, P F

    1999-01-01

    The subunit C1 of the carotenoid-binding protein, V600, of the chondrophore Velella velella has been purified and crystallized. The crystals, which were grown by the vapour-diffusion method from ammonium sulfate as the major precipitant, diffract beyond 3 A and show little radiation damage over long periods (greater than 100 h) on a Cu Kalpha rotating-anode X-ray source. The space group of the crystals is P212121 with cell dimensions a = 42.0, b = 80.9, c = 110. 6 A. PMID:10089420

  6. Purification, crystallization and room-temperature X-ray diffraction of inositol dehydrogenase LcIDH2 from Lactobacillus casei BL23

    PubMed Central

    Bertwistle, Drew; Vogt, Linda; Aamudalapalli, Hari Babu; Palmer, David R. J.; Sanders, David A. R.

    2014-01-01

    Lactobacillus casei BL23 contains two genes, iolG1 and iolG2, homologous with inositol dehydrogenase encoding genes from many bacteria. Inositol dehydrogenase catalyzes the oxidation of inositol with concomitant reduction of NAD+. The protein encoded by iolG2, LcIDH2, has been purified to homogeneity, crystallized and cryoprotected for diffraction at 77 K. The crystals had a high mosaicity and poor processing statistics. Subsequent diffraction measurements were performed without cryoprotectant at room temperature. These crystals were radiation-resistant and a full diffraction data set was collected at room temperature to 1.6 Å resolution. PMID:25005103

  7. Purification, crystallization and room-temperature X-ray diffraction of inositol dehydrogenase LcIDH2 from Lactobacillus casei BL23.

    PubMed

    Bertwistle, Drew; Vogt, Linda; Aamudalapalli, Hari Babu; Palmer, David R J; Sanders, David A R

    2014-07-01

    Lactobacillus casei BL23 contains two genes, iolG1 and iolG2, homologous with inositol dehydrogenase encoding genes from many bacteria. Inositol dehydrogenase catalyzes the oxidation of inositol with concomitant reduction of NAD+. The protein encoded by iolG2, LcIDH2, has been purified to homogeneity, crystallized and cryoprotected for diffraction at 77 K. The crystals had a high mosaicity and poor processing statistics. Subsequent diffraction measurements were performed without cryoprotectant at room temperature. These crystals were radiation-resistant and a full diffraction data set was collected at room temperature to 1.6 Å resolution. PMID:25005103

  8. Stochasticity of convection in Giga-LES data

    NASA Astrophysics Data System (ADS)

    De La Chevrotière, Michèle; Khouider, Boualem; Majda, Andrew J.

    2016-09-01

    The poor representation of tropical convection in general circulation models (GCMs) is believed to be responsible for much of the uncertainty in the predictions of weather and climate in the tropics. The stochastic multicloud model (SMCM) was recently developed by Khouider et al. (Commun Math Sci 8(1):187-216, 2010) to represent the missing variability in GCMs due to unresolved features of organized tropical convection. The SMCM is based on three cloud types (congestus, deep and stratiform), and transitions between these cloud types are formalized in terms of probability rules that are functions of the large-scale environment convective state and a set of seven arbitrary cloud timescale parameters. Here, a statistical inference method based on the Bayesian paradigm is applied to estimate these key cloud timescales from the Giga-LES dataset, a 24-h large-eddy simulation (LES) of deep tropical convection (Khairoutdinov et al. in J Adv Model Earth Syst 1(12), 2009) over a domain comparable to a GCM gridbox. A sequential learning strategy is used where the Giga-LES domain is partitioned into a few subdomains, and atmospheric time series obtained on each subdomain are used to train the Bayesian procedure incrementally. Convergence of the marginal posterior densities for all seven parameters is demonstrated for two different grid partitions, and sensitivity tests to other model parameters are also presented. A single column model simulation using the SMCM parameterization with the Giga-LES inferred parameters reproduces many important statistical features of the Giga-LES run, without any further tuning. In particular it exhibits intermittent dynamical behavior in both the stochastic cloud fractions and the large scale dynamics, with periods of dry phases followed by a coherent sequence of congestus, deep, and stratiform convection, varying on timescales of a few hours consistent with the Giga-LES time series. The chaotic variations of the cloud area fractions were

  9. Cloning, expression, purification, crystallization and preliminary crystallographic study of the protein module (BIV2-Helix) in the fusion core of bovine immunodeficiency-like virus (BIV) gp40

    PubMed Central

    Zhao, Xiaodong; Li, Ming; Xu, Yanhui; Lou, Zhiyong; Meng, Zhaohui; Li, Shu; Tian, Bo; Gao, George F.; Rao, Zihe

    2005-01-01

    The fusion core of bovine immunodeficiency virus (BIV) gp40 is proposed to be involved in membrane fusion. However, no crystal structures are yet available. A predicted protein module BIV2-Helix of BIVgp40 has been expressed in Escherichia coli and purified by chromatography. Recombinant BIV2-Helix was crystallized using the hanging-drop vapour-diffusion technique at 291 K. The crystals were grown in MPD and belonged to the primitive rhombohedral space group R3, with unit-cell parameters a = 39.17, b = 39.17, c = 295.05 Å and two molecules per asymmetric unit. X-ray diffraction data were collected to 1.76 Å in the home laboratory from a single crystal. PMID:16511008

  10. Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis

    SciTech Connect

    Ruppert, Martin; Panjikar, Santosh; Barleben, Leif; Stöckigt, Joachim

    2006-03-01

    Raucaffricine glucosidase, an enzyme involved in the biosynthesis of monoterpenoid indole alkaloids in the plant Rauvolfia serpentina, was crystallized by the hanging-drop vapour-diffusion method using PEG4000 as precipitant. The crystals diffract to 2.3 Å resolution and belong to space group I222. Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å.

  11. Purification, Crystallization and Preliminary X-ray Diffraction Analysis of the Phage T4 Vertex Protein Gp24 and its Mutant Forms

    SciTech Connect

    Boeshans,K.; Liu, F.; Peng, G.; Idler, W.; Jang, S.; Marekov, L.; Black, L.; Ahvazi, B.

    2006-01-01

    The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6 {angstrom} resolution compared to wild-type gp24 at 3.80 {angstrom} resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.

  12. Purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of human lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1)

    SciTech Connect

    Ishigaki, Tomoko; Ohki, Izuru; Oyama, Takuji; Machida, Sachiko; Morikawa, Kousuke; Tate, Shin-ichi

    2005-05-01

    Two different fragments of the ligand-binding domain of LOX-1, the major receptor for oxidized low-density lipoprotein (LDL) on endothelial cells, have been crystallized in different forms. Two different fragments of the ligand-binding domain of LOX-1, the major receptor for oxidized low-density lipoprotein (LDL) on endothelial cells, have been crystallized in different forms. One crystal form contains the disulfide-linked dimer, which is the form of the molecule present on the cell surface; the other contains a monomeric form of the receptor that lacks the cysteine residue necessary to form disulfide-linked homodimers. The crystal of the monomeric ligand-binding domain belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 56.79, b = 67.57, c = 79.02 Å. The crystal of the dimeric form belongs to space group C2, with unit-cell parameters a = 70.86, b = 49.56, c = 76.73 Å, β = 98.59°. Data for the dimeric form of the LOX-1 ligand-binding domain have been collected to 2.4 Å. For the monomeric form of the ligand-binding domain, native, heavy-atom derivative and SeMet-derivative crystals have been obtained; their diffraction data have been measured to 3.0, 2.4 and 1.8 Å resolution, respectively.

  13. Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB

    SciTech Connect

    Pyburn, Tasia M.; Yankovskaya, Victoria; Bensing, Barbara A.; Cecchini, Gary; Sullam, Paul M.; Iverson, T.M.

    2012-07-11

    The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspB{sub BR}) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspB{sub BR} buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspB{sub BR} in each buffer. While both sets of conditions supported crystal growth in space group P2{sub 1}2{sub 1}2{sub 1}, the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 {angstrom} for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 {angstrom} for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 {angstrom} resolution. A complete data set has been collected to 1.3 {angstrom} resolution with an overall R{sub merge} value of 0.04 and an R{sub merge} value of 0.33 in the highest resolution shell.

  14. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli.

    PubMed

    Kawahara, Kazuki; Oki, Hiroya; Fukakusa, Shunsuke; Maruno, Takahiro; Kobayashi, Yuji; Motooka, Daisuke; Taniguchi, Tooru; Honda, Takeshi; Iida, Tetsuya; Nakamura, Shota; Ohkubo, Tadayasu

    2015-06-01

    Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map. PMID:26057791

  15. Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of Rv0241c (HtdX) from Mycobacterium tuberculosis H37Rv

    PubMed Central

    Biswas, Rupam; Dutta, Debajyoti; Das, Amit Kumar

    2013-01-01

    Rv0241c (HtdX) is a putative (3R)-hydroxyacyl-CoA dehydratase of Mycobacterium tuberculosis. The htdX gene belongs to a conserved operon and is expressed in mycobacteria in the presence of several fatty-acid synthase II drugs. To elucidate the structure of HtdX, the protein was cloned, overexpressed, purified to homogeneity and crystallized. The protein was crystallized from two conditions: (i) 3 M sodium chloride, 0.1 M Na HEPES pH 8.0 and (ii) 2.5 M sodium chloride, 0.1 M Tris–HCl pH 8.5. A complete diffraction data set was collected from crystals from both conditions. The crystal from the first condition diffracted to 2.3 Å resolution and belonged to space group I41, with unit-cell parameters a = b = 61.51, c = 143.81 Å. Crystals from the second condition diffracted to 3.1 Å resolution and belonged to space group P43212 or P41212, with unit-cell parameters a = b = 63.67, c = 140.88 Å. Both crystals contained one molecule in the asymmetric unit. PMID:24100560

  16. Effect of additives on the purification of urease

    NASA Astrophysics Data System (ADS)

    Yu, X.; Wang, J.; Ulrich, J.

    2015-12-01

    The effect of additives on the purification of proteins was investigated. The target protein studied here is the enzyme urease. Studies on the purification of urease from jack bean meal were carried out. 32% (v/v) acetone was utilized to extract urease from the jack bean meal. Further purification by crystallization with the addition of 2-mercaptoethanol and EDTA disodium salt dehydrate was carried out. It was found out that the presence of additives can affect the selectivity of the crystallization. Increases in both purity and yield of the urease after crystallization were observed in the presence of additives, which were proven using both SDS-PAGE and activity. Urease crystals with a yield of 69.9% and a purity of 85.1% were obtained in one crystallization step in the presence of additives. Furthermore, the effect of additives on the thermodynamics and kinetics of urease crystallization was studied.

  17. Peering into peer-review at GigaScience.

    PubMed

    Edmunds, Scott C

    2013-01-01

    Fostering and promoting more open and transparent science is one of the goals of GigaScience. One of the ways we have been doing this is by throwing light on the peer-review process and carrying out open peer-review as standard. In this editorial, we provide our rationale for undertaking this policy, give examples of our positive experiences to date, and encourage others to open up the normally opaque publication process. PMID:23587291

  18. Analysis of toughening mechanisms in the Strombus gigas shell.

    PubMed

    DiPette, Scott; Ural, Ani; Santhanam, Sridhar

    2015-08-01

    A finite element analysis of the fracture mechanisms in the Strombus gigas conch shell is presented in this work. The S. gigas shell has a complex microarchitecture that consists of three main macroscopic layers of calcium carbonate: the inner, middle, and outer layers. Each layer is composed of lamellae of calcium carbonate, held together by a cohesive organic protein. As a result of this elaborate architecture, the S. gigas shell exhibits a much greater damage tolerance than the calcium carbonate by itself, with a work of fracture reported to be three magnitudes of order greater. The two main energy dissipating factors that contribute to this are multiple, parallel cracking along first-order interfaces in the inner and outer layers and crack bridging through the second-order interfaces of the middle layer. Finite element analysis was conducted to simulate and replicate flexural strength and work-of-fracture results obtained in the literature for both dry and wet physical bend test specimens. Several parameters were varied including protein strength and fracture toughness, initial protein damage, and the relative heights of macroscopic layers in order to create a model that predicted published, experimental results. The simulations indicate that having some initially weakened protein interfaces is key to matching the parallel cracking in the inner layer of the physical specimens. The wet models exhibit significantly higher work of fracture compared to the dry specimens in large part due to a crack growth resistance behavior in the middle layer, which was successfully modeled. The parametric studies that have been performed on the finite element models provide guidelines for manufacturing the ideal S. gigas-inspired, biomimetic composite.

  19. Analysis of toughening mechanisms in the Strombus gigas shell.

    PubMed

    DiPette, Scott; Ural, Ani; Santhanam, Sridhar

    2015-08-01

    A finite element analysis of the fracture mechanisms in the Strombus gigas conch shell is presented in this work. The S. gigas shell has a complex microarchitecture that consists of three main macroscopic layers of calcium carbonate: the inner, middle, and outer layers. Each layer is composed of lamellae of calcium carbonate, held together by a cohesive organic protein. As a result of this elaborate architecture, the S. gigas shell exhibits a much greater damage tolerance than the calcium carbonate by itself, with a work of fracture reported to be three magnitudes of order greater. The two main energy dissipating factors that contribute to this are multiple, parallel cracking along first-order interfaces in the inner and outer layers and crack bridging through the second-order interfaces of the middle layer. Finite element analysis was conducted to simulate and replicate flexural strength and work-of-fracture results obtained in the literature for both dry and wet physical bend test specimens. Several parameters were varied including protein strength and fracture toughness, initial protein damage, and the relative heights of macroscopic layers in order to create a model that predicted published, experimental results. The simulations indicate that having some initially weakened protein interfaces is key to matching the parallel cracking in the inner layer of the physical specimens. The wet models exhibit significantly higher work of fracture compared to the dry specimens in large part due to a crack growth resistance behavior in the middle layer, which was successfully modeled. The parametric studies that have been performed on the finite element models provide guidelines for manufacturing the ideal S. gigas-inspired, biomimetic composite. PMID:25955562

  20. Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1

    SciTech Connect

    Akioka, Makoto; Nakano, Hiroaki; Horikiri, Aya; Tsujimoto, Yoshiyuki; Matsui, Hiroshi; Shimizu, Tetsuya; Nakatsu, Toru; Kato, Hiroaki; Watanabe, Kunihiko

    2006-12-01

    Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out. To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination.

  1. Purification, crystallization and preliminary X-ray analysis of truncated and mutant forms of VP4 protease from infectious pancreatic necrosis virus

    SciTech Connect

    Lee, Jaeyong; Feldman, Anat R.; Chiu, Elaine; Chan, Charlena; Kim, You-Na; Delmas, Bernard; Paetzel, Mark

    2006-12-01

    Various truncated and mutant forms of the protease VP4 from infectious pancreatic necrosis virus were used to generate two different crystal forms of VP4 which diffracted to beyond 2.4 Å resolution. In viruses belonging to the Birnaviridae family, virus protein 4 (VP4) is the viral protease responsible for the proteolytic maturation of the polyprotein encoding the major capsid proteins (VP2 and VP3). Infectious pancreatic necrosis virus (IPNV), the prototype of the aquabirnavirus genus, is the causative agent of a contagious disease in fish which has a large economic impact on aquaculture. IPNV VP4 is a 226-residue (24.0 kDa) serine protease that utilizes a Ser/Lys catalytic dyad mechanism (Ser633 and Lys674). Several truncated and mutant forms of VP4 were expressed in a recombinant expression system, purified and screened for crystallization. Two different crystal forms diffract beyond 2.4 Å resolution. A triclinic crystal derived from one mutant construct has unit-cell parameters a = 41.7, b = 69.6, c = 191.6 Å, α = 93.0, β = 95.1, γ = 97.7°. A hexagonal crystal with space group P6{sub 1}22/P6{sub 5}22 derived from another mutant construct has unit-cell parameters a = 77.4, b = 77.4, c = 136.9 Å.

  2. Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-Ib [APH(2′′)-Ib

    SciTech Connect

    Walanj, Rupa; Young, Paul; Baker, Heather M.; Baker, Edward N.; Metcalf, Peter; Chow, Joseph W.; Lerner, Stephen; Vakulenko, Sergei; Smith, Clyde A.

    2005-04-01

    APH(2′′)-Ib is an enzyme responsible for high-level gentamicin resistance in E. faecium isolates. Native crystals of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2′′)-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2{sub 1}, with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2′′)-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding.

  3. The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa

    SciTech Connect

    Gohain, Neelakshi; Thomashow, Linda S.; Mavrodi, Dmitri V.; Blankenfeldt, Wulf

    2006-10-01

    PhzS, an FAD-dependent monooxygenase that catalyzes a reaction involved in the biosynthesis of the virulence factor pyocyanin in P. aeruginosa, was cloned, overexpressed and crystallized. Data collection from native and seleno-l-methionine-labelled crystals is reported. The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystals belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 Å, α = γ = 90, β = 110.5°. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 Å. Seleno-l-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 Å, α = γ = 90, β = 110.5° and the diffraction limit is 2.7 Å.

  4. Purification, crystallization and preliminary structural characterization of the N-terminal region of the human formin-homology protein FHOD1

    SciTech Connect

    Schulte, Antje Rak, Alexey; Pylypenko, Olena; Ludwig, Diana; Geyer, Matthias

    2007-10-01

    The N-terminal region (1–339) of the human FHOD1 protein has been crystallized in two different crystal forms. A crystal of the (C31S,C71S) mutant diffracted to around 2.3 Å resolution. Formins are key regulators of actin cytoskeletal dynamics that constitute a diverse protein family that is present in all eukaryotes examined. They typically consist of more than 1000 amino acids and are defined by the presence of two conserved regions, namely the formin homology 1 and 2 domains. Additional conserved domains comprise a GTPase-binding domain for activation, a C-terminal autoregulation motif and an N-terminal recognition domain. In this study, the N-terminal region (residues 1–339) of the human formin homology domain-containing protein 1 (FHOD1) was purified and crystallized from 20%(w/v) PEG 4000, 10%(v/v) glycerol, 0.3 M magnesium chloride and 0.1 M Tris–HCl pH 8.0. Native crystals belong to space group P1, with unit-cell parameters a = 35.4, b = 73.9, c = 78.7 Å, α = 78.2, β = 86.2, γ = 89.7°. They contain two monomers of FHOD1 in the asymmetric unit and diffract to a resolution of 2.3 Å using a synchrotron-radiation source.

  5. Purification, Crystallization and Preliminary X-ray Characterization of Prunin-1, a Major Component of the Almond (Prunus dulcis) Allergen Amandin

    SciTech Connect

    Albillos, Silvia M.; Jin, Tengchuan; Howard, Andrew; Zhang, Yuzhu; Kothary, Mahendra H.; Fu, Tong-Jen

    2008-08-04

    The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond (Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 {angstrom} and belong to the tetragonal space group P4{sub 1}22, with unit cell parameters of a = b = 150.912 {angstrom}, c = 165.248 {angstrom}. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

  6. Purification, crystallization and preliminary X-ray characterization of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.

    PubMed

    Albillos, Silvia M; Jin, Tengchuan; Howard, Andrew; Zhang, Yuzhu; Kothary, Mahendra H; Fu, Tong-Jen

    2008-07-01

    The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond ( Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 A and belong to the tetragonal space group P4(1)22, with unit cell parameters of a = b = 150.912 A, c = 165.248 A. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

  7. Giant bivalves (Tridacna gigas) as recorders of ENSO variability

    NASA Astrophysics Data System (ADS)

    Welsh, Kevin; Elliot, Mary; Tudhope, Alexander; Ayling, Bridget; Chappell, John

    2011-07-01

    We compare monthly resolved oxygen isotope records derived from a giant bivalve shell, Tridacna gigas and massive Porites corals collected along the northern coast of Papua New Guinea. This intercomparison study demonstrates that δ 18O profiles obtained from these different aragonite-secreting organisms collected from within a 30 km range are correlated in great detail and record the timing and amplitude of seasonal and interannual (ENSO-related) variations in sea surface temperature (SST) and water isotopic composition which is closely related to rainfall. Furthermore, the T. gigas record is shown to be close to isotopic equilibrium with the local sea-water, in contrast to the corals which are approximately - 4‰ offset. These results reveal that living and fossil T. gigas clam shells have the potential to yield reliable records of past changes in seasonality and ENSO variability, as well as mean climate conditions. In particular, since the non-porous shells are generally more resistant to diagenesis than coral skeletons, they may provide robust estimates of past tropical climate for periods and locations where unaltered corals are absent.

  8. Impact of atrazine on aneuploidy in pacific oysters, Crassostrea gigas.

    PubMed

    Bouilly, Karine; Leitão, Alexandra; McCombie, Helen; Lapègue, Sylvie

    2003-01-01

    Aneuploidy has previously been described and studied in the Pacific oyster, Crassostrea gigas, and has been shown to be negatively correlated with growth. The present study investigated the effect of atrazine on the level of aneuploidy in this species. Crassostrea gigas adults and juveniles were subjected to different concentrations of atrazine representing a peak value found in a polluted environment (46.5 nM) and a value 10 times higher (465 nM). Although atrazine did not show any effect on the oyster mortality, significant differences in aneuploidy level were observed between the different treatments (9% for the control, 16% for 46.5 nM and 20% for 465 nM atrazine). Moreover, the same levels of aneuploidy were observed at adult and juvenile stages. This is the first reported evidence for an environmental effect on aneuploidy in C. gigas. These results will be useful for the oyster aquaculture industry and management of resources. The lowest atrazine level in the current study represents realistic potential exposure, and the results suggest that studies should be made on other aquatic species at risk of exposure to atrazine in the wild. This widely used compound may be an important factor causing damage to genetic material.

  9. Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeast Saccharomyces cerevisiae

    SciTech Connect

    Rengachari, Srinivasan; Aschauer, Philipp; Sturm, Christian; Oberer, Monika

    2015-01-28

    A soluble variant of the monoglyceride lipase Yju3p was successfully expressed, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space group P2{sub 1}2{sub 1}2{sub 1}), with unit-cell parameters a = 77.2, b = 108.6, c = 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.

  10. Purification, crystallization and preliminary X-ray diffraction analysis of RafE, a sugar-binding lipoprotein from Streptococcus pneumoniae

    SciTech Connect

    Paterson, Neil G. Riboldi-Tunnicliffe, Alan; Mitchell, Timothy J.; Isaacs, Neil W.

    2006-07-01

    The mature form of RafE has been expressed, purified and crystallized. X-ray diffraction data have been collected to 3.65 and 2.90 Å resolution from native and selenomethionine-derivative crystals, respectively. Streptococcus pneumoniae contains a large number of sugar-transport systems and the system responsible for raffinose uptake has recently been identified. The substrate-binding protein component of this system shares strong sequence homology with the multiple sugar metabolism substrate-binding protein MsmE from S. mutans and contains a lipoprotein-attachment site at cysteine residue 23. A truncated form (residues 24–419) of RafE from S. pneumoniae was cloned and overexpressed in Escherichia coli. Native and selenomethionine-labelled protein have been crystallized in the hexagonal space group P6{sub 1}22. Diffraction data have been successfully phased to 2.90 Å using Se SAD data and model building is in progress.

  11. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens

    SciTech Connect

    Ye, Fuzhou; Wang, Chao; Fu, Qinqin; Zhang, Lian-hui; Gao, Yong-gui

    2015-08-25

    The crystallization of the novel virulence factors SghA and SghR is reported. Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction.

  12. Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774

    SciTech Connect

    Gavel, Olga Yu.; Kladova, Anna V.; Bursakov, Sergey A.; Dias, João M.; Texeira, Susana; Shnyrov, Valery L.; Moura, José J. G.; Moura, Isabel; Romão, Maria J.; Trincão, José

    2008-07-01

    Native zinc-containing ATP sulfurylase from D. desulfuricans ATCC 27774 was purified to homogeneity and crystallized. Diffraction data were collected to 2.5 Å resolution. Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC 2.7.7.4; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 Å resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.

  13. Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum {beta}-lactamase conferring severe antibiotic resistance

    SciTech Connect

    Lee, J. H. Sohn, S. G. Jung, H. I. An, Y. J. Lee, S. H.

    2013-07-15

    OXA-17, an extended-spectrum {beta}-lactamase (ESBL) conferring severe antibiotic resistance, hydrolytically inactivates {beta}-lactam antibiotics, inducing a lack of eradication of pathogenic bacteria by oxyimino {beta}-lactams and not helping hospital infection control. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing ESBLs. OXA-17 was purified and crystallized at 298 K. X-ray diffraction data from OXA-17 crystal have been collected to 1.85 A resolution using synchrotron radiation. The crystal of OXA-17 belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 48.37, b = 101.12, and c = 126.07 A. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 54.6%.

  14. Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Salmonella typhimurium

    PubMed Central

    Meshcheryakov, Vladimir A.; Samatey, Fadel A.

    2011-01-01

    FlhB is a key protein in the regulation of protein export by the bacterial flagellar secretion system. It is composed of two domains: an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhBc). FlhBc from Salmonella typhimurium has been successfully crystallized using the vapour-diffusion method. The crystals diffracted to 2.45 Å resolution and belonged to space group P42212, with unit-cell parameters a = b = 49.06, c = 142.94 Å. A selenomethionine-containing variant of FlhBc has also been crystallized in the same space group and was used for initial phase calculation by the multiwavelength anomalous dispersion (MAD) method. PMID:21795800

  15. Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum β-lactamase conferring severe antibiotic resistance

    NASA Astrophysics Data System (ADS)

    Lee, J. H.; Sohn, S. G.; Jung, H. I.; An, Y. J.; Lee, S. H.

    2013-07-01

    OXA-17, an extended-spectrum β-lactamase (ESBL) conferring severe antibiotic resistance, hydrolytically inactivates β-lactam antibiotics, inducing a lack of eradication of pathogenic bacteria by oxyimino β-lactams and not helping hospital infection control. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing ESBLs. OXA-17 was purified and crystallized at 298 K. X-ray diffraction data from OXA-17 crystal have been collected to 1.85 Å resolution using synchrotron radiation. The crystal of OXA-17 belongs to space group P212121, with unit-cell parameters a = 48.37, b = 101.12, and c = 126.07 Å. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 54.6%.

  16. Purification, crystallization and preliminary X-ray analysis of a water-soluble chlorophyll protein from Brassica oleracea L. var. acephala (kale).

    PubMed

    Horigome, Daisuke; Satoh, Hiroyuki; Uchida, Akira

    2003-12-01

    A water-soluble chlorophyll protein (WSCP) with a chlorophyll a:b ratio of 6:1 from Brassica oleracea L. var. acephala (kale) was purified and crystallized by the hanging-drop vapour-diffusion method using PEG 8000 and zinc acetate as precipitants. The crystal belongs to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 162.2, c = 38.7 A. A native data set was collected to 2.80 A resolution at 293 K using Cu Kalpha radiation from a rotating-anode generator. Preliminary analysis via molecular replacement identified one kale WSCP monomer in the asymmetric unit. The crystal packing showed a tetrameric structure for kale WSCP, as suggested by previous biochemical studies of WSCPs from Brassicaceae plants.

  17. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of macrophage growth locus A (MglA) protein from Francisella tularensis

    SciTech Connect

    Subburaman, P.; Austin, B.P.; Shaw, G.X.; Waugh, D.S.; Ji, X.

    2010-11-03

    Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 {angstrom} resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 125, c = 54 {angstrom}.

  18. Expression, purification, crystallization and preliminary X-ray analysis of NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver.

    PubMed

    Yoneda, Kazunari; Fukuda, Yudai; Shibata, Takeshi; Araki, Tomohiro; Nikki, Takahiro; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2012-12-01

    An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 300 as the precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=104.26, b=81.32, c=77.27 Å, β=119.43°, and diffracted to 1.86 Å resolution on beamline NE3A at the Photon Factory. The overall Rmerge was 5.4% and the data completeness was 99.4%. PMID:23192050

  19. Purification, crystallization and preliminary X-ray analysis of the soluble domain of the Na+-pumping cytochrome bo quinol oxidase from Vitreoscilla.

    PubMed

    Kim, Kyung Jin; Kim, Youngchang; Park, Kyung Won; Webster, Dale A; Howard, Andrew J

    2002-08-01

    The 24 kDa CyoA soluble domain of Vitreoscilla cytochrome bo quinol oxidase, which pumps out Na(+) during respiration, has been crystallized from a solution of 2 M ammonium sulfate and 5% 2-propanol. The crystal belongs to cubic space group P4(3)32, with unit-cell parameters a = b = c = 122.2 A, alpha = beta = gamma = 90 degrees and one subunit in the asymmetric unit. A 99.8% complete data set to 3.3 A has been collected at the 17-ID beamline of the Advanced Photon Source. The structure was determined by molecular replacement and refinement is in progress.

  20. Glutamate-5-kinase from Escherichia coli: gene cloning, overexpression, purification and crystallization of the recombinant enzyme and preliminary X-ray studies.

    PubMed

    Pérez-Arellano, Isabel; Gil-Ortiz, Fernando; Cervera, Javier; Rubio, Vicente

    2004-11-01

    Glutamate-5-kinase (G5K) catalyzes the first step of proline (and, in mammals, ornithine) biosynthesis. It is a key regulatory point of these routes, since it is the subject of feedback allosteric inhibition by proline or ornithine. The Escherichia coli gene (proB) for G5K was cloned in pET22, overexpressed in E. coli, purified in a few steps in high yield to 95% homogeneity in the highly active proline-inhibitable form and was shown by cross-linking to be a tetramer. It was crystallized by the hanging-drop vapour-diffusion method at 294 K in the presence of ADP, MgCl(2) and L-glutamate using 1.6 M MgSO(4), 0.1 M KCl in 0.1 M MES pH 6.5 as the crystallization solution. The tetragonal bipyramid-shaped crystals diffracted to 2.5 A resolution using synchrotron radiation. The crystals belong to space group P4(1(3))2(1)2, with unit-cell parameters a = b = 101.1, c = 178.6 A, and contain two monomers in the asymmetric unit, with 58% solvent content.

  1. Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

    SciTech Connect

    Russo, Andrew T.; Watowich, Stanley J.

    2006-06-01

    The C-terminal protease domain of Venezuelan equine encephalitis virus (VEEV) nsP2 has been overexpressed in E. coli, purified and successfully crystallized. Native crystals diffract to beyond 2.5 Å resolution and isomorphous heavy-atom derivatives suitable for phase analysis have been identified. The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichia coli, purified and crystallized. Crystals diffract to beyond 2.5 Å resolution and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way.

  2. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU

    SciTech Connect

    Zhang, Yang-De Li, Hao; Liu, Hui; Pan, Yi-Feng

    2007-02-01

    Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively. The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P2{sub 1}2{sub 1}2{sub 1} and tetragonal P4{sub 1}2{sub 1}2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8*{sub 65–224} structure was determined by molecular replacement.

  3. Purification and crystallization of the catalytic PRONE domain of RopGEF8 and its complex with Rop4 from Arabidopsis thaliana

    SciTech Connect

    Thomas, Christoph; Weyand, Michael; Wittinghofer, Alfred; Berken, Antje

    2006-06-01

    Crystals of the catalytic PRONE domain of the guanine nucleotide exchange factor RopGEF8 and its complex with the Rho-family protein Rop4 from A. thaliana were obtained that diffract to 2.2 and 3.1 Å resolution, respectively. The PRONE domain of the guanine nucleotide exchange factor RopGEF8 (PRONE8) was purified and crystallized free and in complex with the Rho-family protein Rop4 using the hanging-drop vapour-diffusion method. PRONE8 crystals were obtained using NaCl as precipitating agent and belong to the hexagonal space group P6{sub 5}22. Native and anomalous data sets were collected using synchrotron radiation at 100 K to 2.2 and 2.8 Å resolution, respectively. Crystals of the Rop4–PRONE8 complex belonging to space group P6{sub 3} were obtained using Tacsimate and PEG 3350 as precipitating agents and diffracted to 3.1 Å resolution.

  4. Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Der f 21 (rDer f 21) from Dermatophagoides farinae.

    PubMed

    Pang, Sze Lei; Ho, Kok Lian; Waterman, Jitka; Teh, Aik Hong; Chew, Fook Tim; Ng, Chyan Leong

    2015-11-01

    Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.

  5. Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of the biosynthetic N-acetylornithine aminotransferases from Salmonella typhimurium and Escherichia coli

    SciTech Connect

    Rajaram, V.; Prasad, K.; Ratna Prasuna, P.; Ramachandra, N.; Bharath, S. R.; Savithri, H. S.; Murthy, M. R. N.

    2006-10-01

    Acetylornithine aminotransferases, members of the type I subgroup II family of PLP-dependent enzymes, from S. typhimurium and E. coli have been cloned, overexpressed, purified and crystallized. Acetylornithine aminotransferase (AcOAT) is a type I pyridoxal 5′-phosphate-dependent enzyme catalyzing the conversion of N-acetylglutamic semialdehyde to N-acetylornithine in the presence of α-ketoglutarate, a step involved in arginine metabolism. In Escherichia coli, the biosynthetic AcOAT also catalyzes the conversion of N-succinyl-l-2-amino-6-oxopimelate to N-succinyl-l,l-diaminopimelate, one of the steps in lysine biosynthesis. It is closely related to ornithine aminotransferase. AcOAT was cloned from Salmonella typhimurium and E. coli, overexpressed in E. coli and purified using Ni–NTA affinity column chromatography. The enzymes crystallized in the presence of gabaculine. Crystals of E. coli AcOAT (eAcOAT) only diffracted X-rays to 3.5 Å and were twinned. The crystals of S. typhimurium AcOAT (sAcOAT) diffracted to 1.9 Å and had a dimer in the asymmetric unit. The structure of sAcOAT was solved by the molecular-replacement method.

  6. Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle

    SciTech Connect

    Deane, Janet E.; Cordes, Frank S.; Roversi, Pietro; Johnson, Steven; Kenjale, Roma; Picking, William D.; Picking, Wendy L.; Lea, Susan M.; Blocker, Ariel

    2006-03-01

    A monodisperse truncation mutant of MxiH, the subunit of the S. flexneri type III secretion system needle, has been crystallized. SeMet derivatives and a uranyl derivative have undergone preliminary crystallographic analysis. A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiH{sub CΔ5} and diffraction data were collected to 1.9 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 Å, β = 96.5°. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 Å{sup 3} Da{sup −1} for two molecules per asymmetric unit, corresponding to a solvent content of 33%.

  7. Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum

    SciTech Connect

    Byrnes, Laura J.; Badarau, Adriana; Vakulenko, Sergei B.; Smith, Clyde A.

    2008-02-01

    APH(2′′)-Ic is an enzyme that is responsible for high-level gentamicin resistance in E. gallinarum isolates. Crystals of the wild-type enzyme and three mutants have been prepared and a complete X-ray diffraction data set was collected to 2.15 Å resolution from an F108L crystal. Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2′′)-Ic variants were crystallized in the presence of 14–20%(w/v) PEG 4000, 0.25 M MgCl{sub 2}, 0.1 M Tris–HCl pH 8.5 and 1 mM Mg{sub 2}GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 Å, β = 108.8°. X-ray diffraction data were collected to approximately 2.15 Å resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.

  8. Purification, growth, structure, optical and electrical properties of single crystals of the π-molecular complex of phenothiazine with pyromellitic dianhydride

    NASA Astrophysics Data System (ADS)

    Anthonj, R.; Karl, N.; Robertson, Beverly E.; Stezowski, John J.

    1980-01-01

    Single crystals of the black phenothiazine : pyromellitic dianhydride (PTZ:PMDA) 1:1 donor-acceptor complex, have been grown by sublimation from the zone-refined components. The PTZ:PMDA complex crystalizes with P1¯ symmetry (Z=2), a=7.197(1), b=19.072(5), c=6.886(1) Å, α=84.79(1) °, β=72.98(1) °, γ=85.72(2) ° at T=23 °C. The crystal structure model was refined with 5214 data { (sin ϑ/λ)max=0.8071 Å-1} to give R=0.049 and Rw=0.089. The crystal packing consists of two polar endless ...DADA... stacks related to one another by 1¯. The packing is compared with that in the analogous anthracene : PMDA complex. The PTZ molecule displays a modest fold about the N...S axis (dihedral angle 176.4°), however, the deviation from planarity is small in comparison with that in PTZ crystals. The stack axis is nearly perpendicular to the molecular planes, consequently the CT-dichroism lies essentially in the principal axis system of the indicatrix. The absorption edge is not very sharp, even at 4.2 K; it is located at 900±25 nm. This value is compared to the ionization energy of PTZ and to the energy of the CT transition in anthracene : PMDA. No optical emission could be detected; the pathways of radiationless deactivation are discussed in terms of CT-vibron coupling. An intrinsic dark conductivity of σ˜10-12 Ω-1 cm-1 was found. It is consistent with the value of the band gap (1.7 eV) obtained both from the measured activation energy of the dark conductivity and from the estimated thermal dissociation of CT excitons. Possible implications of the polar stacking on the excitonic energy levels and energy transport are briefly discussed.

  9. Expression, purification, crystallization and X-ray crystallographic studies of different redox states of the active site of thioredoxin 1 from the whiteleg shrimp Litopenaeus vannamei

    PubMed Central

    Campos-Acevedo, Adam A.; Garcia-Orozco, Karina D.; Sotelo-Mundo, Rogerio R.; Rudiño-Piñera, Enrique

    2013-01-01

    Thioredoxin (Trx) is a 12 kDa cellular redox protein that belongs to a family of small redox proteins which undergo reversible oxidation to produce a cystine disulfide bond through the transfer of reducing equivalents from the catalytic site cysteine residues (Cys32 and Cys35) to a disulfide substrate. In this study, crystals of thioredoxin 1 from the Pacific whiteleg shrimp Litopenaeus vannamei (LvTrx) were successfully obtained. One data set was collected from each of four crystals at 100 K and the three-dimensional structures of the catalytic cysteines in different redox states were determined: reduced and oxidized forms at 2.00 Å resolution using data collected at a synchrotron-radiation source and two partially reduced structures at 1.54 and 1.88 Å resolution using data collected using an in-house source. All of the crystals belonged to space group P3212, with unit-cell parameters a = 57.5 (4), b = 57.5 (4), c = 118.1 (8) Å. The asymmetric unit contains two subunits of LvTrx, with a Matthews coefficient (V M) of 2.31 Å3 Da−1 and a solvent content of 46%. Initial phases were determined by molecular replacement using the crystallographic model of Trx from Drosophila melanogaster as a template. In the present work, LvTrx was overexpressed in Escherichia coli, purified and crystallized. Structural analysis of the different redox states at the Trx active site highlights its reactivity and corroborates the existence of a dimer in the crystal. In the crystallographic structures the dimer is stabilized by several interactions, including a disulfide bridge between Cys73 of each LvTrx monomer, a hydrogen bond between the side chain of Asp60 of each monomer and several hydrophobic interactions, with a noncrystallographic twofold axis. PMID:23695560

  10. Moderate establishment success of Pacific oyster, Crassostrea gigas, on a sheltered intertidal mussel bed

    NASA Astrophysics Data System (ADS)

    Holm, Mark Wejlemann; Davids, Jens Kristian; Dolmer, Per; Vismann, Bent; Hansen, Benni Winding

    2015-10-01

    The Pacific oyster (Crassostrea gigas Thunberg 1793) is introduced into marine ecosystems worldwide. In Denmark, C. gigas was introduced into the micro tidal Limfjord, around 1972 for aquaculture. This study describes the population structure of C. gigas at Agger Tange in 2007, 2009, 2010 and 2011. Here, C. gigas use beds of Blue mussels (Mytilus edulis L.) as primary habitat. The mean abundance (± 1 SD) of C. gigas was unchanged during our study (45 ± 2 indv. m- 2), while it increased for M. edulis from 2010 to 2011 (934 ± 610 to 1434 ± 750 indv. m- 2, respectively). In 2009, a newly settled cohort of C. gigas was present, but in the succeeding years no or negligible recruitment was recorded. However, age cohort analyses, based on individual shell size at different ages, suggest successful recruitment in three out of seven years. A comparison with the course of the bioinvasion in List Tidal Basin, suggests that the population at Agger Tange is not in the expansion phase of the bioinvasion, despite of frequent settlement, high shell growth rates and relatively high abundance. So far, C. gigas has had moderate establishment success. We conclude that C. gigas is still in the establishment phase, but that this is prolonged, presumably due to low food availability.

  11. Exhaust gas purification device

    SciTech Connect

    Fujiwara, H.; Hibi, T.; Sayo, S.; Sugiura, Y.; Ueda, K.

    1980-02-19

    The exhaust gas purification device includes an exhaust manifold , a purification cylinder connected with the exhaust manifold through a first honey-comb shaped catalyst, and a second honeycomb shaped catalyst positioned at the rear portion of the purification cylinder. Each catalyst is supported by steel wool rings including coarse and dense portions of steel wool. The purification device further includes a secondary air supplying arrangement.

  12. Hybridization Between Natural Extract of Angelica gigas Nakai and Inorganic Nanomaterial of Layered Double Hydroxide via Reconstruction Reaction.

    PubMed

    Kim, Tae-Hyun; Kim, Hyoung-Jun; Choi, Ae-Jin; Choi, Hyun-Jin; Oh, Jae-Min

    2016-01-01

    We have hybridized layered double hydroxide (LDH) with Angelica gigas Nakai root extract (AGNR) through reversible dehydration-rehydration reaction which is known as reconstruction. LDHs having well-ordered hydrotalcite-like crystal structure and average size 250 ± 20 nm were prepared by hydrothermal method. The root of Angelica gigas Nakai, which has been utilized in the treatment of female disorders as herbal medicine, was treated with methanol to obtain extract. Pristine LDHs were calcined at 400 °C for 8 hours to obtain layered double oxide (LDO), which was further dispersed into extract solution with various AGNR/LDO weight ratios, 0.11, 0.21 and 0.43. The extract content in each hybrid increased in proportion to initial AGNR/LDO ratio, showing the highest content of ~12%. The zeta potential of LDH shifted from +44 mV to +20 mV upon hybridization with extract, which was attributed to the adsorption of negatively charged organic moieties in AGNR on LDH surface. The scanning electron microscopic (SEM) results exhibited that the random stacking of LDH nanolayers resulted in LDH-AGNR hybrid with house-of-cards structure, of which inter-particle cavity serves nano-reservoir for natural extract. According to quantitative analyses, it was revealed that the content of active components in AGNR increased when they were hybridized with LDHs compared with those in AGNR alone.

  13. Hybridization Between Natural Extract of Angelica gigas Nakai and Inorganic Nanomaterial of Layered Double Hydroxide via Reconstruction Reaction.

    PubMed

    Kim, Tae-Hyun; Kim, Hyoung-Jun; Choi, Ae-Jin; Choi, Hyun-Jin; Oh, Jae-Min

    2016-01-01

    We have hybridized layered double hydroxide (LDH) with Angelica gigas Nakai root extract (AGNR) through reversible dehydration-rehydration reaction which is known as reconstruction. LDHs having well-ordered hydrotalcite-like crystal structure and average size 250 ± 20 nm were prepared by hydrothermal method. The root of Angelica gigas Nakai, which has been utilized in the treatment of female disorders as herbal medicine, was treated with methanol to obtain extract. Pristine LDHs were calcined at 400 °C for 8 hours to obtain layered double oxide (LDO), which was further dispersed into extract solution with various AGNR/LDO weight ratios, 0.11, 0.21 and 0.43. The extract content in each hybrid increased in proportion to initial AGNR/LDO ratio, showing the highest content of ~12%. The zeta potential of LDH shifted from +44 mV to +20 mV upon hybridization with extract, which was attributed to the adsorption of negatively charged organic moieties in AGNR on LDH surface. The scanning electron microscopic (SEM) results exhibited that the random stacking of LDH nanolayers resulted in LDH-AGNR hybrid with house-of-cards structure, of which inter-particle cavity serves nano-reservoir for natural extract. According to quantitative analyses, it was revealed that the content of active components in AGNR increased when they were hybridized with LDHs compared with those in AGNR alone. PMID:27398576

  14. A convenient purification and preconcentration of peptides with alpha-cyano-4-hydroxycinnamic acid matrix crystals in a pipette tip for matrix-assisted laser desorption/ionization mass spectrometry.

    PubMed

    Rehulková, Helena; Chalupová, Jana; Sebela, Marek; Rehulka, Pavel

    2010-01-01

    Peptide samples derived from enzymatic in-gel digestion of proteins resolved by gel electrophoresis often contain high amount of salts originating from reaction and separation buffers. Different methods are used for desalting prior to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), e.g. reversed-phase pipette tip purification, on-target washing, adding co-matrices, etc. As a suitable matrix for MALDI MS of peptides, alpha-cyano-4-hydroxycinnamic acid (CHCA) is frequently used. Crystalline CHCA shows the ability to bind peptides on its surface and because it is almost insoluble in acidic water solutions, the on-target washing of peptide samples can significantly improve MALDI MS signals. Although the common on-target washing represents a simple, cheap and fast procedure, only a small portion of the available peptide solution is efficiently used for the subsequent MS analysis. The present approach is a combination of the on-target washing principle carried out in a narrow-end pipette tip (e.g. GELoader tip) and preconcentration of peptides from acidified solution by passing it through small CHCA crystals captured inside the tip on a glass microfiber frit. The results of MALDI MS analysis using CHCA-tip peptide preconcentration are comparable with the use of homemade POROS R2 pipette tip microcolumns. Advantages and limitations of this approach are discussed.

  15. A convenient purification and preconcentration of peptides with alpha-cyano-4-hydroxycinnamic acid matrix crystals in a pipette tip for matrix-assisted laser desorption/ionization mass spectrometry.

    PubMed

    Rehulková, Helena; Chalupová, Jana; Sebela, Marek; Rehulka, Pavel

    2010-01-01

    Peptide samples derived from enzymatic in-gel digestion of proteins resolved by gel electrophoresis often contain high amount of salts originating from reaction and separation buffers. Different methods are used for desalting prior to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), e.g. reversed-phase pipette tip purification, on-target washing, adding co-matrices, etc. As a suitable matrix for MALDI MS of peptides, alpha-cyano-4-hydroxycinnamic acid (CHCA) is frequently used. Crystalline CHCA shows the ability to bind peptides on its surface and because it is almost insoluble in acidic water solutions, the on-target washing of peptide samples can significantly improve MALDI MS signals. Although the common on-target washing represents a simple, cheap and fast procedure, only a small portion of the available peptide solution is efficiently used for the subsequent MS analysis. The present approach is a combination of the on-target washing principle carried out in a narrow-end pipette tip (e.g. GELoader tip) and preconcentration of peptides from acidified solution by passing it through small CHCA crystals captured inside the tip on a glass microfiber frit. The results of MALDI MS analysis using CHCA-tip peptide preconcentration are comparable with the use of homemade POROS R2 pipette tip microcolumns. Advantages and limitations of this approach are discussed. PMID:19927305

  16. Cloning, expression, purification, crystallization and preliminary X-ray studies of a pyridoxine 5′-phosphate oxidase from Mycobacterium smegmatis

    SciTech Connect

    Jackson, Colin J. Taylor, Matthew C.; Tattersall, David B.; French, Nigel G.; Carr, Paul D.; Ollis, David L.; Russell, Robyn J.; Oakeshott, John G.

    2008-05-01

    Good-quality crystals of selenomethionine-substituted Msmeg-3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 Å using synchrotron radiation. Pyridoxine 5′-phosphate oxidases (PNPOxs) are known to catalyse the terminal step in pyridoxal 5′-phosphate biosynthesis in a flavin mononucleotide-dependent manner in humans and Escherichia coli. Recent reports of a putative PNPOx from Mycobacterium tuberculosis, Rv1155, suggest that the cofactor or catalytic mechanism may differ in Mycobacterium species. To investigate this, a putative PNPOx from M. smegmatis, Msmeg-3380, has been cloned. This enzyme has been recombinantly expressed in E. coli and purified to homogeneity. Good-quality crystals of selenomethionine-substituted Msmeg-3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 Å using synchrotron radiation.

  17. Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeast Saccharomyces cerevisiae

    PubMed Central

    Rengachari, Srinivasan; Aschauer, Philipp; Sturm, Christian; Oberer, Monika

    2015-01-01

    The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space group P212121), with unit-cell parameters a = 77.2, b = 108.6, c = 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%. PMID:25664804

  18. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens

    PubMed Central

    Ye, Fuzhou; Wang, Chao; Fu, Qinqin; Zhang, Lian-hui; Gao, Yong-gui

    2015-01-01

    Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction. PMID:26323299

  19. Overproduction, purification and crystallization of a chondroitin sulfate A-binding DBL domain from a Plasmodium falciparum var2csa-encoded PfEMP1 protein

    SciTech Connect

    Higgins, Matthew K.

    2008-03-01

    A chondroitin sulfate A-binding DBL important in placental malaria has been overproduced, purified and crystallized. Diffraction data were collected to 1.9 Å resolution. The PfEMP1 proteins of the malaria parasite Plasmodium falciparum are inserted into the membrane of infected red blood cells, where they mediate adhesion to a variety of human receptors. The DBL domains of the var2csa-encoded PfEMP1 protein play a critical role in malaria of pregnancy, tethering infected cells to the surface of the placenta through interactions with the glycosaminoglycan carbohydrate chondroitin sulfate A (CSA). A CSA-binding DBL domain has been overproduced in a bacterial expression system, purified and crystallized. Native data sets extending to 1.9 Å resolution have been collected and phasing is under way.

  20. Cloning, expression, purification, crystallization and preliminary crystallographic studies of BceC, a UDP-glucose dehydrogenase from Burkholderia cepacia IST408

    PubMed Central

    Rocha, Joana; Popescu, Alma O.; Sá-Correia, Isabel; Fialho, Arsénio M.; Frazão, Carlos

    2010-01-01

    Bacteria of the Burkholderia cepacia complex (Bcc) have emerged as important opportunistic pathogens, establishing lung infections in immunocompromised or cystic fibrosis patients. Bcc uses polysaccharide-biofilm production in order to evade the host immune response. The biofilm precursor UDP-glucuronic acid is produced by a twofold NAD+-dependent oxidation of UDP-glucose. In B. cepacia IST408 this enzymatic reaction is performed by the UDP-glucose dehydrogenase BceC, a 470-residue enzyme, the production and crystallization of which are described here. The crystals belonged to the orthorhombic space group P212121 and contained four molecules in the asymmetric unit. Their crystallo­graphic analysis at 2.09 Å resolution and a molecular-replacement study are reported. PMID:20208157

  1. Cloning, purification, crystallization and X-ray crystallographic analysis of the periplasmic sensing domain of Pseudomonas fluorescens chemotactic transducer of amino acids type A (CtaA).

    PubMed

    Ud-Din, Abu Iftiaf Md Salah; Roujeinikova, Anna

    2016-09-01

    Chemotaxis towards nutrients plays a crucial role in root colonization by Pseudomonas fluorescens. The P. fluorescens chemotactic transducer of amino acids type A (CtaA) mediates movement towards amino acids present in root exudates. In this study, the periplasmic sensory domain of CtaA has been crystallized by the hanging-drop vapor diffusion method using ammonium sulfate as a precipitating agent. A complete data set was collected to 1.9 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belong to space group I222 or I212121, with unit-cell parameters a = 67.2, b = 76.0, c = 113.3 Å. This is an important step towards elucidation of the structural basis of how CtaA recognizes its signal molecules and transduces the signal across the membrane. PMID:27251445

  2. Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490963–1138 domain of TamB from Escherichia coli

    PubMed Central

    Josts, Inokentijs; Grinter, Rhys; Kelly, Sharon M.; Mosbahi, Khedidja; Roszak, Aleksander; Cogdell, Richard; Smith, Brian O.; Byron, Olwyn; Walker, Daniel

    2014-01-01

    TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963–1138 domain of TamB was overexpressed in Escherichia coli K-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space group P3121, with unit-cell parameters a = b = 57.34, c = 220.74 Å, and diffracted to 2.1 Å resolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future. PMID:25195908

  3. Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties.

    PubMed

    Don Paul, Craig; Traore, Daouda A K; Byres, Emma; Rossjohn, Jamie; Devenish, Rodney J; Kiss, Csaba; Bradbury, Andrew; Wilce, Matthew C J; Prescott, Mark

    2011-10-01

    Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, α = 90.96, β = 89.92, γ = 104.03°. The Matthews coefficient (V(M) = 2.26 Å(3) Da(-1)) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules.

  4. Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

    PubMed Central

    Russo, Andrew T.; Watowich, Stanley J.

    2006-01-01

    The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichia coli, purified and crystallized. Crystals diffract to beyond 2.5 Å resolution and belong to the orthorhombic space group P212121. Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way. PMID:16754969

  5. Expression, purification, crystallization and preliminary X-ray crystallographic studies of the trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45

    SciTech Connect

    Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2005-01-01

    The trehalulose synthase MutB from P. mesoacidophila MX-45 has been crystallized in two different crystal forms and diffraction data have been collected to 1.6 and 1.8 Å, respectively. The trehalulose synthase (MutB) from Pseudomonas mesoacidophila MX-45, belonging to glycoside hydrolase family 13, catalyses the isomerization of sucrose to trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) and isomaltulose (α-d-glucosylpyranosyl-1,6-d-fructofuranose) as main products and glucose and fructose in residual amounts from the hydrolytic reaction. To date, a three-dimensional structure of a sucrose isomerase that produces mainly trehalulose, as is the case for MutB, has been lacking. Crystallographic studies of this 64 kDa enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of sucrose decomposition, isomerization and of the selectivity of this enzyme that leads to the formation of different products. The MutB protein has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Two different crystal forms have been obtained: one diffracts X-rays to 1.6 Å resolution using synchrotron radiation and belongs to space group P1, with unit-cell parameters a = 63.8, b = 72.0, c = 82.2 Å, α = 67.5, β = 73.1, γ = 70.8°, while the other form diffracts to 1.8 Å resolution using synchrotron radiation and belongs to space group P2{sub 1}, with unit-cell parameters a = 63.7, b = 85.9, c = 119.7 Å, β = 97.7°. A molecular-replacement solution has been found using the structure of the isomaltulose synthase (PalI) from Klebsiella sp. LX3 as a search model.

  6. Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system

    PubMed Central

    Roversi, Pietro; Johnson, Steven; Field, Terry; Deane, Janet E.; Galyov, Edouard E.; Lea, Susan M.

    2006-01-01

    A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å3 Da−1 (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P21212, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å3 Da−1 (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P21212 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P21212 crystals diffract to 3.3 Å resolution. A K2PtCl4 derivative of the P21212 form was also obtained and data were collected to 2.7 Å with radiation of wavelength λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections. PMID:16946464

  7. Expression, Purification And Crystallization of Native And Selenomethionine Labeled Mycobacterium Tuberculosis FDG1 (Rv0407) Using a Mycobacterium Smegmatis Expression System

    SciTech Connect

    Bashiri, G.; Squire, C.J.; Baker, E.N.; Moreland, N.J.

    2009-06-01

    FGD1 is an F{sub 420}-dependent glucose-6-phosphate dehydrogenase from Mycobacterium tuberculosis that has been shown to be essential for activation of the anti-TB compound PA-824. Initial attempts to produce recombinant FGD1 using Escherichia coli as a host was unsuccessful, but when the alternative host Mycobacterium smegmatis was used, soluble protein yields of 7 mg/L of culture were achieved. Both native and selenomethionine-substituted FGD1 were obtained by culturing M. smegmatis in autoinduction media protocols originally developed for E. coli. Using these media afforded the advantages of decreased handling, as cultures did not require monitoring of optical density and induction, and reduced cost by removing the need for expensive ADC enrichment normally used in mycobacterial cultures. Selenomethionine was efficiently incorporated at levels required for multiwavelength anomalous diffraction experiments used in crystal structure determination. As far as we are aware this is the first protocol for preparation of selenomethionine-substituted protein in mycobacteria. Native and selenomethionine-labeled FGD1 were successfully crystallized by vapor diffusion, with the crystals diffracting to 2.1 AA resolution.

  8. Expression, purification, crystallization and preliminary phasing of the heteromerization domain of the tRNA-export and aminoacylation cofactor Arc1p from yeast

    SciTech Connect

    Simader, Hannes; Suck, Dietrich

    2006-04-01

    The heteromerization domain of an aminoacyl-tRNA synthetase cofactor from yeast was crystallized, complete selenomethionine MAD data were collected to 2.8 Å resolution and preliminary phasing reveals the presence of 20 monomers in the asymmetric unit. Eukaryotic aminoacyl-tRNA synthetases (aaRSs) must be integrated into an efficient tRNA-export and shuttling machinery. This is reflected by the presence of additional protein–protein interaction domains and a correspondingly higher degree of complex formation in eukaryotic aaRSs. However, the structural basis of interaction between eukaryotic aaRSs and associated protein cofactors has remained elusive. The N-terminal heteromerization domain of the tRNA aminoacylation and export cofactor Arc1p has been cloned from yeast, expressed and purified. Crystals have been obtained belonging to space group C2, with unit-cell parameters a = 222.32, b = 89.46, c = 126.79 Å, β = 99.39°. Calculated Matthews coefficients are compatible with the presence of 10–25 monomers in the asymmetric unit. A complete multiple-wavelength anomalous dispersion data set has been collected from a selenomethionine-substituted crystal at 2.8 Å resolution. Preliminary phasing reveals the presence of 20 monomers organized in five tetramers per asymmetric unit.

  9. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SpyCEP, a candidate antigen for a vaccine against Streptococcus pyogenes.

    PubMed

    Abate, Francesca; Malito, Enrico; Falugi, Fabiana; Margarit Y Ros, Immaculada; Bottomley, Matthew James

    2013-10-01

    Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen against which an effective vaccine does not yet exist. The S. pyogenes protein SpyCEP (S. pyogenes cell-envelope proteinase) is a surface-exposed subtilisin-like serine protease of 1647 amino acids. In addition to its auto-protease activity, SpyCEP is capable of cleaving interleukin 8 and related chemokines, contributing to GAS immune-evasion strategies. SpyCEP is immunogenic and confers protection in animal models of GAS infections. In order to structurally characterize this promising vaccine candidate, several SpyCEP protein-expression constructs were designed, cloned, produced in Escherichia coli, purified by affinity chromatography and subjected to crystallization trials. Crystals of a selenomethionyl form of a near-full-length SpyCEP ectodomain were obtained. The crystals diffracted X-rays to 3.3 Å resolution and belonged to space group C2, with unit-cell parameters a=139.2, b=120.4, c=104.3 Å, β=111°.

  10. Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii.

    PubMed

    Park, Jeong Soon; Lee, Woo Cheol; Choi, Saehae; Yeo, Kwon Joo; Song, Jung Hyun; Han, Young Hyun; Lee, Je Chul; Kim, Seung Il; Jeon, Young Ho; Cheong, Chaejoon; Kim, Hye Yeon

    2011-12-01

    Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2(1), with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å(3) Da(-1).

  11. Purification and Crystallization of a Multimodular Heterotrimeric Complex Containing Both Type I and Type II Cohesin-dockerin Interactions from the Cellulosome of Clostridium thermocellum

    SciTech Connect

    M Currie; J Adams; S Ali; S Smith; Z Jia

    2011-12-31

    The multimodular scaffoldin subunit CipA is the central component of the cellulosome, a multienzyme plant cell-wall-degrading complex, from Clostridium thermocellum. It captures secreted cellulases and hemicellulases and anchors the entire complex to the cell surface via high-affinity calcium-dependent interactions between cohesin and dockerin modules termed type I and type II interactions. The crystallization of a heterotrimeric complex comprising the type II cohesin module from the cell-surface protein SdbA, a trimodular C-terminal fragment of the scaffoldin CipA and the type I dockerin module from the CelD cellulase is reported. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 119.37, b = 186.31, c = 191.17 {angstrom}. The crystals diffracted to 2.7 {angstrom} resolution with four or eight molecules of the ternary protein complex in the asymmetric unit.

  12. Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

    SciTech Connect

    Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

    2006-12-01

    Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222{sub 1} and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.

  13. Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle

    PubMed Central

    Deane, Janet E.; Cordes, Frank S.; Roversi, Pietro; Johnson, Steven; Kenjale, Roma; Picking, William D.; Picking, Wendy L.; Lea, Susan M.; Blocker, Ariel

    2006-01-01

    A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiHCΔ5 and diffraction data were collected to 1.9 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 Å, β = 96.5°. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 Å3 Da−1 for two molecules per asymmetric unit, corresponding to a solvent content of 33%. PMID:16511329

  14. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase from Streptococcus agalactiae NEM316

    PubMed Central

    Nagarajan, Revathi; Ponnuraj, Karthe

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues. Streptococcus agalactiae, also known as group B streptococcus (GBS), binds onto the host using its surface adhesins and causes sepsis and pneumonia in neonates. GAPDH is one of the surface adhesins of GBS binding to human plasminogen and is a virulent factor associated with host colonization. Although the surface-associated GAPDH has been shown to bind to a variety of host extracellular matrix (ECM) molecules in various bacteria, the molecular mechanism underlying their interaction is not fully understood. To investigate this, structural studies on GAPDH of S. agalactiae were initiated. The gapC gene of S. agalactiae NEM316 encoding GAPDH protein was cloned into pET-28a vector, overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity. The purified protein was crystallized using the hanging-drop vapour-diffusion method. The GAPDH crystals obtained in two different crystallization conditions diffracted to 2.8 and 2.6 Å resolution, belonging to two different space groups P21 and P212121, respectively. The structure was solved by molecular replacement and structure refinement is now in progress. PMID:25005093

  15. Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of a major group 7 allergen, Der f 7, from the dust mite Dermatophagoides farinae.

    PubMed

    Tan, Kang Wei; Kumar, Sundramurthy; Chew, Fook Tim; Mok, Yu Keung

    2011-12-01

    Der f 7 is a major group 7 allergen from the dust mite Dermatophagoides farinae that shows 86% sequence identity to the homologous allergen Der p 7 from D. pteronyssinus. Der f 7 was successfully overexpressed in an Escherichia coli expression system and purified to homogeneity using Ni-NTA affinity and size-exclusion column chromatography. SeMet-labelled Der f 7 was crystallized by the hanging-drop vapour-diffusion method using a reservoir solution consisting of 0.1 M bis-tris pH 7.4 and 28% polyethylene glycol monomethyl ether 2000 at 293 K. X-ray diffraction data were collected to 2.24 Å resolution using synchrotron radiation. The crystals belonged to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell parameters a = 50.19, b = 58.67, c = 123.81 Å. Based on the estimated Matthews coefficient (2.16 Å(3) Da(-1)), two molecules of Der f 7 could be present in the asymmetric unit of the crystal lattice.

  16. Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX).

    PubMed

    Campos, Bruna Medeia; Liberato, Marcelo Vizona; Polikarpov, Igor; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

    2015-03-01

    In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space group P3121, with unit-cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single-wavelength anomalous diffraction method.

  17. Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX).

    PubMed

    Campos, Bruna Medeia; Liberato, Marcelo Vizona; Polikarpov, Igor; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

    2015-03-01

    In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space group P3121, with unit-cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single-wavelength anomalous diffraction method. PMID:25760706

  18. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SpyCEP, a candidate antigen for a vaccine against Streptococcus pyogenes

    PubMed Central

    Abate, Francesca; Malito, Enrico; Falugi, Fabiana; Margarit Y Ros, Immaculada; Bottomley, Matthew James

    2013-01-01

    Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen against which an effective vaccine does not yet exist. The S. pyogenes protein SpyCEP (S. pyogenes cell-envelope proteinase) is a surface-exposed subtilisin-like serine protease of 1647 amino acids. In addition to its auto-protease activity, SpyCEP is capable of cleaving interleukin 8 and related chemokines, contributing to GAS immune-evasion strategies. SpyCEP is immunogenic and confers protection in animal models of GAS infections. In order to structurally characterize this promising vaccine candidate, several SpyCEP protein-expression constructs were designed, cloned, produced in Escherichia coli, purified by affinity chromatography and subjected to crystallization trials. Crystals of a selenomethionyl form of a near-full-length SpyCEP ectodomain were obtained. The crystals diffracted X-rays to 3.3 Å resolution and belonged to space group C2, with unit-cell parameters a = 139.2, b = 120.4, c = 104.3 Å, β = 111°. PMID:24100558

  19. Purification, crystallization and preliminary crystallographic analysis of a natural complex of phospholipase A2 from Echis carinatus (saw-scaled viper).

    PubMed

    Nagpal, A; Chandra, V; Kaur, P; Singh, T P

    1999-06-01

    A novel complex of phospholipase A2 complexed with another venom protein has been isolated and purified from saw-scaled viper (Echis carinatus) venom. The molecular weights of the two components are 16 and 14 kDa, respectively. The complex was purified using an Affigel blue column and an anion-exchange (DEAE Sephacel) column. Long diamond-shaped crystals were obtained by hanging-drop vapour diffusion. The protein complex was dissolved at a concentration of 10 mg ml-1 in 20 mM sodium cacodylate, 1 mM CaCl2 and 2% dioxane at pH 6.0. The reservoir contained the same buffer with 7%(w/v) PEG 4000. Crystals appeared within 2-3 weeks. Native data to 2.9 A resolution have been obtained at 291 K. The crystals belong to the monoclinic space group P21 with unit-cell parameters a = 74.47, b = 47.87, c = 106.39 A, beta = 104.5 degrees and contain two molecules per asymmetric unit. Structure determination by molecular replacement is in progress. PMID:10329797

  20. A Glycoprotein in Shells of Conspecifics Induces Larval Settlement of the Pacific Oyster Crassostrea gigas

    PubMed Central

    Vasquez, Hebert Ely; Hashimoto, Kyotaro; Yoshida, Asami; Hara, Kenji; Imai, Chisato Chris; Kitamura, Hitoshi; Satuito, Cyril Glenn

    2013-01-01

    Settlement of larvae of Crassostrea gigas on shell chips (SC) prepared from shells of 11 different species of mollusks was investigated. Furthermore, the settlement inducing compound in the shell of C. gigas was extracted and subjected to various treatments to characterize the chemical cue. C. gigas larvae settled on SC of all species tested except on Patinopecten yessoensis and Atrina pinnata. In SC of species that induced C. gigas larvae to settle, settlement was proportionate to the amount of SC supplied to the larvae. When compared to C. gigas SC, all species except Crassostrea nippona showed lower settlement inducing activities, suggesting that the cue may be more abundant or in a more available form to the larvae in shells of conspecific and C. nippona than in other species. The settlement inducing activity of C. gigas SC remained intact after antibiotic treatment. Extraction of C. gigas SC with diethyl ether (Et2O-ex), ethanol (EtOH-ex), and water (Aq-ex) did not induce larval settlement of C. gigas larvae. However, extraction of C. gigas SC with 2N of hydrochloric acid (HCl-ex) induced larval settlement that was at the same level as the SC. The settlement inducing compound in the HCl-ex was stable at 100°C but was destroyed or degraded after pepsin, trypsin, PNGase F and trifluoromethanesulfonic acid treatments. This chemical cue eluted between the molecular mass range of 45 and 150 kDa after gel filtration and revealed a major band at 55 kDa on the SDS-PAGE gel after staining with Stains-all. Thus, a 55 kDa glycoprotein component in the organic matrix of C. gigas shells is hypothesized to be the chemical basis of larval settlement on conspecifics. PMID:24349261

  1. Expression, purification, crystallization and preliminary X-ray analysis of the Met244Ala variant of catalase–peroxidase (KatG) from the haloarchaeon Haloarcula marismortui

    SciTech Connect

    Ten-i, Tomomi; Kumasaka, Takashi; Higuchi, Wataru; Tanaka, Satoru; Yoshimatsu, Katsuhiko; Fujiwara, Taketomo; Sato, Takao

    2007-11-01

    The Met244Ala variant of the H. marismortui KatG enzyme was expressed in haloarchaeal host cells and purified to homogeneity. The variant was crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate and NaCl as precipitants. The reddish-brown rod-shaped crystals obtained belong to the monoclinic space group C2, with unit-cell parameters a = 315.24, b = 81.04, c = 74.77 Å, β = 99.81°. The covalent modification of the side chains of Trp95, Tyr218 and Met244 within the active site of Haloarcula marismortui catalase–peroxidase (KatG) appears to be common to all KatGs and has been demonstrated to be particularly significant for its bifunctionality [Smulevich et al. (2006 ▶), J. Inorg. Biochem.100, 568–585; Jakopitsch, Kolarich et al. (2003 ▶), FEBS Lett.552, 135–140; Jakopitsch, Auer et al. (2003 ▶), J. Biol. Chem.278, 20185–20191; Jakopitsch et al. (2004 ▶), J. Biol. Chem.279, 46082–46095; Regelsberger et al. (2001 ▶), Biochem. Soc. Trans.29, 99–105; Ghiladi, Knudsen et al. (2005 ▶), J. Biol. Chem.280, 22651–22663; Ghiladi, Medzihradzky et al. (2005 ▶), Biochemistry, 44, 15093–15105]. The Met244Ala variant of the H. marismortui KatG enzyme was expressed in haloarchaeal host cells and purified to homogeneity. The variant showed a complete loss of catalase activity, whereas the peroxidase activity of this mutant was highly enhanced owing to an increase in its affinity for the peroxidatic substrate. The variant was crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate and NaCl as precipitants. The reddish-brown rod-shaped crystals obtained belong to the monoclinic space group C2, with unit-cell parameters a = 315.24, b = 81.04, c = 74.77 Å, β = 99.81°. A crystal frozen using lithium sulfate as the cryoprotectant diffracted to beyond 2.0 Å resolution. Preliminary X-ray analysis suggests the presence of a dimer in the asymmetric unit.

  2. Isolation and purification of moniliformin.

    PubMed

    Steyn, M; Thiel, P G; van Schalkwyk, G C

    1978-05-01

    A bulk purification procedure is described for moniliformin, a mycotoxin produced by Fusarium moniliforme. The method involves methanol extraction of suitably molded maize, aqueous extraction of the methanol-free residue, ion exchange chromatography with a NaCl concentration gradient, desalination, and crystallization. A pKa value of 1.70 as well as molar absorptivities of 19100 (lambda (H2O) max 229 nm), 5600 (lambda (H2O) max 260 nm), and 4700 (lambda (MeOH) max 260 nm) L/mole/cm are reported for moniliformin.

  3. Purification, crystallization and preliminary crystallographic analysis of DehI, a group I α-haloacid dehalogenase from Pseudomonas putida strain PP3

    SciTech Connect

    Schmidberger, Jason W.; Wilce, Jackie A.; Weightman, Andrew J.; Wilce, Matthew C. J.

    2008-07-01

    The α-haloacid dehalogenase DehI from P. putida strain PP3 was cloned into a vector with an N-terminal His tag and expressed in E. coli Nova Blue strain. Purified protein was crystallized in a primitive monoclinic form and a complete native data set was collected and analysed. Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II α-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from d-haloalkanoic acids and in some cases also the l-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I α-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2{sub 1}), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 Å, α = 90, β = 93.7, γ = 90°, and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary.

  4. Photothermal characterization of thermally treated shells of Strombus Gigas

    NASA Astrophysics Data System (ADS)

    Hernández-Ayala, A.; Quintana, P.; Alvarado-Gil, J. J.; Aldana, D.

    2005-06-01

    The thermal properties of the marine shells of the mollusk Strombus gigas are studied using photoacoustic techniques. In order to generate changes in the layered structure of the shells, they were thermally treated in the range from ambient temperature up to 400ºC. Our results show that the thermal diffusivity and conductivity have a maximum at 200ºC due to the degradation of the organic matrix. At higher temperatures the thermal diffusivity and conductivity decrease due to the calcium carbonate structural phase transition from aragonite to calcite.

  5. Expression, refolding, purification and crystallization of the sensory domain of the TlpC chemoreceptor from Helicobacter pylori for structural studies.

    PubMed

    Liu, Yu Chih; Roujeinikova, Anna

    2015-03-01

    Helicobacter pylori infections are associated with gastritis, duodenal and gastric ulcers and gastric adenocarcinoma. Bacterial chemotaxis, mediated by four different chemoreceptors (also termed transducer-like proteins (Tlp)), plays an important role in initial colonization and development of disease. Chemoreceptor sensory domains of H. pylori share no significant sequence similarity with those of Escherichia coli or any other non-Epsilonproteobacteria. The structural basis of how chemical signals are recognized by chemoreceptors of H. pylori is poorly understood mainly due to the lack of a robust procedure to purify their sensory domains in a soluble form. This study reports a method for extraction of the periplasmic sensory domain of transducer-like protein C (TlpC) from inclusion bodies and refolding to yield 5mg pure crystallizable protein per 1l of bacterial culture. Purified protein was monomeric in solution by size-exclusion chromatography and folded according to the circular dichroism spectrum. Crystals have been grown by the hanging-drop vapor-diffusion method using PEG 4000 as a precipitating agent. The crystals belonged to space group C2, with unit-cell parameters a=189.3, b=103.2, c=61.8Å, β=98.3. A complete X-ray diffraction data set has been collected to 2.2 Å resolution using cryocooling conditions and synchrotron radiation. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains three monomers.

  6. Purification, crystallization and preliminary X-ray diffraction analysis of the IL-20-IL-20R1-IL-20R2 complex

    SciTech Connect

    Logsdon, Naomi J.; Allen, Christopher E.; Rajashankar, Kanagalaghatta R.; Walter, Mark R.

    2012-02-08

    Interleukin-20 (IL-20) is an IL-10-family cytokine that regulates innate and adaptive immunity in skin and other tissues. In addition to protecting the host from various external pathogens, dysregulated IL-20 signaling has been shown to contribute to the pathogenesis of human psoriasis. IL-20 signals through two cell-surface receptor heterodimers, IL-20R1-IL-20R2 and IL-22R1-IL-20R2. In this report, crystals of the IL-20-IL-20R1-IL-20R2 ternary complex have been grown from polyethylene glycol solutions. The crystals belonged to space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = 111, c = 135 {angstrom}, and diffracted X-rays to 3 {angstrom} resolution. The crystallographic asymmetric unit contains one IL-20-IL-20R1-IL-20R2 complex, corresponding to a solvent content of approximately 54%.

  7. Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-d-galactarate (KDG) dehydratase from Agrobacterium tumefaciens

    PubMed Central

    Taberman, Helena; Andberg, Martina; Parkkinen, Tarja; Richard, Peter; Hakulinen, Nina; Koivula, Anu; Rouvinen, Juha

    2014-01-01

    d-Galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-d-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of d-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-l-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications. PMID:24419616

  8. Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain

    SciTech Connect

    Radzimanowski, Jens; Beyreuther, Konrad; Sinning, Irmgard; Wild, Klemens

    2008-05-01

    Alzheimer’s disease is characterized by proteolytic processing of the amyloid precursor protein (APP), which releases the aggregation-prone amyloid-β (Aβ) peptide and liberates the intracellular domain (AICD) that interacts with various adaptor proteins. The crystallized AICD–Fe65-PTB2 complex is of central importance for APP translocation, nuclear signalling, processing and Aβ generation. Alzheimer’s disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid β peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 Å resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

  9. Expression, refolding, purification and crystallization of the sensory domain of the TlpC chemoreceptor from Helicobacter pylori for structural studies.

    PubMed

    Liu, Yu Chih; Roujeinikova, Anna

    2015-03-01

    Helicobacter pylori infections are associated with gastritis, duodenal and gastric ulcers and gastric adenocarcinoma. Bacterial chemotaxis, mediated by four different chemoreceptors (also termed transducer-like proteins (Tlp)), plays an important role in initial colonization and development of disease. Chemoreceptor sensory domains of H. pylori share no significant sequence similarity with those of Escherichia coli or any other non-Epsilonproteobacteria. The structural basis of how chemical signals are recognized by chemoreceptors of H. pylori is poorly understood mainly due to the lack of a robust procedure to purify their sensory domains in a soluble form. This study reports a method for extraction of the periplasmic sensory domain of transducer-like protein C (TlpC) from inclusion bodies and refolding to yield 5mg pure crystallizable protein per 1l of bacterial culture. Purified protein was monomeric in solution by size-exclusion chromatography and folded according to the circular dichroism spectrum. Crystals have been grown by the hanging-drop vapor-diffusion method using PEG 4000 as a precipitating agent. The crystals belonged to space group C2, with unit-cell parameters a=189.3, b=103.2, c=61.8Å, β=98.3. A complete X-ray diffraction data set has been collected to 2.2 Å resolution using cryocooling conditions and synchrotron radiation. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains three monomers. PMID:25462804

  10. Expression, purification, crystallization and preliminary X-ray studies of the outer membrane efflux proteins OprM and OprN from Pseudomonas aeruginosa.

    PubMed

    Broutin, Isabelle; Benabdelhak, Houssain; Moreel, Xavier; Lascombe, Marie-Bernard; Lerouge, Dimitri; Ducruix, Arnaud

    2005-03-01

    OprM and OprN belong to the outer membrane factor family proteins. These approximately 52 kDa proteins are part of the tripartite efflux pumps found in Pseudomonas aeruginosa and are responsible in part for the antibiotic resistance observed in these bacteria. Both proteins have been expressed in Escherichia coli as His-tag proteins and purified accordingly by affinity chromatography in the presence of n-octyl-beta-D-glucopyranoside detergent. OprM and OprN were crystallized using PEG 20 000/ammonium citrate and ammonium sulfate as precipitating agents, respectively. Crystals belong to space group C2, with unit-cell parameters a = 152.6, b = 87.9, c = 355.9 A, beta = 98.9 degrees and a = 151.3, b = 87.6, c = 356.5 A, beta = 98.1 degrees for OprM and OprN, respectively. Using the ESRF synchrotron-radiation source, OprM diffraction data extended to 3.4 A.

  11. Expression, purification, crystallization and preliminary X-ray studies of the outer membrane efflux proteins OprM and OprN from Pseudomonas aeruginosa

    PubMed Central

    Broutin, Isabelle; Benabdelhak, Houssain; Moreel, Xavier; Lascombe, Marie-Bernard; Lerouge, Dimitri; Ducruix, Arnaud

    2005-01-01

    OprM and OprN belong to the outer membrane factor family proteins. These ∼52 kDa proteins are part of the tripartite efflux pumps found in Pseudomonas aeruginosa and are responsible in part for the antibiotic resistance observed in these bacteria. Both proteins have been expressed in Escherichia coli as His-tag proteins and purified accordingly by affinity chromatography in the presence of n-octyl-β-d-glucopyranoside detergent. OprM and OprN were crystallized using PEG 20 000/ammonium citrate and ammonium sulfate as precipitating agents, respectively. Crystals belong to space group C2, with unit-cell parameters a = 152.6, b = 87.9, c = 355.9 Å, β = 98.9° and a = 151.3, b = 87.6, c = 356.5 Å, β = 98.1° for OprM and OprN, respectively. Using the ESRF synchrotron-radiation source, OprM diffraction data extended to 3.4 Å. PMID:16511029

  12. Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties

    PubMed Central

    Don Paul, Craig; Traore, Daouda A. K.; Byres, Emma; Rossjohn, Jamie; Devenish, Rodney J.; Kiss, Csaba; Bradbury, Andrew; Wilce, Matthew C. J.; Prescott, Mark

    2011-01-01

    Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, α = 90.96, β = 89.92, γ = 104.03°. The Matthews coefficient (V M = 2.26 Å3 Da−1) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules. PMID:22102044

  13. Purification, crystallization and preliminary X-ray analysis of the inverse F-BAR domain of the human srGAP2 protein.

    PubMed

    Wang, Hongpeng; Zhang, Yan; Zhang, Zhenyi; Jin, Wei Lin; Wu, Geng

    2014-01-01

    Bin-Amphiphysin-Rvs (BAR) domain proteins play essential roles in diverse cellular processes by inducing membrane invaginations or membrane protrusions. Among the BAR superfamily, the `classical' BAR and Fes/CIP4 homology BAR (F-BAR) subfamilies of proteins usually promote membrane invaginations, whereas the inverse BAR (I-BAR) subfamily generally incur membrane protrusions. Despite possessing an N-terminal F-BAR domain, the srGAP2 protein regulates neurite outgrowth and neuronal migration by causing membrane protrusions reminiscent of the activity of I-BAR domain proteins. In this study, the inverse F-BAR (IF-BAR) domain of human srGAP2 was overexpressed, purified and crystallized. The crystals of the srGAP2 IF-BAR domain protein diffracted to 3.50 Å resolution and belonged to space group P2(1). These results will facilitate further structural determination of the srGAP2 IF-BAR domain and the ultimate elucidation of its peculiar behaviour of inducing membrane protrusions rather than membrane invaginations.

  14. Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome.

    PubMed

    Campos, Bruna Medeia; Alvarez, Thabata Maria; Liberato, Marcelo Vizona; Polikarpov, Igor; Gilbert, Harry J; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

    2014-09-01

    In recent years, owing to the growing global demand for energy, dependence on fossil fuels, limited natural resources and environmental pollution, biofuels have attracted great interest as a source of renewable energy. However, the production of biofuels from plant biomass is still considered to be an expensive technology. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases for polysaccharide degradation, is attracting growing attention. Aiming at the identification of new CBMs, a sugarcane soil metagenomic library was analyzed and an uncharacterized CBM (CBM_E1) was identified. In this study, CBM_E1 was expressed, purified and crystallized. X-ray diffraction data were collected to 1.95 Å resolution. The crystals, which were obtained by the sitting-drop vapour-diffusion method, belonged to space group I23, with unit-cell parameters a = b = c = 88.07 Å. PMID:25195898

  15. Purification, crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A2 with vasoconstrictor activity from Agkistrodon halys pallas venom.

    PubMed

    Zou, Zhisong; Zeng, Fuxing; Zhang, Lu; Niu, Liwen; Teng, Maikun; Li, Xu

    2012-11-01

    Phospholipases A2 (PLA2s) are the major component of snake venoms and exert a variety of relevant toxic actions such as neurotoxicity and myotoxicity, amongst others. An acidic PLA2, here named AhV_aPA, was purified from Agkistrodon halys pallas venom by means of a three-step chromatographic procedure. AhV_aPA migrated as a single band on SDS-PAGE gels, with a molecular weight of about 14 kDa. Like other acidic aPLA2s, AhV_aPA has high enzymatic activity. Tension measurements of mouse thoracic aortic rings remarkably indicated that AhV_aPA could induce a further contractile response on the 60 mM K+-induced contraction, with an EC50 of 369 nmol l(-1). Rod-shaped crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 2.30 Å. The crystals belonged to space group P222, with unit-cell parameters a=44.27, b=68.39, c=81.54 Å. PMID:23143242

  16. Purification, crystallization and preliminary X-ray diffraction analysis of inner membrane complex (IMC) subcompartment protein 1 (ISP1) from Toxoplasma gondii.

    PubMed

    Tonkin, Michelle L; Brown, Shannon; Beck, Josh R; Bradley, Peter J; Boulanger, Martin J

    2012-07-01

    The protozoan parasites of the Apicomplexa phylum are devastating global pathogens. Their success is largely due to phylum-specific proteins found in specialized organelles and cellular structures. The inner membrane complex (IMC) is a unique apicomplexan structure that is essential for motility, invasion and replication. The IMC subcompartment proteins (ISP) have recently been identified in Toxoplasma gondii and shown to be critical for replication, although their specific mechanisms are unknown. Structural characterization of TgISP1 was pursued in order to identify the fold adopted by the ISPs and to generate detailed insight into how this family of proteins functions during replication. An N-terminally truncated form of TgISP1 was purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis. Two crystal forms of TgISP1 belonging to space groups P4(1)32 or P4(3)32 and P2(1)2(1)2(1) diffracted to 2.05 and 2.1 Å resolution, respectively.

  17. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the pectin methylesterase from the sugar cane weevil Sphenophorus levis

    PubMed Central

    Evangelista, Danilo Elton; Schutzer de Godoy, Andre; Fonseca Pereira de Paula, Fernando; Henrique-Silva, Flavio; Polikarpov, Igor

    2014-01-01

    Pectin methylesterase removes the methyl groups from the main chain of pectin, the major component of the middle lamella of the plant cell wall. The enzyme is involved in plant cell-wall development, is part of the enzymatic arsenal used by microorganisms to attack plants and also has a wide range of applications in the industrial sector. Therefore, there is a considerable interest in studies of the structure and function of this enzyme. In this work, the pectin methylesterase from Sphenophorus levis was produced in Pichia pastoris and purified. Crystals belonging to the monoclinic space group C2, with unit-cell parameters a = 122.181, b = 82.213, c = 41.176 Å, β = 97.48°, were obtained by the sitting-drop vapour-diffusion method and an X-ray diffraction data set was collected to 2.1 Å resolution. Structure refinement and model building are in progress. PMID:24598920

  18. Purification of silicon for photovoltaic applications

    NASA Astrophysics Data System (ADS)

    Delannoy, Yves

    2012-12-01

    Solar grade silicon, as a starting material for crystallization to produce solar cells, is discussed here in terms of impurities whose maximum content is estimated from recent literature and conferences. A review of the production routes for each category of solar-grade silicon (undoped, compensated or heavily compensated) is proposed with emphasis on the metallurgical route. Some recent results are proposed concerning segregation, showing that directional solidification systems can be used for solidification even at high solidification rate (15 cm/h). Results on inductive plasma purification, where boron is evacuated as HBO in a gas phase blown from an inductive plasma torch, are shown to apply as well to arc plasmas and purification by moist gas. Special attention is paid to the history of impurities in the purification processes, showing that impure auxiliary phases (silicon tetrachloride, slag, aluminum, etc.) often need their own purification process to enable their recycling, which has to be considered to evaluate the cost (financial, energetic and environmental) of the purification route.

  19. Refinement of the nickel site structure in Desulfovibrio gigas hydrogenase using range-extended EXAFS spectroscopy.

    PubMed

    Gu, Weiwei; Jacquamet, L; Patil, D S; Wang, H X; Evans, D J; Smith, M C; Millar, M; Koch, S; Eichhorn, D M; Latimer, M; Cramer, S P

    2003-01-01

    We have reexamined the Ni EXAFS of oxidized, inactive (as-isolated) and H(2) reduced Desulfovibrio gigas hydrogenase. Better spatial resolution was achieved by analyzing the data over a 50% wider k-range than was previously available. A lower k(min) was obtained using the FEFF code for phase shifts and amplitudes. A higher k(max) was obtained by removing an interfering Cu signal from the raw spectra using multiple energy fluorescence detection. The larger k-range allowed us to better resolve the Ni-S bond lengths and to define more accurately the Ni-O and Ni-Fe bond lengths. We find that as-isolated, hydrogenase has two Ni-S bonds at approximately 2.2 A, but also 1-2 Ni-S bonds in the 2.35+/-0.05 A range. A Ni-O interaction is evident at 1.91 A. The as-isolated Ni-Fe distance cannot be unambiguously determined. Upon H(2) reduction, two short Ni-S bonds persist at approximately 2.2 A, but the remaining Ni-S bonds lengthen to 2.47+/-0.05 A. Good simulations are obtained with a Ni-Fe distance at 2.52 A, in agreement with crystal structures of the reduced enzyme. Although not evident in the crystal structures, an improvement in the fit is obtained by inclusion of one Ni-O interaction at 2.03 A. Implications of these distances for the spin-state of H(2) reduced H(2)ase are discussed. PMID:12538051

  20. Mitochondrial genome of the endangered marine gastropod Strombus gigas Linnaeus, 1758 (Mollusca: Gastropoda).

    PubMed

    Márquez, Edna J; Castro, Erick R; Alzate, Juan F

    2016-01-01

    The queen conch Strombus gigas is an endangered marine gastropod of significant economic importance across the Greater Caribbean region. This work reports for the first time the complete mitochondrial genome of S. gigas, obtained by FLX 454 pyrosequencing. The mtDNA genome encodes for 13 proteins, 22 tRNAs and 2 ribosomal RNAs. In addition, the coding sequences and gene synteny were similar to other previously reported mitogenomes of gastropods.

  1. Mitochondrial genome of the endangered marine gastropod Strombus gigas Linnaeus, 1758 (Mollusca: Gastropoda).

    PubMed

    Márquez, Edna J; Castro, Erick R; Alzate, Juan F

    2016-01-01

    The queen conch Strombus gigas is an endangered marine gastropod of significant economic importance across the Greater Caribbean region. This work reports for the first time the complete mitochondrial genome of S. gigas, obtained by FLX 454 pyrosequencing. The mtDNA genome encodes for 13 proteins, 22 tRNAs and 2 ribosomal RNAs. In addition, the coding sequences and gene synteny were similar to other previously reported mitogenomes of gastropods. PMID:25186797

  2. Adult Pacific Oyster (Crassostrea gigas) May Have Light Sensitivity.

    PubMed

    Wu, Changlu; Wang, Jiao; Yang, Yanjian; Li, Zhuang; Guo, Ting; Li, Yongchuan; Wang, Xiaotong

    2015-01-01

    Light-sensitivity is an important aspect of mollusk survival as it plays a vital role in reproduction and predator avoidance. In the Pacific oyster Crassostrea gigas light sensitivity has been demonstrated in the larval stage but has not yet been conclusively demonstrated in adult oysters. In this paper we describe an experiment which was undertaken to determine if adult Pacific oysters were sensitive to light. One LED flashlight was used to shine light onto adult oysters while they were filtering seawater through their shell openings. We found that the degree of opening increased gradually during the light period but rapidly decreased when the flashlight was turned off in the treated group but not in the control group. These results suggest that adult Pacific oyster may be sensitive to light.

  3. Impact and fracture analysis of fish scales from Arapaima gigas.

    PubMed

    Torres, F G; Malásquez, M; Troncoso, O P

    2015-06-01

    Fish scales from the Amazonian fish Arapaima gigas have been characterised to study their impact and fracture behaviour at three different environmental conditions. Scales were cut in two different directions to analyse the influence of the orientation of collagen layers. The energy absorbed during impact tests was measured for each sample and SEM images were taken after each test in order to analyse the failure mechanisms. The results showed that scales tested at cryogenic temperatures display fragile behaviour, while scales tested at room temperature did not fracture. Different failure mechanisms have been identified, analysed and compared with the failure modes that occur in bone. The impact energy obtained for fish scales was two to three times higher than the values reported for bone in the literature. PMID:25842120

  4. Adult Pacific Oyster (Crassostrea gigas) May Have Light Sensitivity

    PubMed Central

    Yang, Yanjian; Li, Zhuang; Guo, Ting; Li, Yongchuan; Wang, Xiaotong

    2015-01-01

    Light-sensitivity is an important aspect of mollusk survival as it plays a vital role in reproduction and predator avoidance. In the Pacific oyster Crassostrea gigas light sensitivity has been demonstrated in the larval stage but has not yet been conclusively demonstrated in adult oysters. In this paper we describe an experiment which was undertaken to determine if adult Pacific oysters were sensitive to light. One LED flashlight was used to shine light onto adult oysters while they were filtering seawater through their shell openings. We found that the degree of opening increased gradually during the light period but rapidly decreased when the flashlight was turned off in the treated group but not in the control group. These results suggest that adult Pacific oyster may be sensitive to light. PMID:26474058

  5. Impact and fracture analysis of fish scales from Arapaima gigas.

    PubMed

    Torres, F G; Malásquez, M; Troncoso, O P

    2015-06-01

    Fish scales from the Amazonian fish Arapaima gigas have been characterised to study their impact and fracture behaviour at three different environmental conditions. Scales were cut in two different directions to analyse the influence of the orientation of collagen layers. The energy absorbed during impact tests was measured for each sample and SEM images were taken after each test in order to analyse the failure mechanisms. The results showed that scales tested at cryogenic temperatures display fragile behaviour, while scales tested at room temperature did not fracture. Different failure mechanisms have been identified, analysed and compared with the failure modes that occur in bone. The impact energy obtained for fish scales was two to three times higher than the values reported for bone in the literature.

  6. The Borexino purification system

    NASA Astrophysics Data System (ADS)

    Benziger, Jay

    2014-05-01

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

  7. The Trilogy is Complete - GigaGalaxy Zoom Phase 3

    NASA Astrophysics Data System (ADS)

    2009-09-01

    The third image of ESO's GigaGalaxy Zoom project has just been released online, completing this eye-opening dive into our galactic home in outstanding fashion. The latest image follows on from views, released over the last two weeks, of the sky as seen with the unaided eye and through an amateur telescope. This third instalment provides another breathtaking vista of an astronomical object, this time a 370-million-pixel view of the Lagoon Nebula of the quality and depth needed by professional astronomers in their quest to understand our Universe. The newly released image extends across a field of view of more than one and a half square degree - an area eight times larger than that of the full Moon - and was obtained with the Wide Field Imager attached to the MPG/ESO 2.2-metre telescope at the La Silla Observatory in Chile. This 67-million-pixel camera has already created several of ESO's iconic pictures. The intriguing object depicted here - the Lagoon Nebula - is located four to five thousand light-years away towards the constellation of Sagittarius (the Archer). The nebula is a giant interstellar cloud, 100 light-years across, where stars are forming. The scattered dark patches seen all over the nebula are huge clouds of gas and dust that are collapsing under their own weight and which will soon give birth to clusters of young, glowing stars. Some of the smallest clouds are known as "globules" and the most prominent ones have been catalogued by the astronomer Edward Emerson Barnard. The Lagoon Nebula hosts the young open stellar cluster known as NGC 6530. This is home for 50 to 100 stars and twinkles in the lower left portion of the nebula. Observations suggest that the cluster is slightly in front of the nebula itself, though still enshrouded by dust, as revealed by reddening of the starlight, an effect that occurs when small dust particles scatter light. The name of the Lagoon Nebula derives from the wide lagoon-shaped dark lane located in the middle of the

  8. Zooming to the centre of the Milky Way - GigaGalaxy Zoom phase 2

    NASA Astrophysics Data System (ADS)

    2009-09-01

    The second of three images of ESO's GigaGalaxy Zoom project has just been released online. It is a new and wonderful 340-million-pixel vista of the central parts of our home galaxy as seen from ESO's Paranal Observatory with an amateur telescope. This 34 by 20-degree wide image provides us with a view as experienced by amateur astronomers around the world. However, its incredible beauty and appeal owe much to the quality of the observing site and the skills of Stéphane Guisard, the world-renowned astrophotographer, who is also an ESO engineer. This second image directly benefits from the quality of Paranal's sky, one of the best on the planet, where ESO's Very Large Telescope is located. In addition, Guisard has drawn on his professional expertise as an optical engineer specialising in telescopes, a rare combination in the world of astrophotographers. Guisard, as head of the optical engineering team at Paranal, is responsible for ensuring that the Very Large Telescope has the best optical performance possible. To create this stunning, true-colour mosaic of the Galactic Centre region, Guisard assembled about 1200 individual images, totalling more than 200 hours of exposure time, collected over 29 nights, during Guisard's free time, while working during the day at Paranal [1]. The image shows the region spanning the sky from the constellation of Sagittarius (the Archer) to Scorpius (the Scorpion). The very colourful Rho Ophiuchi and Antares region is a prominent feature to the right, although much darker areas, such as the Pipe and Snake nebulae also stand out. The dusty lane of our Milky Way runs obliquely through the image, dotted with remarkable bright, reddish nebulae, such as the Lagoon and the Trifid Nebulae, as well as NGC 6357 and NGC 6334. This dark lane also hosts the very centre of our Galaxy, where a supermassive black hole is lurking. "The area I have depicted in this image is an incredibly rich region of the sky, and the one I find most beautiful

  9. The shell organic matrix of the crossed lamellar queen conch shell (Strombus gigas).

    PubMed

    Osuna-Mascaró, Antonio; Cruz-Bustos, Teresa; Benhamada, Sana; Guichard, Nathalie; Marie, Benjamin; Plasseraud, Laurent; Corneillat, Marion; Alcaraz, Gérard; Checa, Antonio; Marin, Frédéric

    2014-02-01

    In molluscs, the shell organic matrix comprises a large set of biomineral-occluded proteins, glycoproteins and polysaccharides that are secreted by the calcifying mantle epithelium, and are supposed to display several functions related to the synthesis of the shell. In the present paper, we have characterized biochemically the shell matrix associated to the crossed-lamellar structure of the giant queen conch Strombus gigas. The acid-soluble (ASM) and acid-insoluble (AIM) matrices represent an extremely minor fraction of the shell. Both are constituted of polydisperse and of few discrete proteins among which three fractions, obtained by preparative SDS-PAGE and named 1P3, 2P3 and 3P3, are dominant and were further characterized. Compared to other matrices, the acid-soluble matrix is weakly glycosylated (3%) and among the discrete components, only 3P3 seems noticeably glycosylated. The monosaccharide composition of the ASM shows that mannose represents the main monosaccharide. To our knowledge, this is the first report of a high ratio of this sugar in a skeletal matrix. Furthermore, the ASM interacts with the in vitro crystallization of calcium carbonate, but this interaction is moderate. It differs from that of the isolated 1P3 fraction but is similar to that of the 2P3 and 3P3 fractions. At last, antibodies developed from the 3P3 fraction were used to localize this fraction within the shell by immunogold. This study is the first one aiming at characterizing the organic matrix associated to the crossed-lamellar structure of the queen conch shell.

  10. The shell organic matrix of the crossed lamellar queen conch shell (Strombus gigas).

    PubMed

    Osuna-Mascaró, Antonio; Cruz-Bustos, Teresa; Benhamada, Sana; Guichard, Nathalie; Marie, Benjamin; Plasseraud, Laurent; Corneillat, Marion; Alcaraz, Gérard; Checa, Antonio; Marin, Frédéric

    2014-02-01

    In molluscs, the shell organic matrix comprises a large set of biomineral-occluded proteins, glycoproteins and polysaccharides that are secreted by the calcifying mantle epithelium, and are supposed to display several functions related to the synthesis of the shell. In the present paper, we have characterized biochemically the shell matrix associated to the crossed-lamellar structure of the giant queen conch Strombus gigas. The acid-soluble (ASM) and acid-insoluble (AIM) matrices represent an extremely minor fraction of the shell. Both are constituted of polydisperse and of few discrete proteins among which three fractions, obtained by preparative SDS-PAGE and named 1P3, 2P3 and 3P3, are dominant and were further characterized. Compared to other matrices, the acid-soluble matrix is weakly glycosylated (3%) and among the discrete components, only 3P3 seems noticeably glycosylated. The monosaccharide composition of the ASM shows that mannose represents the main monosaccharide. To our knowledge, this is the first report of a high ratio of this sugar in a skeletal matrix. Furthermore, the ASM interacts with the in vitro crystallization of calcium carbonate, but this interaction is moderate. It differs from that of the isolated 1P3 fraction but is similar to that of the 2P3 and 3P3 fractions. At last, antibodies developed from the 3P3 fraction were used to localize this fraction within the shell by immunogold. This study is the first one aiming at characterizing the organic matrix associated to the crossed-lamellar structure of the queen conch shell. PMID:24291423

  11. GaN Initiative for Grid Applications (GIGA)

    SciTech Connect

    Turner, George

    2015-07-03

    For nearly 4 ½ years, MIT Lincoln Laboratory (MIT/LL) led a very successful, DoE-funded team effort to develop GaN-on-Si materials and devices, targeting high-voltage (>1 kV), high-power, cost-effective electronics for grid applications. This effort, called the GaN Initiative for Grid Applications (GIGA) program, was initially made up of MIT/LL, the MIT campus group of Prof. Tomas Palacios (MIT), and the industrial partner M/A Com Technology Solutions (MTS). Later in the program a 4th team member was added (IQE MA) to provide commercial-scale GaN-on-Si epitaxial materials. A basic premise of the GIGA program was that power electronics, for ubiquitous utilization -even for grid applications - should be closer in cost structure to more conventional Si-based power electronics. For a number of reasons, more established GaN-on-SiC or even SiC-based power electronics are not likely to reach theses cost structures, even in higher manufacturing volumes. An additional premise of the GIGA program was that the technical focus would be on materials and devices suitable for operating at voltages > 1 kV, even though there is also significant commercial interest in developing lower voltage (< 1 kV), cost effective GaN-on-Si devices for higher volume applications, like consumer products. Remarkable technical progress was made during the course of this program. Advances in materials included the growth of high-quality, crack-free epitaxial GaN layers on large-diameter Si substrates with thicknesses up to ~5 μm, overcoming significant challenges in lattice mismatch and thermal expansion differences between Si and GaN in the actual epitaxial growth process. Such thick epilayers are crucial for high voltage operation of lateral geometry devices such as Schottky barrier (SB) diodes and high electron mobility transistors (HEMTs). New “Normally-Off” device architectures were demonstrated – for safe operation of power electronics circuits. The trade-offs between lateral and

  12. Phylogeny of forkhead genes in three spiralians and their expression in Pacific oyster Crassostrea gigas

    NASA Astrophysics Data System (ADS)

    Yang, Mei; Xu, Fei; Liu, Jun; Que, Huayong; Li, Li; Zhang, Guofan

    2014-11-01

    The Fox genes encode a group of transcription factors that contain a forkhead domain, which forms a structure known as a winged helix. These transcription factors play a crucial role in several key biological processes, including development. High-degree identity in the canonical forkhead domain has been used to divide Fox proteins into 23 families (FoxA to FoxS). We surveyed the genome of three spiralians, the oyster Crassostrea gigas, the limpet Lottia gigantea, and the annelid Capitella teleta. We identified 25 C. gigas fox genes, 21 L. gigantea fox genes, and 25 C. teleta fox genes. The C. gigas fox and L. gigantea fox genes represented 19 of the 23 families, whereas FoxI, Q1, R, and S were missing. The majority of the Fox families were observed within the C. teleta fox genes, with the exception of FoxR and S. In addition, the foxAB-like gene, foxY-like gene, and foxH gene were also present in the three genomes. The conserved FoxC-FoxL1 cluster, observed in mammals, was also found in C. gigas. The diversity of temporal expression patterns observed across the developmental process implies the C. gigas fox genes exert a wide range of functions. Further functional studies are required to gain insight into the evolution of Fox genes in bilaterians.

  13. Proteomic analysis of hemolymph from poly(I:C)-stimulated Crassostrea gigas.

    PubMed

    Green, Timothy J; Chataway, Timothy; Melwani, Aroon R; Raftos, David A

    2016-01-01

    Synthetic double stranded RNA (Poly(I:C)) injection of Crassostrea gigas results in a systemic antiviral response involving many evolutionary conserved antiviral effectors (ISGs). Compared to mammals, the timing of C. gigas ISG expression to viral or poly(I:C) injection is delayed (>12 h p.i.). It could be interpreted that a cytokine is responsible for the systemic, but delayed expression of C. gigas ISGs. We therefore analysed the acellular fraction of C. gigas hemolymph by two-dimensional electrophoresis (2-DE) to identify hemolymph proteins induced by poly(I:C). Poly(I:C) injection increased the relative intensity of four protein spots. These protein spots were identified by tandem mass spectrometry (LC-MS/MS) as a small heat shock protein (sHSP), poly(I:C)-inducible protein 1 (PIP1) and two isoforms of C1q-domain containing protein (C1qDC). RT-qPCR analysis confirmed that the genes encoding these proteins are induced in hemocytes of C. gigas injected with poly(I:C) (p < 0.05). Proteomic data from this experiment corroborates previous microarray and whole transcriptome studies that have reported up-regulation of C1qDC and sHSP during mass mortality events among farmed oysters. PMID:26578249

  14. The GigaFitter: Performance at CDF and Perspectives for Future Applications

    NASA Astrophysics Data System (ADS)

    Amerio, S.; Annovi, A.; Bettini, M.; Bucciantonio, M.; Catastini, P.; Crescioli, F.; Dell'Orso, M.; Giannetti, P.; Lucchesi, D.; Nicoletto, M.; Piendibene, M.; Volpi, G.

    2010-04-01

    The Silicon Vertex Trigger (SVT) is a processor developed at CDF experiment to perform online fast and precise track reconstruction. SVT is made of two pipelined processors, the Associative Memory, finding low precision tracks, and the Track Fitter, refining the track quality whith high precision fits. We will describe the architecture and the performances of a next generation track fitter, the GigaFitter, developed to reduce the degradation of the SVT efficiency due to the increasing instantaneous luminosity. The GigaFitter reduces the track parameter reconstruction to a few clock cycles and can perform many fits in parallel, thus allowing high resolution tracking at very high rate. The core of the GigaFitter is implemented in a modern Xilinx Virtex-5 FPGA chip, rich in powerful DSP arrays. The FPGA is housed on a mezzanine board which receives the data from a subset of the tracking detector and transfers the fitted tracks to a Pulsar motherboard for the final corrections. Instead of the current 12 boards, one per sector of the detector, the final system will be much more compact, consisting of a single GigaFitter Pulsar board equipped with four mezzanine cards receiving the data from the entire tracking detector. Moreover, the GigaFitter modular structure is adequate to scale for much better performances and is general enough to be easily adapted to future High Energy Physics (HEP) experiments and applications outside HEP.

  15. Stress-induced immune changes in the oyster Crassostrea gigas.

    PubMed

    Lacoste, Arnaud; Malham, Shelagh K; Gélébart, Florence; Cueff, Anne; Poulet, Serge A

    2002-01-01

    Information concerning the effect of stress on invertebrate immune functions are scarce. The present study investigated the consequences of a 15-min mechanical disturbance on immune parameters in oysters Crassostrea gigas. As indicated by noradrenaline and dopamine measurements, the mechanical disturbance caused a transient state of stress in oysters. The number of circulating hemocytes, the migratory and phagocytic activities and reactive oxygen species production of hemocytes were measured before, during and after application of the stressor. Results show that all immune functions were significantly downregulated during stress and a transient period of immunostimulation was observed 30-240 min after the end of the disturbance. Taken together, these results suggest that stress can exert a profound influence on oyster immune functions and they may explain why stress and the outbreak of disease are often linked in shellfish culture. Furthermore, the present study strongly suggests that checking the stress status of animals may be necessary to avoid biases when studying oyster immune responses in vivo.

  16. Terrestrial ecology of semi-aquatic giant gartersnakes (Thamnophis gigas)

    USGS Publications Warehouse

    Halstead, Brian J.; Skalos, Shannon M.; Wylie, Glenn D.; Casazza, Michael L.

    2015-01-01

    Wetlands are a vital component of habitat for semiaquatic herpetofauna, but for most species adjacent terrestrial habitats are also essential. We examined the use of terrestrial environments by Giant Gartersnakes (Thamnophis gigas) to provide behavioral information relevant to conservation of this state and federally listed threatened species. We used radio telemetry data collected 1995–2011 from adults at several sites throughout the Sacramento Valley, California, USA, to examine Giant Gartersnake use of the terrestrial environment. We found Giant Gartersnakes in terrestrial environments more than half the time during the summer, with the use of terrestrial habitats increasing to nearly 100% during brumation. While in terrestrial habitats, we found Giant Gartersnakes underground more than half the time in the early afternoon during summer, and the probability of being underground increased to nearly 100% of the time at all hours during brumation. Extreme temperatures also increased the probability that we would find Giant Gartersnakes underground. Under most conditions, we found Giant Gartersnakes to be within 10 m of water at 95% of observations. For females during brumation and individuals that we found underground, however, the average individual had a 10% probability of being located > 20 m from water. Individual variation in each of the response variables was extensive; therefore, predicting the behavior of an individual was fraught with uncertainty. Nonetheless, our estimates provide resource managers with valuable information about the importance of protecting and carefully managing terrestrial habitats for conserving a rare semiaquatic snake.

  17. Stress-induced immune changes in the oyster Crassostrea gigas.

    PubMed

    Lacoste, Arnaud; Malham, Shelagh K; Gélébart, Florence; Cueff, Anne; Poulet, Serge A

    2002-01-01

    Information concerning the effect of stress on invertebrate immune functions are scarce. The present study investigated the consequences of a 15-min mechanical disturbance on immune parameters in oysters Crassostrea gigas. As indicated by noradrenaline and dopamine measurements, the mechanical disturbance caused a transient state of stress in oysters. The number of circulating hemocytes, the migratory and phagocytic activities and reactive oxygen species production of hemocytes were measured before, during and after application of the stressor. Results show that all immune functions were significantly downregulated during stress and a transient period of immunostimulation was observed 30-240 min after the end of the disturbance. Taken together, these results suggest that stress can exert a profound influence on oyster immune functions and they may explain why stress and the outbreak of disease are often linked in shellfish culture. Furthermore, the present study strongly suggests that checking the stress status of animals may be necessary to avoid biases when studying oyster immune responses in vivo. PMID:11687258

  18. Direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase.

    PubMed

    Correia dos Santos, Margarida M; Sousa, Patrícia M P; Gonçalves, M Lurdes S; Romão, M João; Moura, Isabel; Moura, José J G

    2004-04-01

    This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase (DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.

  19. Study of atrazine effects on Pacific oyster, Crassostrea gigas, haemocytes.

    PubMed

    Gagnaire, B; Renault, T; Bouilly, K; Lapegue, S; Thomas-Guyon, H

    2003-01-01

    Shellfish farming is an important economic activity around the world. This activity often takes place in areas subjected to various recurring pollutions. The recrudescent use of herbicides in agriculture including atrazine implies pollutant transfer towards aquatic environment in estuarine areas. Harmful effects of such substances on animals in marine environment, particularly on cultured bivalves, are poorly documented. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their way of life. They filter large volumes of seawater and may therefore accumulate and concentrate contaminants within their tissues. Moreover, development of techniques allowing effect analysis of such compounds on bivalve biology may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, influence of atrazine on defence mechanisms was analyzed in Pacific oysters, Crassostrea gigas. Atrazine was tested in vitro and in vivo on oyster haemocytes, and its effects were analyzed by flow cytometry. Haemocyte viability, cell cycle and cellular activities were monitored. Atrazine induced no significant effect in oyster under tested conditions except for peroxidase activity.

  20. High Infestation by Dawestrema cycloancistrioides in Arapaima gigas Cultured in the Amazon Region, Peru

    PubMed Central

    Mathews, Patrick D.; Malheiros, Antonio F.; Vasquez, Narda D.; Chavez, Milton D.

    2014-01-01

    The aim of this study was to evaluate the presence of Dawestrema cycloancistrioides in semi-intensive fish farming of fingerlings of Arapaima gigas. Between September and November 2013, 60 individuals of A. gigas born in captivity, were collected in three concrete ponds, from a semi-intensive fish farm in the Peruvian Amazon. For the study of sclerotized structures, parasites were fixed in a solution of ammonium picrate glycerine and mounted in Canada balsam. To visualize internal structures, parasites were fixed in hot formaldehyde solution (4%) for staining with Gomori's trichrome. The parasitic indexes calculated were prevalence, mean intensity, and mean abundance. This study identified a high infestation of a monogenean D. cycloancistrioides in gills of A. gigas. The prevalence was 100%. The mean intensity and mean abundance of the parasite were 144.9 of parasites per individual. This study confirms the necessity of constant monitoring of fish in order to reduce fish mortality. PMID:26464924

  1. Genome sequence of the model sulfate reducer Desulfovibrio gigas: a comparative analysis within the Desulfovibrio genus*

    PubMed Central

    Morais-Silva, Fabio O; Rezende, Antonio Mauro; Pimentel, Catarina; Santos, Catia I; Clemente, Carla; Varela–Raposo, Ana; Resende, Daniela M; da Silva, Sofia M; de Oliveira, Luciana Márcia; Matos, Marcia; Costa, Daniela A; Flores, Orfeu; Ruiz, Jerónimo C; Rodrigues-Pousada, Claudina

    2014-01-01

    Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however, not yet available. The sequencing of the D. gigas genome provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. Comparison with genomes of other Desulfovibrio spp. reveals the presence of two different CRISPR/Cas systems in D. gigas. Phylogenetic analysis using conserved protein sequences (encoded by rpoB and gyrB) indicates two main groups of Desulfovibrio spp, being D. gigas more closely related to D. vulgaris and D. desulfuricans strains. Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase, and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh. PMID:25055974

  2. Ribonucleic acid purification.

    PubMed

    Martins, R; Queiroz, J A; Sousa, F

    2014-08-15

    Research on RNA has led to many important biological discoveries and improvement of therapeutic technologies. From basic to applied research, many procedures employ pure and intact RNA molecules; however their isolation and purification are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality. The current techniques to isolate and purify RNA molecules still have several limitations and the requirement for new methods able to improve RNA quality to meet regulatory demands is growing. In fact, as basic research improves the understanding of biological roles of RNAs, the biopharmaceutical industry starts to focus on them as a biotherapeutic tools. Chromatographic bioseparation is a high selective unit operation and is the major option in the purification of biological compounds, requiring high purity degree. In addition, its application in biopharmaceutical manufacturing is well established. This paper discusses the importance and the progress of RNA isolation and purification, considering RNA applicability both in research and clinical fields. In particular and in view of the high specificity, affinity chromatography has been recently applied to RNA purification processes. Accordingly, recent chromatographic investigations based on biorecognition phenomena occurring between RNA and amino acids are focused. Histidine and arginine have been used as amino acid ligands, and their ability to isolate different RNA species demonstrated a multipurpose applicability in molecular biology analysis and RNA therapeutics preparation, highlighting the potential contribution of these methods to overcome the challenges of RNA purification. PMID:24951289

  3. Succinonitrile Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The Succinonitrile (SCN) Purification Facility provides succinonitrile and succinonitrile alloys to several NRA selected investigations for flight and ground research at various levels of purity. The purification process employed includes both distillation and zone refining. Once the appropriate purification process is completed, samples are characterized to determine the liquidus and/or solidus temperature, which is then related to sample purity. The lab has various methods for measuring these temperatures with accuracies in the milliKelvin to tenths of milliKelvin range. The ultra-pure SCN produced in our facility is indistinguishable from the standard material provided by NIST to well within the stated +/- 1.5mK of the NIST triple point cells. In addition to delivering material to various investigations, our current activities include process improvement, characterization of impurities and triple point cell design and development. The purification process is being evaluated for each of the four vendors to determine the efficacy of each purification step. We are also collecting samples of the remainder from distillation and zone refining for analysis of the constituent impurities. The large triple point cells developed will contain SCN with a melting point of 58.0642 C +/- 1.5mK for use as a calibration standard for Standard Platinum Resistance Thermometers (SPRTs).

  4. Purification of genuine multipartite entanglement

    SciTech Connect

    Huber, Marcus; Plesch, Martin

    2011-06-15

    In tasks where multipartite entanglement plays a central role, state purification is, due to inevitable noise, a crucial part of the procedure. We consider a scenario exploiting the multipartite entanglement in a straightforward multipartite purification algorithm and compare it to bipartite purification procedures combined with state teleportation. While complete purification requires an infinite amount of input states in both cases, we show that for an imperfect output fidelity the multipartite procedure exhibits a major advantage in terms of input states used.

  5. [Ingestion and digestion of seven species of microalgae by larvae of Strombus gigas (Mesogastropoda: Strombidae)].

    PubMed

    Patiño Súarez, V; Aldana Aranda, D

    2000-12-01

    The potential nutritional value of seven microalgal diets as measured by their ingestibility and digestibility to queen conch Strombus gigas larvae was tested with 30 day old larvae reared at 28 degrees C and fed at 1000 cells x ml(-1). The algae were Tetraselmis suecica, Tetraselmis chuii Isochrysis aff. galbana, Dunaliella tertiolecta, Chlamydomonas coccoides, Chaetoceros sp. and Thalassiosira fluviatilis. Ingestion and digestion were measured by the four nutritional stages studied with epifluorescence microscopy with live larvae. Temporal and absolute indices showed that larvae fed Chaetoceros sp. and T. fluviatilis had lower ingestion and digestion levels. The other algae are recommend to feed S. gigas larvae.

  6. [Ingestion and digestion of seven species of microalgae by larvae of Strombus gigas (Mesogastropoda: Strombidae)].

    PubMed

    Patiño Súarez, V; Aldana Aranda, D

    2000-12-01

    The potential nutritional value of seven microalgal diets as measured by their ingestibility and digestibility to queen conch Strombus gigas larvae was tested with 30 day old larvae reared at 28 degrees C and fed at 1000 cells x ml(-1). The algae were Tetraselmis suecica, Tetraselmis chuii Isochrysis aff. galbana, Dunaliella tertiolecta, Chlamydomonas coccoides, Chaetoceros sp. and Thalassiosira fluviatilis. Ingestion and digestion were measured by the four nutritional stages studied with epifluorescence microscopy with live larvae. Temporal and absolute indices showed that larvae fed Chaetoceros sp. and T. fluviatilis had lower ingestion and digestion levels. The other algae are recommend to feed S. gigas larvae. PMID:15266796

  7. Water purification in Borexino

    SciTech Connect

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

    2013-08-08

    Astroparticle Physics and Underground experiments searching for rare nuclear events, need high purity materials to act as detectors or detector shielding. Water has the advantage of being cheap, dense and easily available. Most of all, water can be purified to the goal of obatining a high level of radiopurity. Water Purification can be achieved by means of a combination of processes, including filtration, reverse osmosis, deionization and gas stripping. The Water Purification System for the Borexino experiment, will be described together with its main performances.

  8. Literature review of giant gartersnake (Thamnophis gigas) biology and conservation

    USGS Publications Warehouse

    Halstead, Brian J.; Wylie, Glenn D.; Casazza, Michael L.

    2015-01-01

    This report reviews the available literature on giant gartersnakes (Thamnophis gigas) to compile existing information on this species and identify knowledge gaps that, if addressed, would help to inform conservation efforts for giant gartersnakes.  Giant gartersnakes comprise a species of semi-aquatic snake precinctive to wetlands in the Central Valley of California.  The diversion of surface water and conversion of wetlands to agricultural and other land uses resulted in the loss of more than 90 percent of natural giant gartersnake habitats.  Because of this habitat loss, giant gartersnakes are now listed by the United States and California Endangered Species Acts as Threatened.  Most extant populations occur in the rice-growing regions of the Sacramento Valley, which comprises the northern portion of the giant gartersnake’s former range.  The huge demand for water in California for agriculture, industry, recreation, and other human consumption, combined with periodic severe drought, places remaining giant gartersnake habitats at increased risk of degradation and loss.  This literature review summarizes the available information on giant gartersnake distribution, habitat relations, behavior, demography, and other aspects of its biology relevant to conservation.  This information is then compiled into a graphical conceptual model that indicates the importance of different aspects of giant gartersnake biology for maintaining positive population growth, and identifies those areas for which important information relevant for conservation is lacking.  Directing research efforts toward these aspects of giant gartersnake ecology will likely result in improvements to conserving this unique species while meeting the high demands for water in California.

  9. Literature review of giant gartersnake (Thamnophis gigas) biology and conservation

    USGS Publications Warehouse

    Halstead, Brian J.; Wylie, Glenn D.; Casazza, Michael L.

    2015-08-03

    This report reviews the available literature on giant gartersnakes (Thamnophis gigas) to compile existing information on this species and identify knowledge gaps that, if addressed, would help to inform conservation efforts for giant gartersnakes.  Giant gartersnakes comprise a species of semi-aquatic snake precinctive to wetlands in the Central Valley of California.  The diversion of surface water and conversion of wetlands to agricultural and other land uses resulted in the loss of more than 90 percent of natural giant gartersnake habitats.  Because of this habitat loss, giant gartersnakes are now listed by the United States and California Endangered Species Acts as Threatened.  Most extant populations occur in the rice-growing regions of the Sacramento Valley, which comprises the northern portion of the giant gartersnake’s former range.  The huge demand for water in California for agriculture, industry, recreation, and other human consumption, combined with periodic severe drought, places remaining giant gartersnake habitats at increased risk of degradation and loss.  This literature review summarizes the available information on giant gartersnake distribution, habitat relations, behavior, demography, and other aspects of its biology relevant to conservation.  This information is then compiled into a graphical conceptual model that indicates the importance of different aspects of giant gartersnake biology for maintaining positive population growth, and identifies those areas for which important information relevant for conservation is lacking.  Directing research efforts toward these aspects of giant gartersnake ecology will likely result in improvements to conserving this unique species while meeting the high demands for water in California.

  10. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  11. The Global Invertebrate Genomics Alliance (GIGA): developing community resources to study diverse invertebrate genomes.

    PubMed

    Bracken-Grissom, Heather; Collins, Allen G; Collins, Timothy; Crandall, Keith; Distel, Daniel; Dunn, Casey; Giribet, Gonzalo; Haddock, Steven; Knowlton, Nancy; Martindale, Mark; Medina, Mónica; Messing, Charles; O'Brien, Stephen J; Paulay, Gustav; Putnam, Nicolas; Ravasi, Timothy; Rouse, Greg W; Ryan, Joseph F; Schulze, Anja; Wörheide, Gert; Adamska, Maja; Bailly, Xavier; Breinholt, Jesse; Browne, William E; Diaz, M Christina; Evans, Nathaniel; Flot, Jean-François; Fogarty, Nicole; Johnston, Matthew; Kamel, Bishoy; Kawahara, Akito Y; Laberge, Tammy; Lavrov, Dennis; Michonneau, François; Moroz, Leonid L; Oakley, Todd; Osborne, Karen; Pomponi, Shirley A; Rhodes, Adelaide; Santos, Scott R; Satoh, Nori; Thacker, Robert W; Van de Peer, Yves; Voolstra, Christian R; Welch, David Mark; Winston, Judith; Zhou, Xin

    2014-01-01

    Over 95% of all metazoan (animal) species comprise the "invertebrates," but very few genomes from these organisms have been sequenced. We have, therefore, formed a "Global Invertebrate Genomics Alliance" (GIGA). Our intent is to build a collaborative network of diverse scientists to tackle major challenges (e.g., species selection, sample collection and storage, sequence assembly, annotation, analytical tools) associated with genome/transcriptome sequencing across a large taxonomic spectrum. We aim to promote standards that will facilitate comparative approaches to invertebrate genomics and collaborations across the international scientific community. Candidate study taxa include species from Porifera, Ctenophora, Cnidaria, Placozoa, Mollusca, Arthropoda, Echinodermata, Annelida, Bryozoa, and Platyhelminthes, among others. GIGA will target 7000 noninsect/nonnematode species, with an emphasis on marine taxa because of the unrivaled phyletic diversity in the oceans. Priorities for selecting invertebrates for sequencing will include, but are not restricted to, their phylogenetic placement; relevance to organismal, ecological, and conservation research; and their importance to fisheries and human health. We highlight benefits of sequencing both whole genomes (DNA) and transcriptomes and also suggest policies for genomic-level data access and sharing based on transparency and inclusiveness. The GIGA Web site (http://giga.nova.edu) has been launched to facilitate this collaborative venture.

  12. The Global Invertebrate Genomics Alliance (GIGA): Developing Community Resources to Study Diverse Invertebrate Genomes

    PubMed Central

    2014-01-01

    Over 95% of all metazoan (animal) species comprise the “invertebrates,” but very few genomes from these organisms have been sequenced. We have, therefore, formed a “Global Invertebrate Genomics Alliance” (GIGA). Our intent is to build a collaborative network of diverse scientists to tackle major challenges (e.g., species selection, sample collection and storage, sequence assembly, annotation, analytical tools) associated with genome/transcriptome sequencing across a large taxonomic spectrum. We aim to promote standards that will facilitate comparative approaches to invertebrate genomics and collaborations across the international scientific community. Candidate study taxa include species from Porifera, Ctenophora, Cnidaria, Placozoa, Mollusca, Arthropoda, Echinodermata, Annelida, Bryozoa, and Platyhelminthes, among others. GIGA will target 7000 noninsect/nonnematode species, with an emphasis on marine taxa because of the unrivaled phyletic diversity in the oceans. Priorities for selecting invertebrates for sequencing will include, but are not restricted to, their phylogenetic placement; relevance to organismal, ecological, and conservation research; and their importance to fisheries and human health. We highlight benefits of sequencing both whole genomes (DNA) and transcriptomes and also suggest policies for genomic-level data access and sharing based on transparency and inclusiveness. The GIGA Web site (http://giga.nova.edu) has been launched to facilitate this collaborative venture. PMID:24336862

  13. The Global Invertebrate Genomics Alliance (GIGA): developing community resources to study diverse invertebrate genomes.

    PubMed

    Bracken-Grissom, Heather; Collins, Allen G; Collins, Timothy; Crandall, Keith; Distel, Daniel; Dunn, Casey; Giribet, Gonzalo; Haddock, Steven; Knowlton, Nancy; Martindale, Mark; Medina, Mónica; Messing, Charles; O'Brien, Stephen J; Paulay, Gustav; Putnam, Nicolas; Ravasi, Timothy; Rouse, Greg W; Ryan, Joseph F; Schulze, Anja; Wörheide, Gert; Adamska, Maja; Bailly, Xavier; Breinholt, Jesse; Browne, William E; Diaz, M Christina; Evans, Nathaniel; Flot, Jean-François; Fogarty, Nicole; Johnston, Matthew; Kamel, Bishoy; Kawahara, Akito Y; Laberge, Tammy; Lavrov, Dennis; Michonneau, François; Moroz, Leonid L; Oakley, Todd; Osborne, Karen; Pomponi, Shirley A; Rhodes, Adelaide; Santos, Scott R; Satoh, Nori; Thacker, Robert W; Van de Peer, Yves; Voolstra, Christian R; Welch, David Mark; Winston, Judith; Zhou, Xin

    2014-01-01

    Over 95% of all metazoan (animal) species comprise the "invertebrates," but very few genomes from these organisms have been sequenced. We have, therefore, formed a "Global Invertebrate Genomics Alliance" (GIGA). Our intent is to build a collaborative network of diverse scientists to tackle major challenges (e.g., species selection, sample collection and storage, sequence assembly, annotation, analytical tools) associated with genome/transcriptome sequencing across a large taxonomic spectrum. We aim to promote standards that will facilitate comparative approaches to invertebrate genomics and collaborations across the international scientific community. Candidate study taxa include species from Porifera, Ctenophora, Cnidaria, Placozoa, Mollusca, Arthropoda, Echinodermata, Annelida, Bryozoa, and Platyhelminthes, among others. GIGA will target 7000 noninsect/nonnematode species, with an emphasis on marine taxa because of the unrivaled phyletic diversity in the oceans. Priorities for selecting invertebrates for sequencing will include, but are not restricted to, their phylogenetic placement; relevance to organismal, ecological, and conservation research; and their importance to fisheries and human health. We highlight benefits of sequencing both whole genomes (DNA) and transcriptomes and also suggest policies for genomic-level data access and sharing based on transparency and inclusiveness. The GIGA Web site (http://giga.nova.edu) has been launched to facilitate this collaborative venture. PMID:24336862

  14. Effects of gamma irradiation on the yields of volatile extracts of Angelica gigas Nakai

    NASA Astrophysics Data System (ADS)

    Seo, Hye-Young; Kim, Jun-Hyoung; Song, Hyun-Pa; Kim, Dong-Ho; Byun, Myung-Woo; Kwon, Joog-Ho; Kim, Kyong-Su

    2007-11-01

    The study was carried out to determine the effects of gamma irradiation on the volatile flavor components including essential oils, of Angelica gigas Nakai. The volatile organic compounds from non- and irradiated A. gigas Nakai at doses of 1, 3, 5, 10 and 20 kGy were extracted by a simultaneous steam distillation and extraction (SDE) method and identified by GC/MS analysis. A total of 116 compounds were identified and quantified from non- and irradiated A. gigas Nakai. The major volatile compounds were identified 2,4,6-trimethyl heptane, α-pinene, camphene, α-limonene, β-eudesmol, α-murrolene and sphatulenol. Among these compounds, the amount of essential oils in non-irradiated sample were 77.13%, and the irradiated samples at doses of 1, 3, 5, 10 and 20 kGy were 84.98%, 83.70%, 83.94%, 82.84% and 82.58%, respectively. Oxygenated terpenes such as β-eudesmol, α-eudesmol, and verbenone were increased after irradiation but did not correlate with the irradiation dose. The yields of active substances such as essential oil were increased after irradiation; however, the yields of essential oils and the irradiation dose were not correlated. Thus, the profile of composition volatiles of A. gigas Nakai did not change with irradiation.

  15. Seawater temperature effect on metal accumulation and toxicity in the subantarctic Macquarie Island isopod, Exosphaeroma gigas.

    PubMed

    Lewis, Alexander; King, Catherine K; Hill, Nicole A; Cooper, Ashley; Townsend, Ashley T; Mondon, Julie A

    2016-08-01

    Very little is currently known of subantarctic nearshore invertebrates' sensitivity to environmental metals and the role of temperature in this relationship. This study investigated Cu and Zn toxicity in the common subantarctic intertidal isopod, Exosphaeroma gigas, and the influence of temperature on Cu toxicity and bioaccumulation kinetics. Adult E. gigas are insensitive to Cu and Zn at concentrations of 3200 and 7400μg/L respectively in non-renewal tests at 5.5°C (ambient subtidal temperature) over 14days. Under renewed exposures over the same temperature and time period the LC50 for copper was 2204μg/L. A 10-fold increase in Cu body burden occurred relative to zinc, indicating E. gigas has different strategies for regulating the two metals. Copper toxicity and time to mortality both increased with elevated temperature. However, temperature did not significantly affect Cu uptake rate and efflux rate constants derived from biodynamic modelling at lower Cu concentrations. These results may be attributable to E. gigas being an intertidal species with physiological mechanisms adapted to fluctuating environmental conditions. Cu concentrations required to elicit a toxicity response indicates that E. gigas would not be directly threatened by current levels of Cu or Zn present in Macquarie Island intertidal habitats, with the associated elevated temperature fluctuations. This study provides evidence that the sensitivity of this subantarctic intertidal species to metal contaminants is not as high as expected, and which has significance for the derivation of relevant guidelines specific to this distinct subpolar region of the world. PMID:27367827

  16. Fishery biology of jumbo flying squid Dosidicus gigas off Costa Rica Dome

    NASA Astrophysics Data System (ADS)

    Chen, Xinjun; Li, Jianghua; Liu, Bilin; Li, Gang; Lu, Huajie

    2014-06-01

    The jumbo flying squid ( Dosidicus gigas) population was surveyed with the help of Chinese squid jigging vessels off the Costa Rica Dome (4°-11°N, 90°-100°W) in 2009 and 2010. The daily catch of D. gigas in the two survey cruises ranged from 0 to 5.5 t and was mostly obtained from the areas bounded by 6°-9°N and 91°-94°W and by 6°30'-7°30'N and 96°-97°W. The sea surface temperature in the areas yielding the most catch ranged from 27.5 to 29°C. The sex ratio of the total catch was 3.75:1 (female: male). The mantle length of the squid ranged from 211 to 355 mm (male) and from 204 to 429 mm (female) with an average of 297.9 and 306.7 mm, respectively. In the relationship of the mantle length (mm) and body weight (g) of the squid, there was no significant difference between sexes. The female and male were at a similar maturity, and most individuals are maturing or have matured with a few females being spent. The size (mantle length) and age at the first sexual maturity were 297 mm and 195 d in females, and less than 211 mm and 130 d in males, respectively. Most of the sampled stomachs (70.6%) had no food remains. The major preys of the squids were fish, cephalopods and crustaceans, with the most abundant Myctophum orientale and D. gigas. The preys in more than 65% of the non-empty sampled stomachs evidenced the cannibalism of D. gigas. The results improved current understanding of the fishery biology of D. gigas off the Costa Rica Dome, which may facilitate the assessment and management of relative fishery resources.

  17. Purification, crystallization and preliminary X-ray diffraction of the N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/P{sub i} carrier SCaMC1

    SciTech Connect

    Yang, Qin Brüschweiler, Sven; Chou, James J.

    2013-12-24

    The N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/P{sub i} carrier SCaMC1 was crystallized in the presence of Ca{sup 2+}. X-ray diffraction data were collected to 2.9 Å resolution from crystals which belonged to space group P6{sub 2}22.

  18. Expression at 279 K, purification, crystallization and preliminary X-ray crystallographic analysis of a novel cold-active β-1,4-D-mannanase from the Antarctic springtail Cryptopygus antarcticus.

    PubMed

    Kim, Min-Kyu; An, Young Jun; Jeong, Chang-Sook; Song, Jung Min; Kang, Mee Hye; Lee, Youn-Ho; Cha, Sun-Shin

    2013-09-01

    The CaMan gene product from Cryptopygus antarcticus, which belongs to the glycoside hydrolase family 5 type β-1,4-D-mannanases, has been crystallized using a precipitant solution consisting of 0.1 M Tris-HCl pH 8.5, 25%(w/v) polyethylene glycol 3350 by the microbatch crystallization method at 295 K. The CaMan protein crystal belonged to space group P212121, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 61.29%. CaMan-mannopentaose (M5) complex crystals that were isomorphous to the CaMan crystals were obtained using the same mother liquor containing 1 mM M5. PMID:23989150

  19. Expression at 279 K, purification, crystallization and preliminary X-ray crystallographic analysis of a novel cold-active β-1,4-d-mannanase from the Antarctic springtail Cryptopygus antarcticus

    PubMed Central

    Kim, Min-Kyu; An, Young Jun; Jeong, Chang-Sook; Song, Jung Min; Kang, Mee Hye; Lee, Youn-Ho; Cha, Sun-Shin

    2013-01-01

    The CaMan gene product from Cryptopygus antarcticus, which belongs to the glycoside hydrolase family 5 type β-1,4-d-mannanases, has been crystallized using a precipitant solution consisting of 0.1 M Tris–HCl pH 8.5, 25%(w/v) polyethylene glycol 3350 by the microbatch crystallization method at 295 K. The CaMan protein crystal belonged to space group P212121, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 61.29%. CaMan–mannopentaose (M5) complex crystals that were isomorphous to the CaMan crystals were obtained using the same mother liquor containing 1 mM M5. PMID:23989150

  20. A Novel and Fast Purification Method for Nucleoside Transporters.

    PubMed

    Hao, Zhenyu; Thomsen, Maren; Postis, Vincent L G; Lesiuk, Amelia; Sharples, David; Wang, Yingying; Bartlam, Mark; Goldman, Adrian

    2016-01-01

    Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification. PMID:27376071

  1. A Novel and Fast Purification Method for Nucleoside Transporters

    PubMed Central

    Hao, Zhenyu; Thomsen, Maren; Postis, Vincent L. G.; Lesiuk, Amelia; Sharples, David; Wang, Yingying; Bartlam, Mark; Goldman, Adrian

    2016-01-01

    Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification. PMID:27376071

  2. Affinity Purification of Antibodies.

    PubMed

    Hnasko, Robert M; McGarvey, Jeffery A

    2015-01-01

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

  3. Innovative uses of GigaPan Technology for Onsite and Distance Education

    NASA Astrophysics Data System (ADS)

    Bentley, C.; Schott, R. C.; Piatek, J. L.; Richards, B.

    2013-12-01

    GigaPans are gigapixel panoramic images that can be viewed at a wide range of magnifications, allowing users to explore them in various degrees of detail from the smallest scale to the full image extent. In addition to panoramic images captured with the GigaPan camera mount ('Dry Falls' - http://www.gigapan.com/gigapans/89093), users can also upload annotated images (For example, 'Massanutten sandstone slab with trace fossils (annotated)', http://www.gigapan.com/gigapans/124295) and satellite images (For example, 'Geology vs. Topography - State of Connecticut', http://www.gigapan.com/gigapans/111265). Panoramas with similar topics have been gathered together on the site in galleries, both user-generated and site-curated (For example, http://www.gigapan.com/galleries?categories=geology&page=1). Further innovations in display technology have also led to the development of improved viewers (for example, the annotations in the image linked above can be explored via paired viewers at http://coursecontent.nic.edu/bdrichards/gigapixelimages/callanview) GigaPan panoramas can be created through use of the GigaPan robotic camera mount and a digital camera (different models of the camera mount are available and work with a wide range of cameras). The camera mount can be used to create high-resolution pans ranging in scale from hand sample to outcrop up to landscape via the stitching software included with the robotic mount. The software can also be used to generate GigaPan images from other sources, such as thin section or satellite images, so these images can also be viewed with the online viewer. GigaPan images are typically viewed via a web-based interface that allows the user to interact with the image from the limits of the image detail up to the full panorama. After uploading, information can be added to panoramas with both text captions and geo-referencing (geo-located panoramas can then be viewed in Google Earth). Users can record specific locations and zoom levels in

  4. Water Purification Systems

    NASA Technical Reports Server (NTRS)

    1992-01-01

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

  5. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

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

  6. Californium purification and electrodeposition

    SciTech Connect

    Burns, Jonathan D.; Van Cleve, Shelley M.; Smith, Edward Hamilton; Boll, Rose Ann

    2014-11-30

    The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of the feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO3 and HCl, and Eichrom TEVA resin in NH4SCN. The TEVA NH4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.

  7. Probabilistic theories with purification

    SciTech Connect

    Chiribella, Giulio; D'Ariano, Giacomo Mauro; Perinotti, Paolo

    2010-06-15

    We investigate general probabilistic theories in which every mixed state has a purification, unique up to reversible channels on the purifying system. We show that the purification principle is equivalent to the existence of a reversible realization of every physical process, that is, to the fact that every physical process can be regarded as arising from a reversible interaction of the system with an environment, which is eventually discarded. From the purification principle we also construct an isomorphism between transformations and bipartite states that possesses all structural properties of the Choi-Jamiolkowski isomorphism in quantum theory. Such an isomorphism allows one to prove most of the basic features of quantum theory, like, e.g., existence of pure bipartite states giving perfect correlations in independent experiments, no information without disturbance, no joint discrimination of all pure states, no cloning, teleportation, no programming, no bit commitment, complementarity between correctable channels and deletion channels, characterization of entanglement-breaking channels as measure-and-prepare channels, and others, without resorting to the mathematical framework of Hilbert spaces.

  8. Californium purification and electrodeposition

    DOE PAGES

    Burns, Jonathan D.; Van Cleve, Shelley M.; Smith, Edward Hamilton; Boll, Rose Ann

    2014-11-30

    The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of themore » feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO3 and HCl, and Eichrom TEVA resin in NH4SCN. The TEVA NH4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.« less

  9. Sclerochronology of Holocene oyster shells (Crassostrea gigas) from the West Coast of Bohai Sea, China

    NASA Astrophysics Data System (ADS)

    Fan, C.; Koeniger, P.; Wang, H.; Frechen, M.

    2009-04-01

    Sclerochronology, the study of periodic increments in skeletal organisms, can decipher the life history and environmental records preserved in fossil shells. Although there have been a number of studies that apply isotopic analyses to shells in open ocean and fresh water, investigations for brackish environments are rare. One of the common inhabitants in estuaries is the Crassostrea oyster. Kirby et al. (1998) demonstrated a close correspondence between the ligamental increments of convex and concave bands and yearly ^18O cycles; Andrus and Crowe (2000) found a close correspondence between translucent growth bands on the cross-section of the hinge and yearly ^18O cycles. They conclude that the morphological features on hinge and growth bands on the cross-section are formed annually and can be used to determine accurately age and growth rate in this species. However, Surge et al. (2001) could not find that these morphologic features have seasonal significance in the C. virginica shells. Therefore, these concave ridges are not reliable independent proxies of seasonality. These studies were carried out with C. virginica shells; none was studied with nature C. gigas, which was widely distributed along the Pacific coastal area. C. gigas has been introduced from its native home to all over the world, ranging from North America to Australia and Europe; it has become an important commercial harvest in many of these places. Buried Holocene oyster shells of C. gigas were sampled from a huge buried oyster reef on the West of Bohai Sea, China. One of these shells was selected for high resolution micro-sampling and stable isotope analyses testing the assumption that C. gigas ligamental increments are annual in nature. We analyzed 236 consecutive samples from the shell to show that morphologic features both on hinge and cross-section are annual by comparing them to the ^18O profiles. We tested the assumption that the morphologic features of C.gigas are delineated by convex tops

  10. Functionalized liposome purification via Liposome Extruder Purification (LEP).

    PubMed

    Alves, Nathan J; Cusick, William; Stefanick, Jared F; Ashley, Jonathan D; Handlogten, Michael W; Bilgicer, Basar

    2013-09-01

    Liposome Extruder Purification (LEP) allows for the rapid purification of diverse liposome formulations using the same extrusion apparatus employed during liposome formation. The LEP process provides a means for purifying functionalized liposomes from non-conjugated drug or protein contaminants with >93% liposome recovery and >93% contaminant removal in a single step.

  11. Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase

    PubMed Central

    2009-01-01

    Background Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available. Description In the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.html. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster. Conclusion A publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as

  12. Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas (Thunberg)

    NASA Astrophysics Data System (ADS)

    Wang, Yiyan; Sun, Hushan; Wang, Yanjie; Yan, Dongchun; Wang, Lei

    2015-03-01

    In this study, a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase, and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules (YGI, YGII, and YGIII) that differed in electron density and acid phosphatase reaction were identified in early cleavage, morula, blastula, gastrula, trochophore, and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation, indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI (free of acid phosphatase reaction) with YGII (rich in acid phosphatase reaction) could be the way by which yolk degradation is triggered.

  13. Attenuated reproduction of Strombus gigas by an Apicomplexa: Emeriidae-like parasite in the digestive gland.

    PubMed

    Baqueiro Cardenas, Erick; Montero, Jorge; Frenkiel, Liliane; Aldana Aranda, Dalila

    2012-07-01

    An intense and generalized sporozoan infection was detected in every population of the queen conch, Strombus gigas through the Caribbean. In this contribution we establish the relationship between occurrences of an Apicomplexa: Emeriidae-like organism and reproductive activity at San Andres archipelago, Colombia. Occurrence of the parasites was estimated counting the feeding stage Merozoites and cysts Sporozoites at 40× magnification. Nonmetric multidimensional scaling analysis (NMDS) was made to correlate the parasites stages abundance with frequency of the reproductive stages. Gametogenesis and spawning were always low coinciding with high numbers of Merozoites, a positive correlation was established between parasite abundance with reabsorption and undifferentiated stages, and negative correlation was observed between parasite abundance with maturity and spawning stages. The nonmetric multidimensional scaling (NMDS) shows that gametogenesis, maturity and spawning increase as the number of parasites decrease, factor that could be threatening reproduction of S. gigas through the Caribbean.

  14. Attenuated reproduction of Strombus gigas by an Apicomplexa: Emeriidae-like parasite in the digestive gland.

    PubMed

    Baqueiro Cardenas, Erick; Montero, Jorge; Frenkiel, Liliane; Aldana Aranda, Dalila

    2012-07-01

    An intense and generalized sporozoan infection was detected in every population of the queen conch, Strombus gigas through the Caribbean. In this contribution we establish the relationship between occurrences of an Apicomplexa: Emeriidae-like organism and reproductive activity at San Andres archipelago, Colombia. Occurrence of the parasites was estimated counting the feeding stage Merozoites and cysts Sporozoites at 40× magnification. Nonmetric multidimensional scaling analysis (NMDS) was made to correlate the parasites stages abundance with frequency of the reproductive stages. Gametogenesis and spawning were always low coinciding with high numbers of Merozoites, a positive correlation was established between parasite abundance with reabsorption and undifferentiated stages, and negative correlation was observed between parasite abundance with maturity and spawning stages. The nonmetric multidimensional scaling (NMDS) shows that gametogenesis, maturity and spawning increase as the number of parasites decrease, factor that could be threatening reproduction of S. gigas through the Caribbean. PMID:22484565

  15. Oxygen Sag and Stream Purification.

    ERIC Educational Resources Information Center

    Neal, Larry; Herwig, Roy

    1978-01-01

    Presents a literature review of water quality related to oxygen sag and stream purification, covering publications of 1976-77. This review includes: (1) self-purification models; (2) oxygen demand; and (3) reaeration and oxygen transfer. A list of 60 references is also presented. (HM)

  16. Process for purification of silicon

    NASA Technical Reports Server (NTRS)

    Rath, H. J.; Sirtl, E.; Pfeiffer, W.

    1981-01-01

    The purification of metallurgically pure silicon having a silicon content of more than 95% by weight is accomplished by leaching with an acidic solution which substantially does not attack silicon. A mechanical treatment leading to continuous particle size reduction of the granulated silicon to be purified is combined with the chemical purification step.

  17. Genome and Transcriptome Analyses Provide Insight into the Euryhaline Adaptation Mechanism of Crassostrea gigas

    PubMed Central

    Zhang, Linlin; Li, Chunyan; Li, Li; She, Zhicai; Huang, Baoyu; Zhang, Guofan

    2013-01-01

    Background The Pacific oyster, Crassostrea gigas, has developed special mechanisms to regulate its osmotic balance to adapt to fluctuations of salinities in coastal zones. To understand the oyster’s euryhaline adaptation, we analyzed salt stress effectors metabolism pathways under different salinities (salt 5, 10, 15, 20, 25, 30 and 40 for 7 days) using transcriptome data, physiology experiment and quantitative real-time PCR. Results Transcriptome data uncovered 189, 480, 207 and 80 marker genes for monitoring physiology status of oysters and the environment conditions. Three known salt stress effectors (involving ion channels, aquaporins and free amino acids) were examined. The analysis of ion channels and aquaporins indicated that 7 days long-term salt stress inhibited voltage-gated Na+/K+ channel and aquaporin but increased calcium-activated K+ channel and Ca2+ channel. As the most important category of osmotic stress effector, we analyzed the oyster FAAs metabolism pathways (including taurine, glycine, alanine, beta-alanine, proline and arginine) and explained FAAs functional mechanism for oyster low salinity adaptation. FAAs metabolism key enzyme genes displayed expression differentiation in low salinity adapted individuals comparing with control which further indicated that FAAs played important roles for oyster salinity adaptation. A global metabolic pathway analysis (iPath) of oyster expanded genes displayed a co-expansion of FAAs metabolism in C. gigas compared with seven other species, suggesting oyster’s powerful ability regarding FAAs metabolism, allowing it to adapt to fluctuating salinities, which may be one important mechanism underlying euryhaline adaption in oyster. Additionally, using transcriptome data analysis, we uncovered salt stress transduction networks in C. gigas. Conclusions Our results represented oyster salt stress effectors functional mechanisms under salt stress conditions and explained the expansion of FAAs metabolism pathways as

  18. Identification and functional characterization of two executioner caspases in Crassostrea gigas.

    PubMed

    Qu, Tao; Huang, Baoyu; Zhang, Linlin; Li, Li; Xu, Fei; Huang, Wen; Li, Chunyan; Du, Yishuai; Zhang, Guofan

    2014-01-01

    Caspase-3 and caspase-7 are two key effector caspases that play important roles in apoptotic pathways that maintain normal tissue and organ development and homeostasis. However, little is known about the sequence, structure, activity, and function of effector caspases upon apoptosis in mollusks, especially marine bivalves. In this study, we investigated the possible roles of two executioner caspases in the regulation of apoptosis in the Pacific oyster Crassostrea gigas. A full-length caspase-3-like gene named Cgcaspase-3 was cloned from C.gigas cDNA, encoding a predicted protein containing caspase family p20 and p10 domain profiles and a conserved caspase active site motif. Phylogenetic analysis demonstrated that both Cgcaspase-3 and Cgcaspase-1 may function as effector caspases clustered in the invertebrate branch. Although the sequence identities between the two caspases was low, both enzymes possessed executioner caspase activity and were capable of inducing cell death. These results suggested that Cgcaspase-3 and Cgcaspase-1 were two effector caspases in C. gigas. We also observed that nucleus-localized Cgcaspase-3, may function as a caspase-3-like protein and cytoplasm-localized Cgcaspase-1 may function as a caspase-7-like protein. Both Cgcaspase-3 and Cgcaspase-1 mRNA expression increased after larvae settled on the substratum, suggesting that both caspases acted in several tissues or organs that degenerated after oyster larvae settlement. The highest caspase expression levels were observed in the gills indicating that both effector caspases were likely involved in immune or metabolic processes in C. gigas. PMID:24551213

  19. Two-Step Vapor/Liquid/Solid Purification

    NASA Technical Reports Server (NTRS)

    Holland, L. R.

    1986-01-01

    Vertical distillation system combines in single operation advantages of multiple zone refining with those of distillation. Developed specifically to load Bridgman-Stockbarger (vertical-solidification) growth ampoules with ultrapure tellurium and cadmium, system, with suitable modifications, serves as material refiner. In first phase of purification process, ampoule heated to drive off absorbed volatiles. Second phase, evaporator heated to drive off volatiles in charge. Third phase, slowly descending heater causes distillation from evaporator to growing crystal in ampoule.

  20. Stanols as a tool to track the origin of microbial contamination of oysters, Crassostrea gigas, in shellfish areas.

    NASA Astrophysics Data System (ADS)

    Harrault, Loïc; Jardé, Emilie; Jeanneau, Laurent; Petitjean, Patrice

    2013-04-01

    Runoff of cattle manures (cows, pigs, sheeps) or discharge of effluent from wastewater treatment plants (WWTP) into aquatic ecosystems can lead to microbiological contamination of waters and living organisms. In coastal ecosystems and particularly in shellfish harvesting areas, the presence of pathogen microorganisms in waters induces fecal contamination of filter feeding bivalves (oysters, mussels, scallops…), therefore leading to human health risks associated to the consumption of these contaminated organisms. Watershed management plans that aim at limiting these risks require the development of tools able to identify fecal contamination sources. The fecal indicator bacteria used in the regulations to determine fecal contamination are not source specific since they are found in the feces of most warm-blooded animals. Thus, microbiological biomarkers have been developed in association with chemical biomarkers as Microbial Source Tracking (MST) methods. Fecal stanols, by-products of sterols obtained by human and animal microbial gut flora, are found in considerable amounts in feces with different relative proportions depending on their animal or human source. Recently, in association with microbiological biomarkers, the stanol fingerprint of contaminated waters has been successfully used to determine the main source of fecal contamination (cow, pig or human sources) in rural watersheds (Brittany, France). Up to now, the use of the stanol fingerprint to track the fecal contamination in shellfish tissues, especially bivalves, has been limited to the analysis of coprostanol, a stanol commonly associated to human contamination. Therefore, whether the stanol fingerprint can be used as a MST method in bivalves or not is still unknown. The first aim of this study was to compare several organic extraction procedures of stanols in the oyster Crassostrea gigas to determine a reliable method for stanol fingerprint analysis in bivalves. Solvent extraction and purification

  1. Expression, purification, crystallization, data collection and preliminary biochemical characterization of methicillin-resistant Staphylococcus aureus Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase

    SciTech Connect

    Seetharamappa, Jaldappagari; Oke, Muse; Liu, Huanting; McMahon, Stephen A.; Johnson, Kenneth A.; Carter, Lester; Dorward, Mark; Zawadzki, Michal; Overton, Ian M.; Niekirk, C. A. Johannes van; Graham, Shirley; Botting, Catherine H.; Taylor, Garry L.; White, Malcolm F.; Barton, Geoffrey J.; Coote, Peter J.; Naismith, James H.

    2007-05-01

    As part of work on S. aureus, the crystallization of Sar2028, a protein that is upregulated in MRSA, is reported. Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase with a molecular weight of 48 168 Da, was overexpressed in methicillin-resistant Staphylococcus aureus compared with a methicillin-sensitive strain. The protein was expressed in Escherichia coli, purified and crystallized. The protein crystallized in a primitive orthorhombic Laue group with unit-cell parameters a = 83.6, b = 91.3, c = 106.0 Å, α = β = γ = 90°. Analysis of the systematic absences along the three principal axes indicated the space group to be P2{sub 1}2{sub 1}2{sub 1}. A complete data set was collected to 2.5 Å resolution.

  2. Shell thickening and chambering in the oyster Crassostrea gigas: natural and anthropogenic influence of tributyltin contamination.

    PubMed

    Higuera-Ruiz, R; Elorza, J

    2011-04-01

    Abnormal thickening and chambering in Crassostrea gigas oysters have been adopted for many years as bioindicators of available tributyltin (TBT) in coastal waters. Nevertheless, since natural causes can also induce the formation of multiple chambers, a field study and laboratory experimentation has been conducted with 72 examples of C. gigas in successive culture media. This work has enabled differences to be established between natural fine sediment-induced characteristics and the influence of TBT on the shells. External shell deformities have been assessed using three biometric indices, shell thickness index, weight index and volume index. Internal differences have been observed in longitudinal sections of the shell: retraction of growth, stagnation of the adductor muscle scar and thinning of the chambers in the TBT-polluted shell secretion. A new index, the opening chambers index, has been proposed, with a value of less than 1 in the TBT-polluted environment and greater than 1 in shells secreted in an unpolluted production site. These conclusions should be borne in mind when C. gigas is used in biomonitoring programmes.

  3. Thermal stress induces a distinct transcriptome profile in the Pacific oyster Crassostrea gigas.

    PubMed

    Lim, Hyun-Jeong; Kim, Bo-Mi; Hwang, In Joon; Lee, Jeong-Soo; Choi, Ik-Young; Kim, Youn-Jung; Rhee, Jae-Sung

    2016-09-01

    Oysters are frequently subjected to heat stress during tidal emersion/immersion cycles in their habitats due to attachment on the rocky shore. To understand the effect of temperature elevation on the whole transcriptome over time, the Pacific oyster Crassostrea gigas was exposed to seawater temperature 32°C for 72h from the control 20°C. RNA-seq identified differentially expressed stress responsive transcripts upon thermal stress in the gill tissues of C. gigas. The primary effect of heat stress appears to be significantly induced transcription of molecular chaperones, including members of the heat shock protein (hsp) families, while genes typically associated with protein metabolism, such as those involved in protein degradation (e.g. ATP-dependent proteolysis pathway) and biosynthesis (e.g. ribosomal protein genes), were repressed. In particular, several hsp70 isoforms and a small hsp20 maintained prolonged mRNA expressions for 72h. This study provides preliminary insights into the molecular response of C. gigas to heat stress and suggests a basis for future studies examining molecular adaptation or thermotolerance metabolism in the Pacific oyster. PMID:27341139

  4. Functional analysis of Pacific oyster (Crassostrea gigas) β-thymosin: Focus on antimicrobial activity.

    PubMed

    Nam, Bo-Hye; Seo, Jung-Kil; Lee, Min Jeong; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Park, Nam Gyu

    2015-07-01

    An antimicrobial peptide, ∼5 kDa in size, was isolated and purified in its active form from the mantle of the Pacific oyster Crassostrea gigas by C18 reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionisation time-of-flight analysis revealed 4656.4 Da of the purified and unreduced peptide. A comparison of the N-terminal amino acid sequence of oyster antimicrobial peptide with deduced amino acid sequences in our local expressed sequence tag (EST) database of C. gigas (unpublished data) revealed that the oyster antimicrobial peptide sequence entirely matched the deduced amino acid sequence of an EST clone (HM-8_A04), which was highly homologous with the β-thymosin of other species. The cDNA possessed a 126-bp open reading frame that encoded a protein of 41 amino acids. To confirm the antimicrobial activity of C. gigas β-thymosin, we overexpressed a recombinant β-thymosin (rcgTβ) using a pET22 expression plasmid in an Escherichia coli system. The antimicrobial activity of rcgTβ was evaluated and demonstrated using a bacterial growth inhibition test in both liquid and solid cultures. PMID:25842181

  5. Changes in protein expression of pacific oyster Crassostrea gigas exposed in situ to urban sewage.

    PubMed

    Flores-Nunes, Fabrício; Gomes, Tânia; Company, Rui; Moraes, Roberta R M; Sasaki, Silvio T; Taniguchi, Satie; Bicego, Márcia C; Melo, Cláudio M R; Bainy, Afonso C D; Bebianno, Maria J

    2015-11-01

    The composition and concentration of substances in urban effluents are complex and difficult to measure. These contaminants elicit biological responses in the exposed organisms. Proteomic analysis is a powerful tool in environmental toxicology by evidencing alterations in protein expression due to exposure to contaminants and by providing a useful framework for the development of new potential biomarkers. The aim of this study was to determine changes in protein expression signatures (PES) in the digestive gland of oysters Crassostrea gigas transplanted to two farming areas (LIS and RIB) and to one area contaminated by sanitary sewage (BUC) after 14 days of exposure. This species is one of the most cultivated molluscs in the world. The identified proteins are related to the cytoskeleton (CKAP5 and ACT2), ubiquitination pathway conjugation (UBE3C), G protein-coupled receptor and signal transduction (SVEP1), and cell cycle/division (CCNB3). CKAP5 showed higher expression in oysters kept at BUC in comparison with those kept at the farming areas, while ACT2, UBE3C, SVEP1, and CCNB3 were suppressed. The results suggest that these changes might lead to DNA damage, apoptosis, and interference with the immune system in oyster C. gigas exposed to sewage and give initial information on PES of C. gigas exposed to sanitary sewage, which can subsequently be useful in the development of more sensitive tools for biomonitoring coastal areas, particularly those devoted mainly to oyster farming activities.

  6. Liquid Scintillator Purification

    SciTech Connect

    Kishimoto, Y.

    2005-09-08

    The KamLAND collaboration has studied background requirements and purification methods needed to observe the 7Be neutrino from the sun. First we will discuss the present background situation in KamLAND where it is found that the main background components are 210Pb and 85Kr. It is then described how to purify the liquid scintillator. The present status and results on how to remove 210Pb from the liquid scintillator are discussed. Specifically, the detailed analysis of the effects of distillation and adsorption techniques are presented.

  7. Protein production and purification

    PubMed Central

    2010-01-01

    In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus ‘what to try first’ strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators. PMID:18235434

  8. Air/Water Purification

    NASA Technical Reports Server (NTRS)

    1992-01-01

    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.

  9. Water Purification Product

    NASA Technical Reports Server (NTRS)

    2004-01-01

    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.

  10. Purification, crystallization and preliminary X-ray analysis of recombinant betaine aldehyde dehydrogenase 2 (OsBADH2), a protein involved in jasmine aroma, from Thai fragrant rice (Oryza sativa L.)

    PubMed Central

    Kuaprasert, Buabarn; Silprasit, Kun; Horata, Natharinee; Khunrae, Pongsak; Wongpanya, Ratree; Boonyalai, Nonlawat; Vanavichit, Apichart; Choowongkomon, Kiattawee

    2011-01-01

    Fragrant rice (Oryza sativa L.) betaine aldehyde dehydrogenase 2 (OsBADH2) is a key enzyme in the synthesis of fragrance aroma compounds. The extremely low activity of OsBADH2 in catalyzing the oxidation of acetaldehyde is believed to be crucial for the accumulation of the volatile compound 2-acetyl-1-pyrroline (2AP) in many scented plants, including fragrant rice. Recombinant fragrant rice OsBADH2 was expressed in Escherichia coli as an N-terminal hexahistidine fusion protein, purified using Ni Sepharose affinity chromatography and crystallized using the microbatch method. Initial crystals were obtained within 24 h using 0.1 M Tris pH 8.5 with 30%(w/v) PEG 4000 and 0.2 M magnesium chloride as the precipitating agent at 291 K. Crystal quality was improved when the enzyme was cocrystallized with NAD+. Improved crystals were grown in 0.1 M HEPES pH 7.4, 24%(w/v) PEG 4000 and 0.2 M ammonium chloride and diffracted to beyond 2.95 Å resolution after being cooled in a stream of N2 immediately prior to X-ray diffraction experiments. The crystals belonged to space group C2221, with unit-cell parameters a = 66.03, b = 183.94, c = 172.28 Å. An initial molecular-replacement solution has been obtained and refinement is in progress. PMID:22102032

  11. The Kinome of Pacific Oyster Crassostrea gigas, Its Expression during Development and in Response to Environmental Factors.

    PubMed

    Epelboin, Yanouk; Quintric, Laure; Guévélou, Eric; Boudry, Pierre; Pichereau, Vianney; Corporeau, Charlotte

    2016-01-01

    Oysters play an important role in estuarine and coastal marine habitats, where the majority of humans live. In these ecosystems, environmental degradation is substantial, and oysters must cope with highly dynamic and stressful environmental constraints during their lives in the intertidal zone. The availability of the genome sequence of the Pacific oyster Crassostrea gigas represents a unique opportunity for a comprehensive assessment of the signal transduction pathways that the species has developed to deal with this unique habitat. We performed an in silico analysis to identify, annotate and classify protein kinases in C. gigas, according to their kinase domain taxonomy classification, and compared with kinome already described in other animal species. The C. gigas kinome consists of 371 protein kinases, making it closely related to the sea urchin kinome, which has 353 protein kinases. The absence of gene redundancy in some groups of the C. gigas kinome may simplify functional studies of protein kinases. Through data mining of transcriptomes in C. gigas, we identified part of the kinome which may be central during development and may play a role in response to various environmental factors. Overall, this work contributes to a better understanding of key sensing pathways that may be central for adaptation to a highly dynamic marine environment. PMID:27231950

  12. New resources for marine genomics: bacterial artificial chromosome libraries for the Eastern and Pacific oysters (Crassostrea virginica and C. gigas).

    PubMed

    Cunningham, Charles; Hikima, Jun-ichi; Jenny, Matthew J; Chapman, Robert W; Fang, Guang-Chen; Saski, Chris; Lundqvist, Mats L; Wing, Rod A; Cupit, Pauline M; Gross, Paul S; Warr, Greg W; Tomkins, Jeff P

    2006-01-01

    Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at (www.genome.clemson.edu). PMID:16896533

  13. The Kinome of Pacific Oyster Crassostrea gigas, Its Expression during Development and in Response to Environmental Factors

    PubMed Central

    Epelboin, Yanouk; Quintric, Laure; Guévélou, Eric; Boudry, Pierre; Pichereau, Vianney; Corporeau, Charlotte

    2016-01-01

    Oysters play an important role in estuarine and coastal marine habitats, where the majority of humans live. In these ecosystems, environmental degradation is substantial, and oysters must cope with highly dynamic and stressful environmental constraints during their lives in the intertidal zone. The availability of the genome sequence of the Pacific oyster Crassostrea gigas represents a unique opportunity for a comprehensive assessment of the signal transduction pathways that the species has developed to deal with this unique habitat. We performed an in silico analysis to identify, annotate and classify protein kinases in C. gigas, according to their kinase domain taxonomy classification, and compared with kinome already described in other animal species. The C. gigas kinome consists of 371 protein kinases, making it closely related to the sea urchin kinome, which has 353 protein kinases. The absence of gene redundancy in some groups of the C. gigas kinome may simplify functional studies of protein kinases. Through data mining of transcriptomes in C. gigas, we identified part of the kinome which may be central during development and may play a role in response to various environmental factors. Overall, this work contributes to a better understanding of key sensing pathways that may be central for adaptation to a highly dynamic marine environment. PMID:27231950

  14. Repertoire and evolution of TNF superfamily in Crassostrea gigas: implications for expansion and diversification of this superfamily in Mollusca.

    PubMed

    Gao, Dahai; Qiu, Limei; Gao, Qiang; Hou, Zhanhui; Wang, Lingling; Song, Linsheng

    2015-08-01

    Tumor necrosis factor superfamily (TNFSF) members represent a group of cytokines participating in diverse immunological, pathological and developmental pathways. However, compared with deuterostomia and cnidaia, the composition and evolution of TNF homologous in protostomia are still not well understood. In the present study, a total of 81 TNF superfamily (TNFSF) genes from 15 mollusk species, including 23 TNFSF genes from Crassostrea gigas, were surveyed by genome-wide bioinformatics analysis. The phylogenetic analysis showed that 14 out of 23 C. gigas TNFSF genes in five clades exhibited orthologous relationships with Pinctada fucata TNFSF genes. Moreover, there were 15 C. gigas TNFSF genes located in oyster-specific clusters, which were contributed by small-scaled tandem and/or segmental duplication events in oyster. By comparing the sequences of duplicated TNFSF pairs, exon loss and variant in exon/intron length were revealed as the major modes of divergence in gene structure. Most of the duplicated C. gigas TNFSF pairs were evolved under purifying selection with consistent tissue expression patterns, implying functional constraint shaped diversification. This study demonstrated the expansion and early divergence of TNF superfamily in C. gigas, which provides potential insight into revealing the evolution and function of this superfamily in mollusk.

  15. Fluorescent Applications to Crystallization

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

    2006-01-01

    By covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and tests with model proteins have shown that labeling u to 5 percent of the protein molecules does not affect the X-ray data quality obtained . The presence of the trace fluorescent label gives a number of advantages. Since the label is covalently attached to the protein molecules, it "tracks" the protein s response to the crystallization conditions. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a darker background. Non-protein structures, such as salt crystals, do not show up under fluorescent illumination. Crystals have the highest protein concentration and are readily observed against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. Preliminary tests, using model proteins, indicates that we can use high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that more rapid amorphous precipitation kinetics may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Experiments are now being carried out to test this approach using a wider range, of proteins. The trace fluorescently labeled crystals will also

  16. Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490(963-1138) domain of TamB from Escherichia coli.

    PubMed

    Josts, Inokentijs; Grinter, Rhys; Kelly, Sharon M; Mosbahi, Khedidja; Roszak, Aleksander; Cogdell, Richard; Smith, Brian O; Byron, Olwyn; Walker, Daniel

    2014-09-01

    TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963-1138 domain of TamB was overexpressed in Escherichia coli K-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space group P3121, with unit-cell parameters a = b = 57.34, c = 220.74 Å, and diffracted to 2.1 Å resolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future.

  17. Purification, crystallization and preliminary X-ray analysis of an HA17–HA70 (HA2–HA3) complex from Clostridium botulinum type C progenitor toxin

    PubMed Central

    Iwasa, Chikako; Tonozuka, Takashi; Shinoda, Masaya; Sagane, Yoshimasa; Niwa, Koichi; Watanabe, Toshihiro; Yoshida, Hiromi; Kamitori, Shigehiro; Takao, Toshifumi; Oguma, Keiji; Nishikawa, Atsushi

    2014-01-01

    The haemagglutinin (HA) complex of Clostridium botulinum type C toxin is composed of three types of subcomponents: HA33, HA17 and HA70 (also known as HA1, HA2 and HA3, respectively). Here, a 260 kDa HA17–HA70 complex was crystallized. His-tagged HA17 and maltose-binding-protein-tagged HA70 were expressed in Escherichia coli and their complex was affinity-purified using a combination of amylose resin chromatography and nickel–nitrilotri­acetic acid agarose chromatography. Diffraction data were collected to 8.0 Å resolution and the crystal belonged to the tetragonal space group P41212. The molecular-replacement solution indicated that one molecule of HA17 was bound to each HA70 monomer. PMID:24419620

  18. Purification, crystallization and preliminary X-ray diffraction analysis of the human major histocompatibility antigen HLA-B*2703 complexed with a viral peptide and with a self-peptide

    SciTech Connect

    Loll, Bernhard; Biesiadka, Jacek; Saenger, Wolfram

    2005-04-01

    The product of the human leukocyte antigen (HLA) gene HLA-B*2703 differs from that of the prototypical subtype HLA-B*2705 by a single amino acid at heavy-chain residue 59 that is involved in anchoring the peptide N-terminus within the A pocket of the molecule. Two B*2703–peptide complexes were crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as a precipitant. A pocket of the molecule, two HLA-B*2703–peptide complexes were crystallized and data sets were collected to high resolution using synchrotron radiation. The product of the human leukocyte antigen (HLA) gene HLA-B*2703 differs from that of the prototypical subtype HLA-B*2705 by a single amino acid at heavy-chain residue 59 that is involved in anchoring the peptide N-terminus within the A pocket of the molecule. Two B*2703–peptide complexes were crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as a precipitant. The crystals belong to space group P2{sub 1} (pVIPR peptide) or P2{sub 1}2{sub 1}2{sub 1} (pLMP2 peptide). Data sets were collected to 1.55 Å (B*2703–pVIPR) or 2.0 Å (B*2703–pLMP2) resolution using synchrotron radiation. With B*2705–pVIPR as a search model, a clear molecular-replacement solution was found for both B*2703 complexes.

  19. Purification, crystallization and preliminary crystallographic analysis of the ligand-binding regions of the PctA and PctB chemoreceptors from Pseudomonas aeruginosa in complex with amino acids

    PubMed Central

    Rico-Jiménez, Miriam; Muñoz-Martínez, Francisco; Krell, Tino; Gavira, Jose A.; Pineda-Molina, Estela

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen and one of the major model organisms for the study of chemotaxis. The bacterium harbours 26 genes encoding chemoreceptors, most of which have not been annotated with a function. The paralogous chemoreceptors PctA and PctB (Pseudomonas chemotactic transducer A and B) were found to mediate chemotaxis towards l-amino acids. However, the ligand spectrum of the receptors is quite different since the recombinant ligand-binding region (LBR) of PctA binds 17 different l-­amino acids whereas that of PctB recognizes only five. To determine the molecular basis underlying this ligand specificity, PctA-LBR and PctB-LBR have been purified and crystals have been produced after pre-incubation with l-­Ile and l-Arg, respectively. Initial crystallization conditions have been identified by the counter-diffusion method and X-ray data have been collected at 2.5 Å (PctA-LBR bound to l-Ile) and 3.14 Å (PctB-LBR bound to l-Arg) resolution. Crystals belonged to space groups P212121 and P3121, with unit-cell parameters a = 72.2, b = 78.5, c = 116.6 Å and a = b = 111.6, c = 117.4, respectively, for PctA-LBR and PctB-LBR. Molecular-replacement methods will be pursued for structural determination. PMID:24316847

  20. Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG.

    PubMed

    Chaurasia, Priyanka; von Ossowski, Ingemar; Palva, Airi; Krishnan, Vengadesan

    2015-01-01

    SpaD is the predicted backbone-pilin subunit of the SpaFED pilus, whose loci are encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG, a Gram-positive gut-adapted commensal strain with perceived probiotic benefits. In this study, soluble recombinant SpaD protein was overproduced in Escherichia coli and then purified by Ni2+-chelating affinity and gel-filtration chromatography. After limited proteolysis with α-chymotrypsin, good-quality crystals of SpaD were obtained which diffracted beyond 2.0 Å resolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=50.11, b=83.27, c=149.65 Å. For phasing, sodium iodide-derivatized crystals were prepared using the halide quick-soaking method and diffraction data were collected in-house to a resolution of 2.2 Å. An interpretable electron-density map was successfully obtained using single-wavelength anomalous diffraction (SAD).