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Sample records for gigas purification crystallization

  1. Purification and characterization of trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas).

    PubMed

    Fu, Xue-Yan; Xue, Chang-Hu; Miao, Ben-Chun; Liang, Jun-Ni; Li, Zhao-Jie; Cui, Feng-Xia

    2006-02-01

    Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM. PMID:16448210

  2. 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.

  3. 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

  4. Purification of a Multidrug Resistance Transporter for Crystallization Studies.

    PubMed

    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. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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. PMID:25700885

  10. 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…

  11. Purification, crystallization and preliminary X-ray analysis of struthiocalcin 1 from ostrich (Struthio camelus) eggshell

    SciTech Connect

    Reyes-Grajeda, Juan Pablo; Marín-García, Liliana; Stojanoff, Vivian; Moreno, Abel

    2007-11-01

    The purification, crystallization and preliminary X-ray diffraction data of the protein struthiocalcin 1 isolated from ostrich eggshell are reported. The purification, crystallization and preliminary X-ray analysis of struthiocalcin 1 (SCA-1), a protein obtained from the intramineral part of ostrich (Struthio camelus) eggshell, is reported.

  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. Modified purification of mercuric iodide for crystal growth

    NASA Astrophysics Data System (ADS)

    Skinner, N. L.; Ortale, C.; Schieber, M. M.; Vandenberg, L.

    The standard procedure used in our laboratory to purify commercially available mercuric iodide consists of a sequence of steps: (1) repeated sublimation under continous evacuation, followed by (2) melting and recrystallization, and (3) a sublimation process in a closed tube. This paper describes a modification of the standard purification sequence by adding recrystallization of the mercuric iodide in hydrochloric acid. Leaching cation impurities out of mercuric iodide powder with hydrochloric acid has been practiced before by Zaletin, (V.M. Zaletin, I.H. Nozhiua, I.N. Fomin, V.T. Shystov, and N.V. Protasov, Atomic Energy 48, 169 (1980)). Our objective for the hydrochloric acid treatment was to remove nitrates and hydrocarbons which were interfering with the vapor transport during crystal growth. Results of the procedure are presented in terms of total carbon and selected ion content of the treated and untreated material.

  14. Modified purification of mercuric iodide for crystal growth

    NASA Astrophysics Data System (ADS)

    Skinner, N. L.; Ortale, C.; Schieber, M. M.; Van Den Berg, L.

    1988-06-01

    The standard procedure used in our laboratory to purify commercially available mercuric iodide (HgI 2) consists of a sequence of steps: (1) repeated sublimation under continuous evacuation, followed by (2) melting and recrystallization, and (3) a sublimation process in a closed tube. This paper describes a modification of the standard purification sequence by adding recrystallization of the HgI 2 in hydrochloric acid. Leaching cation impurities out of HgI 2 powder with hydrochloric acid has been practised before by Zaletin et al. Our objective for the hydrochloric acid treatment was to remove nitrates and hydrocarbons which were interfering with the vapor transport during crystal growth. Results of the procedure are presented in terms of total carbon and selected ion content of the treated and untreated material.

  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. 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.

  17. 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.

  18. 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.

  19. Preparation of group I introns for biochemical studies and crystallization assays by native affinity purification.

    PubMed

    Vicens, Quentin; Gooding, Anne R; Duarte, Luis F; Batey, Robert T

    2009-01-01

    The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides -- some containing exons -- were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3' overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules. PMID:19710925

  20. 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.

  1. 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

  2. Purification, sequence and crystallization of an anti-tissue factor Fab and its use for the crystallization of tissue factor

    NASA Astrophysics Data System (ADS)

    Ruf, Wolfram; Stura, Enrico A.; LaPolla, Robert J.; Syed, Rashid; Edgington, Thomas S.; Wilson, Ian A.

    1992-08-01

    The crystallization of proteins as Fab-antigen complexes may be advantageous in the crystallization of biologically important proteins, since many different monoclonal antibodies are often available against a given protein. From each of these, Fab fragments can be generated providing new opportunities for crystallization of the protein as a complex. Here we report the screening and identification of a suitable Fab for the crystallization of the soluble extracellular domain of tissue factor, the receptor responsible for the cellular initiation of the coagulation protease cascade. From six specific Fabs, three have been identified that crystallize readily as free Fabs. The refinement of the purification procedure for one of these Fabs (TF8-5G9) was required to provide material which reliably formed complex crystals with the tissue factor extracellular domain. A further improvement in the quality of the complex crystals was achieved by enzymatic removal of sialic acid from tissue factor resulting in a significant reduction of its charge heterogeneity.

  3. 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.

  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. 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.

  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. Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

    PubMed Central

    Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

    2011-01-01

    Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the

  8. 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.

  9. 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.

  10. Purification and characterization of pea seedling amine oxidase for crystallization studies.

    PubMed Central

    McGuirl, M A; McCahon, C D; McKeown, K A; Dooley, D M

    1994-01-01

    Pea (Pisum sativum L.) seedling amine oxidase (EC 1.4.3.6) is the first amine oxidase to be crystallized that diffracts to atomic resolution (2.5 A). Extensive modifications of a published purification procedure were necessary to obtain protein that would give diffraction-quality crystals. Here we report the improved purification and also use this high-purity protein to reexamine some fundamental characteristics of pea seedling amine oxidase. The extinction coefficient at 280 nm (epsilon 1%(280)) and the molecular mass of the protein are investigated by a variety of techniques, yielding epsilon 1%(280) = 20 cm-1 and a mass 150 +/- 6 kD. In addition, the stoichiometry of the metal and organic cofactors, Cu(II) and 6-hydroxy dopa (Topa) quinone, respectively, is examined. The ratio of Cu(II):Topa:protein monomer is found to be 1:1:1. PMID:7824646

  11. 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.

  12. Purification, crystallization and preliminary X-ray structure analysis of the laccase from Ganoderma lucidum

    PubMed Central

    Lyashenko, Andrey V.; Belova, Oksana; Gabdulkhakov, Azat G.; Lashkov, Alexander A.; Lisov, Alexandr V.; Leontievsky, Alexey A.; Mikhailov, Al’bert M.

    2011-01-01

    The ligninolytic enzymes of the basidiomycetes play a key role in the global carbon cycle. A characteristic property of these enzymes is their broad substrate specificity, which has led to their use in various biotechnologies, thus stimulating research into the three-dimensional structures of ligninolytic enzymes. This paper presents the purification, crystallization and preliminary X-ray analysis of the laccase from the ligninolytic basidiomycete Ganoderma lucidum. PMID:21821897

  13. 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.

  14. 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.

  15. 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.

  16. Purification, crystallization and preliminary crystallographic analysis of banyan peroxidase

    PubMed Central

    Sharma, Anurag; Palm, Gottfried J.; Kumari, Moni; Panjikar, Santosh; Jagannadham, M. V.; Hinrichs, Winfried

    2012-01-01

    Plant peroxidases are extensively used in a wide range of biotechnological applications owing to their high environmental and thermal stability. A new peroxidase, named banyan peroxidase, was purified from the latex of Ficus benghalensis and crystallized. X-ray diffraction data were collected from native crystals and from bromide and xenon derivatives to resolutions of up to 1.66 Å in the trigonal space group P3221, with unit-cell parameters a = b = 73.1, c = 164.6 Å. The anomalous signal of the intrinsic iron and calcium ions was sufficient for structure solution by SAD, although the sequence is not yet known. PMID:22869125

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. Purification, crystallization and preliminary crystallographic analysis of mare lactoferrin.

    PubMed

    Sharma, A K; Kathikeyan, S; Kaur, P; Singh, T P; Yadav, M P

    1996-11-01

    Lactoferrin is an iron-binding glycoprotein with a molecular weight of 80 kDa. The protein has two iron binding sites. It has two structural lobes, each housing one Fe(3+) and the synergistic CO(3)(2-) ion. The protein was isolated from the colostrum/milk of mares maintained at National Research Centre on Equines, Hisar, India. The purified samples of the protein were crystallized using a microdialysis method. The protein was dialysed against low ionic strength buffer solution. Several crystal forms were obtained, out of which three were characterized which have cell dimensions as follows. Form I a = 79.8, b = 103.5, c = 112.0 A, space group P2(1)2(1)2(1), with one protein molecule per asymmetric unit and a solvent content of 57%. Form II a = 84.9, b = 99.7, c = 103.5 A, space group P2(1)2(1)2(1) with one molecule per asymmetric unit and a solvent content of 55%. Form III a = 151.0, b = 151.0, c = 240.6 A, space group P4(1)2(1)2 with three molecules in the asymmetric unit and a solvent content of 57%. The intensity data up to 3.8 A resolution for form I, 2.9 A resolution data for form II and 6 A resolution data for form III have been collected. Further calculations are in progress. PMID:15299585

  2. Purification, characterization, and crystallization of Crocodylus siamensis hemoglobin.

    PubMed

    Jandaruang, Jinda; Siritapetawee, Jaruwan; Songsiriritthigul, Chomphunuch; Preecharram, Sutthidech; Azuma, Taoka; Dhiravisit, Apisak; Fukumori, Yoshihiro; Thammasirirak, Sompong

    2014-08-01

    Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P(50)) and 1.96 (n value), and a small Bohr effect (δH(+) = -0.29) at a pH of 6.9-8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O(2) affinity at P(50) of 2.5 mmHg which may assure efficient utilization of the lung O(2) reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10-13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl(2)·6H(2)O] solution at a pH of 7.0-8.5. PMID:24928538

  3. 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.

  4. Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase.

    PubMed

    Chesterman, Chelsy; Jia, Zongchao

    2016-09-01

    Escherichia coli tyrosine kinase (Etk) is a membrane bound kinase in gram-negative bacteria that regulates the export of capsular polysaccharides (CPS). The molecular mechanism behind CPS regulation remains unclear, despite access to a crystal structure of the cytoplasmic kinase domain of Etk. In this study, an efficient protocol to produce full length Etk solubilized in n-dodecyl-β-d-maltoside has been established with high yield. We have determined that detergent solubilized Etk retains kinase activity, but the protein is prone to aggregation, degradation, and has been unsuccessful in protein crystallization trials. In response, we designed and characterized truncations of Etk that do not aggregate and have led to successful crystallization experiments. In this article, we discuss our optimized expression and purification protocol for Etk, the design of Etk protein truncations, and the behavior of Etk during purification in a range of stabilizing detergents. These efforts have successfully produced protein suitable for crystallization. Our results will be a useful guide for future structural and functional studies of the bacterial tyrosine kinase family. PMID:26363120

  5. 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.

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

    PubMed

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

    2005-06-01

    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 A resolution have been obtained at 277 K by the hanging-drop method. PMID:16511103

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

    PubMed Central

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

    2005-01-01

    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. PMID:16511103

  8. 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.

  9. Purification, crystallization and preliminary X-ray diffraction analysis of a fungal saponin-detoxifying enzyme.

    PubMed

    Bamford, Vicki A; Kolade, Ola O; Osbourn, Anne E; Hemmings, Andrew M

    2004-07-01

    Tomatinase, an extracellular enzyme belonging to family 3 of the glycosyl hydrolases, is produced by the fungal tomato-leaf pathogen Septoria lycopersici and detoxifies the saponin alpha-tomatine. An efficient strategy for purification of the enzyme from fungal culture medium has been developed. Single crystals have been grown by vapour diffusion at 289 K from 17.5%(w/v) PEG 4K, 5%(v/v) 2-propanol and 0.1 M sodium acetate pH 4.5 as precipitant. When cryoprotected at 100 K, these crystals diffract to at least 3.0 A and belong to space group P2(1)2(1)2. Based on an estimated molecular weight of 110 kDa for the glycosylated protein and assuming two molecules in the asymmetric unit, the crystals contain approximately 46% solvent. PMID:15213407

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Expression, purification, crystallization and crystallographic analysis of the N-terminal domain of translocated intimin receptor.

    PubMed

    Huang, Bing Yang; Gu, Jiang; Zhang, Yan Fang; Zhou, Jun Jun; Song, Xiao Yong; Lin, Yi; Li, Xin Min; Li, Lu

    2016-01-01

    Translocated intimin receptor (Tir) is an Escherichia coli-encoded protein that is transported into the host cell through a sophisticated bacterial type III secretion system (T3SS). Tir anchors the infected cell membrane twice using both its N- and C-termini from inside the host cytoplasm for signalling. It plays a key role in enterohemorrhagic Escherichia coli (EHEC) infection, attaching and effacing (A/E) lesions and intracellular signal transduction. Here, the overexpression, purification and crystallization of its N-terminal intracellular domain are reported. The crystal belonged to the orthorhombic space group I4122, with unit-cell parameters a = b = 59.79, c = 183.11 Å. The asymmetric unit contained one molecule, with a solvent content of 51% and a VM of 2.55 Å(3) Da(-1). PMID:26750484

  17. Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR).

    PubMed

    Rosenberg, Mark F; Kamis, Alhaji Bukar; Aleksandrov, Luba A; Ford, Robert C; Riordan, John R

    2004-09-10

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active. Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states. These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein. In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter. The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered. PMID:15247233

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Switching between purification and contamination regimes governed by the ionic purity of nanoparticles dispersed in liquid crystals

    NASA Astrophysics Data System (ADS)

    Garbovskiy, Yuriy

    2016-03-01

    This paper reports non-trivial effects of the ionic purity of nanoparticles on the concentration of ions in liquid crystals. Nanoparticles dispersed in liquid crystals can affect the concentration of mobile ions in different ways. 100% pure nanoparticles can only decrease the concentration of ions by means of adsorption/desorption processes. Liquid crystals doped with contaminated nanoparticles exhibit three regimes, namely, the purification, contamination, and no change in the concentration of ions. Switching between these regimes is governed by three dominant factors: the purity of liquid crystals, the purity of nanoparticles, and the ratio of the adsorption rate to the desorption rate.

  4. Furanocoumarins from Dorstenia gigas.

    PubMed

    Franke, K; Porzel, A; Masaoud, M; Adam, G; Schmidt, J

    2001-03-01

    A series of linear and angular prenylated furanocoumarins and a benzofuran derivative were isolated from leaves and twigs of Dorstenia gigas (Moraceae), a plant occurring endemically on Socotra Island (Yemen). The structures were elucidated by spectroscopic methods (NMR, MS, UV) and chemical derivatization. PMID:11281139

  5. 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.

  6. 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.

  7. Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization.

    PubMed

    Kober, Daniel L; Yurtsever, Zeynep; Brett, Thomas J

    2015-01-01

    Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case. PMID:26780656

  8. Expression, purification, crystallization and preliminary crystallographic analysis of human myotubularin-related protein 3

    PubMed Central

    Son, Ji Young; Lee, Jee Un; Yoo, Ki-Young; Shin, Woori; Im, Dong-Won; Kim, Seung Jun; Ryu, Seong Eon; Heo, Yong-Seok

    2014-01-01

    Myotubularin-related proteins are a large family of phosphatases that have the catalytic activity of dephosphorylating the phospholipid molecules phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate. Each of the 14 family members contains a phosphatase catalytic domain, which is inactive in six family members owing to amino-acid changes in a key motif for the activity. All of the members also bear PH-GRAM domains, which have low homologies between them and have roles that are not yet clear. Here, the cloning, expression, purification and crystallization of human myotubularin-related protein 3 encompassing the PH-GRAM and the phosphatase catalytic domain are reported. Preliminary X-ray crystallographic analysis shows that the crystals diffracted to 3.30 Å resolution at a synchrotron X-ray source. The crystals belonged to space group C2, with unit-cell parameters a = 323.3, b = 263.3, c = 149.4 Å, β = 109.7°. PMID:25195900

  9. 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.

  10. Cloning, purification, crystallization and preliminary X-ray studies of HMO2 from Saccharomyces cerevisiae.

    PubMed

    Guo, Zhen; Zhang, Shaocheng; Zhang, Hongpeng; Jin, Li; Zhao, Shasha; Yang, Wei; Tang, Jian; Wang, Deqiang

    2014-01-01

    The high-mobility group protein (HMO2) of Saccharomyces cerevisiae is a component of the chromatin-remodelling complex INO80, which is involved in double-strand break (DSB) repair. HMO2 can also bind DNA to protect it from exonucleolytic cleavage. Nevertheless, little structural information is available regarding these functions of HMO2. Since determination of three-dimensional structure is a powerful means to facilitate functional characterization, X-ray crystallography has been used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of HMO2 from S. cerevisiae are reported. The crystal belonged to space group P222, with unit-cell parameters a = 39.35, b = 75.69, c = 108.03 Å, and diffracted to a resolution of 3.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a VM value of 3.19 Å(3) Da(-1). PMID:24419618

  11. 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.

  12. Application of the effects of ionic strength reducing agents in the purification and crystallization of chitinase A.

    PubMed

    Papanikolau, Yannis; Petratos, Kyriacos

    2002-10-01

    The effects of ionic strength reducing agents may find a large number of applications. Based on these effects, we have redesigned the purification scheme of Chitinase A (ChiA) from Serratia marcescen. This scheme led to reproducibly crystallizable enzyme in both salting-in and salting-out conditions, which are presented here. Herein, we demonstrate some experimental applications of the ionic strength reducing agents theory and, in parallel, provide further evidence of the theory's correctness. Finally, we report a new crystal form produced recently in salting-in crystallization experiments. This form may allow the co-crystallization of ChiA mutants with longer substrates. PMID:12351868

  13. 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

  14. 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

  15. 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.

  16. Recombinant expression, purification, and crystallization of the glutaminyl-tRNA synthetase from Toxoplasma gondii.

    PubMed

    van Rooyen, Jason M; Hakimi, Mohamed-Ali; Belrhali, Hassan

    2015-06-01

    Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous Escherichia coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural-functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex. PMID:25736594

  17. The purification and preparation of high-purity PbCl 2 and ternary alkali lead chloride single crystals

    NASA Astrophysics Data System (ADS)

    Nitsch, K.; Cihlář, A.; Málková, Z.; Rodová, M.; Vaněček, M.

    1993-08-01

    Purification and crystal growth of PbCl 2 and congruently melting ternary alkali lead chlorides, KPb 2Cl 5, RbPbCl 3, RbPb 2Cl 5, and CsPbCl 3, will be described in the present paper. Source materials were purified by the chlorination of molten chlorides with gaseous mixture of chlorinating agents in combination with a subsequent zone-melting. The efficiency of this method is confirmed by chemical analysis before and after purification and by the behaviour of the melts. Large single crystals of PbCl 2, KPb 2Cl 5, RbPb 2Cl 5 and CsPbCl 3, 20 mm in diameter and 50-60 mm long, were grown by the Bridgman method. Their quality was checked by the optical absorption and elastic light scattering measurements.

  18. Purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3].

    PubMed

    Kubiak, Xavier; Pluvinage, Benjamin; Li de la Sierra-Gallay, Inès; Weber, Patrick; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2012-02-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3], a putative NAT isoenzyme that possesses a unique catalytic triad containing a glutamate residue, is reported. The crystal diffracted to 2.42 Å resolution and belonged to the monoclinic space group C121, with unit-cell parameters a = 90.44, b = 44.52, c = 132.98 Å, β = 103.8°. PMID:22297998

  19. 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

  20. Expression, purification, and crystallization of Schizosaccharomyces pombe eIF2B.

    PubMed

    Kashiwagi, Kazuhiro; Shigeta, Tomoaki; Imataka, Hiroaki; Ito, Takuhiro; Yokoyama, Shigeyuki

    2016-03-01

    Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution. PMID:27023709

  1. 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

  2. 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.

  3. Expression, purification, crystallization and preliminary diffraction analysis of CapF, a capsular polysaccharide-synthesis enzyme from Staphylococcus aureus.

    PubMed

    Miyafusa, Takamitsu; Tanaka, Yoshikazu; Kuroda, Makoto; Ohta, Toshiko; Tsumoto, Kouhei

    2008-06-01

    Capsular polysaccharides (CPs) are important virulence factors of Staphylococcus aureus. The biosynthesis of type 5 and type 8 CPs (CP5 and CP8), which are produced by most clinical isolates of S. aureus, is catalyzed by 16 CP-assembling proteins. One of these proteins is the enzyme CapF, which catalyzes the synthesis of UDP-N-acetyl-L-fucosamine, a component of both CP5 and CP8. Here, the cloning, expression, purification, crystallization and diffraction analysis of CapF are reported. Optimization of the crystallization conditions by differential scanning calorimetry afforded a crystal of selenomethionine-substituted CapF that diffracted to a resolution of 2.80 A. The crystal belongs to space group P3(2)21, with unit-cell parameters a = b = 119.6, c = 129.5 A. PMID:18540063

  4. 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.

  5. 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.

  6. 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)

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

    SciTech Connect

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

    2006-04-01

    The expression, purification and crystallization of a viral protease from the blotched snakehead virus (BSNV) are described and the diffraction data collected to 2.5 Å from two different crystal forms are reported. 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 Å. 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 Å.

  8. 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%.

  9. Purification, crystallization and preliminary crystallographic analysis of the CBS-domain protein MJ1004 from Methanocaldococcus jannaschii

    PubMed Central

    Oyenarte, Iker; Lucas, María; Gómez García, Inmaculada; Martínez-Cruz, Luis Alfonso

    2011-01-01

    The purification and preliminary crystallographic analysis of the archaeal CBS-domain protein MJ1004 from Methanocaldococcus jannaschii are described. The native protein was overexpressed, purified and crystallized in the monoclinic space group P21, with unit-cell parameters a = 54.4, b = 53.8, c = 82.6 Å, β = 106.1°. The crystals diffracted X-rays to 2.7 Å resolution using synchrotron radiation. Matthews-volume calculations suggested the presence of two molecules in the asymmetric unit that are likely to correspond to a dimeric species, which is also observed in solution. PMID:21393835

  10. 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

  11. 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.

  12. 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 .

  13. Optimized Purification of a Heterodimeric ABC Transporter in a Highly Stable Form Amenable to 2-D Crystallization

    PubMed Central

    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 (kcat∼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

  14. 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

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

    PubMed Central

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

    2014-01-01

    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

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

    SciTech Connect

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

    2006-03-01

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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. Purification and crystallization of Phd, the antitoxin of the phd/doc operon

    PubMed Central

    Garcia-Pino, Abel; Sterckx, Yann; Vandenbussche, Guy; Loris, Remy

    2010-01-01

    The antitoxin Phd from the phd/doc module of bacteriophage P1 was crystallized in two distinct crystal forms. Crystals of His-tagged Phd contain a C-terminally truncated version of the protein and diffract to 2.20 Å resolution. Crystals of untagged Phd purified from the Phd–Doc complex diffract to 2.25 Å resolution. These crystals are partially merohedrally twinned and contain the full-length version of the protein. PMID:20124714

  5. 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

  6. Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

    PubMed Central

    Klinglmayr, Eva; Wenger, Julia; Mayr, Sandra; Bossy-Wetzel, Ella; Puehringer, Sandra

    2012-01-01

    The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P21212, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis. PMID:23027751

  7. 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.

  8. 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.

  9. Purification, crystallization and preliminary X-ray crystallographic analysis of the CIDE-N domain of Fsp27.

    PubMed

    Wang, Xiaodan; Zhang, Bo; Xu, Duo; Gao, Jinlan; Wang, Linfang; Wang, Zhi; Shan, Yaming; Yu, Xianghui

    2012-12-01

    Fsp27, a member of the CIDE protein family which is selectively expressed in adipocytes, has emerged as a novel regulator for unilocular lipid droplet (LD) formation, lipid metabolism, differentiation of adipocytes and insulin sensitivity. An LD is a subcellular compartment that is used by adipocytes for the efficient storage of fats. The CIDE-N domain of Fsp27 functions as a recruitment platform that induces the correct configuration of the Fsp27 CIDE-C domain to facilitate LD fusion. This study reports the high-yield expression of the mouse Fsp27 CIDE-N domain in Escherichia coli; a two-step purification protocol with high efficiency was established and crystallographic analysis was performed. The purity of the recombinant Fsp27 was >95% as assessed by SDS-PAGE. Crystals were obtained at 291 K using 28% polyethylene glycol 4000 as a precipitant. Diffraction data were collected to 1.92 Å resolution and the crystal belonged to space group P6(5), with unit-cell parameters a=b=63.3, c=37.4 Å, α=β=90, γ=120°. The components of the crystal were identified by ion-trap LC/MS/MS spectrometric analysis. The structure has been solved by molecular replacement and refinement is in progress. PMID:23192040

  10. 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.

  11. 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.

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

    PubMed

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

    2014-03-01

    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 I4122 and cubic P213. 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(3) Da(-1) and a solvent content of 73.23%. PMID:24598926

  13. Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine β-synthase from Apis mellifera

    PubMed Central

    Oyenarte, Iker; Majtan, Tomas; Ereño, June; Corral-Rodríguez, María Angeles; Klaudiny, Jaroslav; Majtan, Juraj; Kraus, Jan P.; Martínez-Cruz, Luis Alfonso

    2012-01-01

    Cystathionine β-synthase (CBS) is a pyridoxal-5′-phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinuria, a multisystemic disease affecting to various extents the vasculature, connective tissues and central nervous system. At present, the crystal structure of CBS from Drosophila melanogaster is the only available structure of the full-length enzyme. Here we describe a cloning, overexpression, purification and preliminary crystallographic analysis of a full-length CBS from Apis mellifera (AmCBS) which maintains 51 and 46% sequence identity with its Drosophila and human homologs, respectively. The AmCBS yielded crystals belonging to space group P212121, with unit-cell parameters a = 85.90, b = 95.87, c = 180.33 Å. Diffraction data were collected to a resolution of 3.0 Å. The crystal structure contained two molecules in the asymmetric unit which presumably correspond to the dimeric species observed in solution. PMID:23143241

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein.

    PubMed

    Rahaman, Siti Nurulnabila A; Mat Yusop, Jastina; Mohamed-Hussein, Zeti-Azura; Ho, Kok Lian; Teh, Aik-Hong; Waterman, Jitka; Ng, Chyan Leong

    2016-03-01

    C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%. PMID:26919524

  19. 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.

  20. Expression, purification, crystallization and preliminary crystallographic analysis of human Rad GTPase

    SciTech Connect

    Yanuar, Arry; Sakurai, Shigeru; Kitano, Ken; Hakoshima, Toshio

    2005-11-01

    Human Rad has been crystallized. A diffraction data set was collected to a resolution of 1.8 Å. Human Rad is a new member of the Ras GTPase superfamily and is overexpressed in human skeletal muscle of individuals with type II diabetes. The GTPase core domain was overexpressed in Escherichia coli and purified for crystallization. Crystals were obtained at 293 K by vapour diffusion using a crystallization robot. The crystals were found to belong to space group P2{sub 1}, with unit-cell parameters a = 52.2, b = 58.6, c = 53.4 Å, β = 97.9°, and contained two Rad molecules in the crystallographic asymmetric unit. A diffraction data set was collected to a resolution of 1.8 Å using synchrotron radiation at SPring-8.

  1. 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°.

  2. Purification, crystallization and preliminary crystallographic analysis of the CBS pair of the human metal transporter CNNM4

    PubMed Central

    Gómez García, Inmaculada; Oyenarte, Iker; Martínez-Cruz, Luis Alfonso

    2011-01-01

    This work describes the purification and preliminary crystallographic analysis of the CBS-pair regulatory domain of the human ancient domain protein 4 (ACDP4), also known as CNNM4. ACDP proteins represent the least-studied members of the eight different types of magnesium transporters that have been identified in mammals to date. In humans the ACDP family includes four members: CNNM1–4. CNNM1 acts as a cytosolic copper chaperone and has been associated with urofacial syndrome, whereas CNNM2 and CNNM4 have been identified as magnesium transporters. Interestingly, mutations in the CNNM4 gene have clinical consequences that are limited to retinal function and biomineralization and are considered to be the cause of Jalili syndrome, which consists of autosomal recessive cone-rod dystrophy and amelogenesis imperfecta. The truncated protein was overexpressed, purified and crystallized in the orthorhombic space group C222. The crystals diffracted X-rays to 3.6 Å resolution using synchrotron radiation. Matthews volume calculations suggested the presence of two molecules in the asymmetric unit, which were likely to correspond to a CBS module of the CBS pair of CNNM4. PMID:21393841

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26423580

  16. 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.

  17. 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.

  18. 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.

  19. Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus

    SciTech Connect

    Tsukazaki, Tomoya; Mori, Hiroyuki; Fukai, Shuya; Numata, Tomoyuki; Perederina, Anna; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo; Vassylyev, Dmitry G.; Nureki, Osamu; Ito, Koreaki

    2006-04-01

    SecDF is a multi-path membrane protein required for efficient protein translocation and integration via translocon. Purification and crystallization of T. thermophilus SecDF have been achieved by exploiting unique crystallization techniques that allowed the collection of a 3.74 Å data set. Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 Å upon X-ray irradiation, revealing that they belonged to space group P4{sub 3}2{sub 1}2. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 Å resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery.

  20. 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%. PMID:25372826

  1. 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.

  2. 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

  3. Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate

    SciTech Connect

    Inaoka, Daniel Ken; Takashima, Eizo; Osanai, Arihiro; Shimizu, Hironari; Nara, Takeshi; Aoki, Takashi; Harada, Shigeharu; Kita, Kiyoshi

    2005-10-01

    The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate. Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R{sub merge} of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V{sub M}) of 2.2 Å{sup 3} Da{sup −1} and a solvent content of 44%.

  4. 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.

  5. 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.

  6. 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. PMID:21139221

  7. 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.

  8. 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.

  9. 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.

  10. Purification, crystallization and preliminary X-ray diffraction analysis of the glyoxalase II from Leishmania infantum

    SciTech Connect

    Trincão, José; Sousa Silva, Marta; Barata, Lídia; Bonifácio, Cecília; Carvalho, Sandra; Tomás, Ana Maria; Ferreira, António E. N.; Cordeiro, Carlos; Ponces Freire, Ana; Romão, Maria João

    2006-08-01

    A glyoxalase II from L. infantum was cloned, purified and crystallized and its structure was solved by X-ray crystallography. In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C222{sub 1} (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 Å) and diffract beyond 2.15 Å using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypano-somatids.

  11. Purification, Crystallization and Preliminary Diffraction Studies of the Sulfolobus solfataricu PCNA Proteins in Different Oligomeric Forms

    SciTech Connect

    Guangxin,X.; Vladena, H.; Hong, L.

    2007-01-01

    PCNA is a ring-shaped protein that encircles DNA and is essential for DNA metabolism, including DNA replication and repair. PCNA is either a homotrimer in eukaryotes and euryarchaeotes or a heterotrimer in some crenarchaeotes. The crenarchaeon Sulfolobus solfataricus encodes three PCNA homologues (PCNA1, PCNA2, and PCNA3). PCNA1 and PCNA2 form a stable dimer. The dimer then recruits PCNA3 to form the trimeric ring-shaped molecule that is typical for all PCNA proteins. We crystallized the PCNA3 monomer, the PCNA1-PCNA2 heterodimer, and the PCNA1-PCNA2-PCNA3 heterotrimer. The crystals diffract X-ray to 1.9, 2.6, and 2.5 Angstroms resolutions, respectively. SAD phasing and molecular replacement solutions have confirmed that the crystals do contain the corresponding monomer, dimer, and trimer.

  12. Purification, Crystallization and Preliminary Diffraction Studies of the Sulfolobus solfataricus PCNA Proteins in Different Oligomeric Forms

    SciTech Connect

    Xing,G.; Hlinkova, V.; Ling, H.

    2007-01-01

    PCNA is a ring-shaped protein that encircles DNA and is essential for DNA metabolism, including DNA replication and repair. PCNA is either a homotrimer in eukaryotes and euryarchaeotes or a heterotrimer in some crenarchaeotes. The crenarchaeon Sulfolobus solfataricus encodes three PCNA homologues (PCNA1, PCNA2, and PCNA3). PCNA1 and PCNA2 form a stable dimer. The dimer then recruits PCNA3 to form the trimeric ring-shaped molecule that is typical for all PCNA proteins. We crystallized the PCNA3 monomer, the PCNA1-PCNA2 heterodimer, and the PCNA1-PCNA2-PCNA3 heterotrimer. The crystals diffract X-ray to 1.9, 2.6, and 2.5 Angstroms resolutions, respectively. SAD phasing and molecular replacement solutions have confirmed that the crystals do contain the corresponding monomer, dimer, and trimer.

  13. 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.

  14. 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.

  15. 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.

  16. Purification, crystallization and preliminary X-ray analysis of Hsp33 from Saccharomyces cerevisiae

    PubMed Central

    Liu, Wei; Zhou, Ye-Yun; Teng, Mai-Kun; Zhou, Cong-Zhao

    2007-01-01

    The heat-shock protein Hsp33 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. A crystal was obtained using the hanging-drop vapour-diffusion method and a data set was collected to 2.7 Å resolution. The crystal belongs to space group P43212, with unit-cell parameters a = b = 96.43, c = 132.22 Å, α = β = γ = 90°. The asymmetric unit is assumed to contain two subunits of Hsp33, with a V M value of 2.96 Å3 Da−1 and a solvent content of 58.41%. PMID:17277453

  17. 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.

  18. 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.

  19. Purification, Crystallization, and Preliminary Crystallographic Analysis of Deoxyuridine Triphosphate Nucleotidohydrolase from Arabidopsis Thaliana

    SciTech Connect

    Bajaj,M.; Moriyama, H.

    2007-01-01

    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 Angstroms resolution using Cu K{alpha} radiation. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 69.90, b = 70.86 Angstroms, c = 75.55 Angstroms . Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a VM of 1.8 Angstroms 3 Da-1.

  20. Purification, crystallization and preliminary crystallographic analysis of peroxidase from the palm tree Chamaerops excelsa.

    PubMed

    Textor, Larissa C; Santos, Jademilson C; Cuadrado, Nazaret Hidalgo; Roig, Manuel G; Zhadan, Galina G; Shnyrov, Valery L; Polikarpov, Igor

    2011-12-01

    Plant peroxidases are presently used extensively in a wide range of biotechnological applications owing to their high environmental and thermal stability. As part of efforts towards the discovery of appealing new biotechnological enzymes, the peroxidase from leaves of the palm tree Chamaerops excelsa (CEP) was extracted, purified and crystallized in its native form. An X-ray diffraction data set was collected at a synchrotron source and data analysis showed that the CEP crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 70.2, b = 100.7, c = 132.3 Å. PMID:22139187

  1. 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. PMID:19342775

  2. Expression, purification, crystallization and preliminary X-ray analysis of Aeromonas hydrophilia metallo-[beta]-lactamase

    SciTech Connect

    Sharma, N.; Toney, J.H.; Fitzgerald, P.M.D.

    2010-07-20

    The CphA metallo-{beta}-lactamase from Aeromonas hydrophilia has been expressed, purified and crystallized by the hanging-drop vapor-diffusion method using ammonium sulfate as the precipitant. The crystals exhibit orthorhombic symmetry (P2{sub 1}2{sub 1}2), with unit-cell parameters a = 40.75, b = 42.05, c = 128.88 {angstrom}. There is one monomer in the asymmetric unit and the solvent content is estimated to be 44% by volume. A data set extending to 1.8 {angstrom} has been measured.

  3. 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

  4. Purification, crystallization and preliminary crystallographic analysis of peroxidase from the palm tree Chamaerops excelsa

    PubMed Central

    Textor, Larissa C.; Santos, Jademilson C.; Hidalgo Cuadrado, Nazaret; Roig, Manuel G.; Zhadan, Galina G.; Shnyrov, Valery L.; Polikarpov, Igor

    2011-01-01

    Plant peroxidases are presently used extensively in a wide range of bio­technological applications owing to their high environmental and thermal stability. As part of efforts towards the discovery of appealing new biotechnological enzymes, the peroxidase from leaves of the palm tree Chamaerops excelsa (CEP) was extracted, purified and crystallized in its native form. An X-­ray diffraction data set was collected at a synchrotron source and data analysis showed that the CEP crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 70.2, b = 100.7, c = 132.3 Å. PMID:22139187

  5. 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

  6. 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.

  7. Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate.

    PubMed

    Inaoka, Daniel Ken; Takashima, Eizo; Osanai, Arihiro; Shimizu, Hironari; Nara, Takeshi; Aoki, Takashi; Harada, Shigeharu; Kita, Kiyoshi

    2005-10-01

    Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD-orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 A3 Da(-1) and a solvent content of 44%. PMID:16511183

  8. 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.

  9. 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.

  10. Purification, crystallization and preliminary crystallographic analysis of cytochrome P450 203A1 from Rhodopseudomonas palustris

    SciTech Connect

    Pang, Xiaoyun; Xu, Feng; Bell, Stephen G.; Guo, Delin; Wong, Luet-Lok; Rao, Zihe

    2007-04-01

    The cytochrome P450 enzyme CYP203A1 from Rhodopseudomonas palustris binds a wide range of highly substituted aromatic compounds and may play an important role in the astonishing metabolic diversity of this organism. Crystals of CYP203A1 that diffract to 2.0 Å resolution have been obtained. Cytochrome P450 enzymes constitute a large family of haemoproteins that catalyze the monooxygenation of a great variety of endogenous and exogenous organic compounds. Cytochrome P450 203A1 (CYP203A1, RPA1009) from the metabolically versatile organism Rhodopseudomonas palustris binds a broad range of substrates, in particular substituted aromatic compounds. Crystals of CYP203A1 suitable for X-ray crystallography have been obtained and diffraction data were collected in-house to 2.0 Å resolution from a single crystal. The crystals belong to space group P222, with unit-cell parameters a = 40.1, b = 95.1, c = 99.0 Å, α = β = γ = 90°. There is one protein molecule per asymmetric unit.

  11. Purification, crystallization and preliminary crystallographic studies on 2-dehydro-3-deoxygalactarate aldolase from Leptospira interrogans

    SciTech Connect

    Li, Xu; Huang, Hua; Song, Xiaomin; Wang, Yanli; Xu, Hang; Teng, Maikun; Gong, Weimin

    2006-12-01

    Preliminary crystallographic studies on 2-dehydro-3-deoxygalactarate aldolase from L. interrogans. 2-Dehydro-3-deoxygalactarate (DDG) aldolase is a member of the class II aldolase family and plays an important role in the pyruvate-metabolism pathway, catalyzing the reversible aldol cleavage of DDG to pyruvate and tartronic semialdehyde. As it is a potential novel antibiotic target, it is necessary to elucidate the catalytic mechanism of DDG aldolase. To determine the crystal structure, crystals of DDG aldolase from Leptospira interrogans were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution using a Cu Kα rotating-anode X-ray source. The crystal belonged to space group C2, with unit-cell parameters a = 293.5, b = 125.6, c = 87.6 Å, β = 100.9°. The V{sub M} is calculated to be 2.4 Å{sup 3} Da{sup −1}, assuming there to be 12 protein molecules in the asymmetric unit.

  12. 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).

  13. Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

    SciTech Connect

    Brzezinski, Krzysztof; Bujacz, Grzegorz; Jaskolski, Mariusz

    2008-07-01

    Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4{sub 3}2{sub 1}2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified. Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation.

  14. 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.

  15. Purification and crystallization of mono-ubiquitylated ubiquitin receptor Rpn10.

    PubMed

    Keren-Kaplan, Tal; Prag, Gali

    2012-09-01

    Protein ubiquitylation controls nearly all cellular pathways in eukaryotes. A repertoire of proteins named ubiquitin (Ub) receptors harbouring ubiquitin-binding domains (UBDs) recognize ubiquitylated proteins. These Ub receptors decode the Ub signal by tethering a UBD or UBDs to a functional domain or domains, thus linking the ubiquitylated target to a specific function. The rapid dynamics of ubiquitylation/deubiquitylation has impeded the characterization of ubiquitylated proteins. To bypass this obstacle, a recently developed synthetic system that reconstructs the entire eukaryotic ubiquitylation cascade in Escherichia coli was used to purify the mono-ubiquitylated form of the regulatory proteasomal non-ATPase subunit (Ub-Rpn10) from Saccharomyces cerevisiae. Here, the first crystallization and data collection of Ub-Rpn10 is reported. Purified Ub-Rpn10 was crystallized in 12%(w/v) PEG 20,000, 0.1 M MES pH 6.5 and yielded thin rhombus-shaped crystals. X-ray analysis revealed that these crystals belonged to the monoclinic system C2, with unit-cell parameters a = 107.3, b = 49.7, c = 81.3 Å, α = γ = 90.0, β = 130.5°. A full synchrotron data set has been collected, merged and scaled with a diffraction limit of 3.14 Å. PMID:22949210

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Cloning, purification, crystallization and X-ray crystallographic analysis of Ignicoccus hospitalis neelaredoxin.

    PubMed

    Pinho, Filipa G; Romão, Célia V; Pinto, Ana F; Saraiva, Lígia M; Huber, Harald; Matias, Pedro M; Teixeira, Miguel; Bandeiras, Tiago M

    2010-05-01

    Superoxide reductases (SORs) are metalloproteins which constitute the most recently identified oxygen-detoxification system in anaerobic and microaerobic bacteria and archaea. SORs are involved in scavenging superoxide radicals from the cell by catalyzing the reduction of superoxide ({\\rm O}_{2};{\\bullet -}) to hydrogen peroxide and are characterized by a catalytic nonhaem iron centre coordinated by four histidine ligands and one cysteine ligand. Ignicoccus hospitalis, a hyperthermophilic crenarchaeon, is known to have a neelaredoxin-type SOR that keeps toxic oxygen species levels under control. Blue crystals of recombinant I. hospitalis oxidized neelaredoxin (14.1 kDa, 124 residues) were obtained. These crystals diffracted to 2.4 A resolution in-house at room temperature and belonged to the hexagonal space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 108, c = 64 A. Cell-content analysis indicated the presence of one monomer in the asymmetric unit. PMID:20445270

  3. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6

    PubMed Central

    Morita, Junko; Kato, Kazuki; Mihara, Emiko; Ishitani, Ryuichiro; Takagi, Junichi; Nishimasu, Hiroshi; Aoki, Junken; Nureki, Osamu

    2014-01-01

    Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8 Å resolution. The crystal belonged to space group P1, with unit-cell parameters a = 63.7, b = 68.8, c = 69.7 Å, α = 60.6, β = 87.0, γ = 68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%. PMID:24915096

  4. 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.

  5. 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}.

  6. 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}.

  7. Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum

    PubMed Central

    Tylichová, Martina; Briozzo, Pierre; Kopečný, David; Ferrero, Julien; Moréra, Solange; Joly, Nathalie; Snégaroff, Jacques; Šebela, Marek

    2008-01-01

    Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Amino­aldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 Å resolution at 100 K. Crystals belong to the monoclinic space group P21, with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 Å, β = 98.1°. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit. PMID:18259056

  8. Purification of Germanium Crystals by Zone Refinement: Theoretical and Experimental Approaches

    NASA Astrophysics Data System (ADS)

    Yang, Gang; Guan, Yutong; Wang, Guojian; Mei, Hao; Jian, Fanyi; Mei, Dongming; Crystal Growth Team

    2015-03-01

    The results of single germanium crystals grown from zone-refined germanium ingots, identified by photon thermal ionization spectroscopy (PTIS), show that there are four main impurities, aluminum (Al), phosphor (P), boron (B) and gallium (Ga) in the crystals. Based the PTIS results, we investigated the influences of zone speed, zone width and the number of passes on effective segregation coefficient of Al, P and Ga in the process of zone refinement, then the further calculation of distribution of Al, P and Ga along the zone refined ingots has been conducted. In terms of trend of impurity distribution, the calculated results have a very good agreement with the experimental results. We report both the theoretical calculations and the experimental results. This work is supported by DOE grant DE-FG02-10ER46709 and the state of South Dakota.

  9. 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

  10. Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus

    PubMed Central

    Pendini, Nicole R.; Polyak, Steve W.; Booker, Grant W.; Wallace, John C.; Wilce, Matthew C. J.

    2008-01-01

    Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 Å resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P42212, with unit-cell parameters a = b = 93.665, c = 131.95. PMID:18540065

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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%.

  19. Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum

    SciTech Connect

    Kwon, Soo-Young; Kang, Beom Sik; Kim, Ghyung-Hwa; Kim, Kyung-Jin

    2007-11-01

    PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 94.72, c = 359.68 Å, γ = 120°. The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.

  20. Expression, purification and preliminary X-ray analysis of crystals of Bacillus subtilis glutamate racemase.

    PubMed

    Taal, Makie A; Sedelnikova, Svetlana E; Ruzheinikov, Sergey N; Baker, Patrick J; Rice, David W

    2004-11-01

    Glutamate racemase (MurI, RacE; E.C.5.1.1.3) catalyses the cofactor-independent conversion of L-glutamate to D-glutamate, an essential step in the synthesis of components of the bacterial cell wall. The gene for RacE from Bacillus subtilis has been cloned and the protein expressed in Escherichia coli, purified and crystallized in the presence of L-glutamate using the hanging-drop method of vapour diffusion with diammonium tartrate as the precipitant. The crystals belong to the monoclinic space group C2, with approximate unit-cell parameters a = 133.6, b = 60.1, c = 126.2 A, beta = 117.6 degrees . Consideration of the possible values of V(M) suggests that the asymmetric unit contains either two (V(M) = 3.75 A(3) Da(-1)) or three (V(M) = 2.5 A(3) Da(-1)) subunits. The crystals diffract X-rays to at least 2.1 A resolution on a synchrotron-radiation source and are suitable for structural studies. Determination of the structure may provide insight into the molecular basis of substrate recognition and catalysis by this enzyme. PMID:15502318

  1. 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.

  2. Purification, crystallization and preliminary X-ray crystallographic analysis of the nucleocapsid protein of Bunyamwera virus

    SciTech Connect

    Rodgers, John W.; Zhou, Qingxian; Green, Todd J.; Barr, John N.; Luo, Ming

    2006-04-01

    The nucleocapsid protein of Bunyamwera virus, the prototypic member of the Bunyaviridae family of segmented negative-sense RNA viruses, has been expressed and crystallized. Complete X-ray diffraction data sets have been collected. Bunyamwera virus (BUNV) is the prototypic member of the Bunyaviridae family of segmented negative-sense RNA viruses. The BUNV nucleocapsid protein has been cloned and expressed in Escherichia coli. The purified protein has been crystallized and a complete data set has been collected to 3.3 Å resolution at a synchrotron source. Crystals of the nucleocapsid protein belong to space group C2, with unit-cell parameters a = 384.7, b = 89.8, c = 89.2 Å, β = 94.4°. Self-rotation function analysis of the X-ray diffraction data has provided insight into the oligomeric state of the protein as well as the orientation of the oligomers in the asymmetric unit. The structure determination of the protein is ongoing.

  3. 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.

  4. 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.

  5. 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.

  6. Expression, Purification, Crystallization and Preliminary X-ray Analysis of Pseudomonas fluorescens AlgK

    SciTech Connect

    Keiski,C.; Yip, P.; Robinson, H.; Burrows, L.; Howell, P.

    2007-01-01

    AlgK is an outer-membrane lipoprotein involved in the biosynthesis of alginate in Pseudomonads and Azotobacter vinelandii. A recombinant form of Pseudomonas fluorescens AlgK with a C-terminal polyhistidine affinity tag has been expressed and purified from the periplasm of Escherichia coli cells and diffraction-quality crystals of AlgK have been grown using the hanging-drop vapour-diffusion method. The crystals grow as flat plates with unit-cell parameters a = 79.09, b = 107.85, c = 119.15 {angstrom}, = 96.97{sup o}. The crystals exhibit the symmetry of space group P2{sub 1} and diffract to a minimum d-spacing of 2.5 {angstrom} at Station X29 of the National Synchrotron Light Source, Brookhaven National Laboratory. On the basis of the Matthews coefficient (V{sub M} = 2.53 {angstrom}{sup 3} Da{sup -1}), four protein molecules are estimated to be present in the asymmetric unit.

  7. Purification, crystallization and preliminary X-ray crystallographic analysis of glycosyltransferase-1 from Bacillus cereus

    PubMed Central

    Hsieh, Yin-Cheng; Chiu, Hsi-Ho; Huang, Yen-Chieh; Fun, Hoong-Kun; Lu, Chia-Yu; Li, Yaw-Kuen; Chen, Chun-Jung

    2014-01-01

    Glycosyltransferases (GTs), which are distributed widely in various organisms, including bacteria, fungi, plants and animals, play a role in synthesizing biological compounds. Glycosyltransferase-1 from Bacillus cereus (BcGT-1), which is capable of transferring glucose to small molecules such as kaempferol and quercetin, has been identified as a member of the family 1 glycosyltransferases which utilize uridine diphosphate glucose (UDP-glucose) as the sugar donor. BcGT-1 (molecular mass 45.5 kDa) has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction of BcGT-1 crystals to 2.10 Å resolution, the crystal belonged to space group P1, with unit-cell parameters a = 54.56, b = 84.81, c = 100.12 Å, α = 78.36, β = 84.66, γ = 84.84°. Preliminary analysis indicates the presence of four BcGT-1 molecules in the asymmetric unit with a solvent content of 50.27%. PMID:25195897

  8. 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.

  9. 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.

  10. Purification, crystallization and preliminary crystallographic analysis of human cystathionine β-synthase

    PubMed Central

    Oyenarte, Iker; Majtan, Tomas; Ereño, June; Corral-Rodríguez, María Angeles; Kraus, Jan P.; Martínez-Cruz, Luis Alfonso

    2012-01-01

    Human cystathionine β-synthase (CBS) is a pyridoxal-5′-phosphate-dependent hemeprotein, whose catalytic activity is regulated by S-adenosylmethionine. CBS catalyzes the β-replacement reaction of homocysteine (Hcy) with serine to yield cystathionine. CBS is a key regulator of plasma levels of the thrombogenic Hcy and deficiency in CBS is the single most common cause of homocystinuria, an inherited metabolic disorder of sulfur amino acids. The properties of CBS enzymes, such as domain organization, oligomerization degree or regulatory mechanisms, are not conserved across the eukaryotes. The current body of knowledge is insufficient to understand these differences and their impact on CBS function and physiology. To overcome this deficiency, we have addressed the crystallization and preliminary crystallographic analysis of a protein construct (hCBS516–525) that contains the full-length CBS from Homo sapiens (hCBS) and just lacks amino-acid residues 516–525, which are located in a disordered loop. The human enzyme yielded crystals belonging to space group I222, with unit-cell parameters a = 124.98, b = 136.33, c = 169.83 Å and diffracting X-rays to a resolution of 3.0 Å. The crystal structure appears to contain two molecules in the asymmetric unit which presumably correspond to a dimeric form of the enzyme. PMID:23143240

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

    PubMed Central

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

    2015-01-01

    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 P212121, 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. PMID:26144240

  12. 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.

  13. Cloning, purification, crystallization and preliminary X-ray diffraction analysis of nitrile hydratase from the themophilic Bacillus smithii SC-­J05-1

    PubMed Central

    Hourai, Shinji; Ishii, Takeshi; Miki, Misao; Takashima, Yoshiki; Mitsuda, Satoshi; Yanagi, Kazunori

    2005-01-01

    Nitrile hydratase (NHase) converts nitriles to the corresponding amides and is recognized as having important industrial applications. Purification, cloning, crystallization and initial crystallographic studies of the NHase from Bacillus smithii SC-J05-1 (Bs NHase) were conducted to analyze the activity, specificity and thermal stability of this hydrolytic enzyme. Bs NHase was purified to homogeneity from microbial cells of B. smithii SC-J05-1 and the nucleotide sequences of both the α- and β-subunits were determined. Purified Bs NHase was used for crystallization and several crystal forms were obtained by the vapour-diffusion method. Microseeding and the addition of magnesium ions were essential components to obtain crystals suitable for X-ray diffraction analysis. PMID:16511211

  14. 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.

  15. Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima

    SciTech Connect

    Lyashenko, Andrey V.; Zhukhlistova, Nadegda E.; Gabdoulkhakov, Azat G.; Zhukova, Yuliya N.; Voelter, Wolfang; Zaitsev, Viatcheslav N.; Bento, Isabel; Stepanova, Elena V.; Kachalova, Galina S.; Koroleva, Ol’ga V.; Cherkashyn, Evgeniy A.; Tishkov, Vladimir I.; Lamzin, Victor S.; Schirwitz, Katja; Betzel, Christian; Mikhailov, Al’bert M.

    2006-10-01

    The crystallization and preliminary X-ray structure at 1.9 Å resolution of the fungal laccase from C. maxima are presented. Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 Å resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccase structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 Å (R factor = 18.953%; R{sub free} = 23.835; r.m.s.d. bond lengths, 0.06 Å; r.m.s.d. bond angles, 1.07°) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002 ▶) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 Å X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO{sub 2} substituents.

  16. 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.

  17. 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.

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

    PubMed Central

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

    2014-01-01

    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 Å3 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. PMID:25195912

  19. Cloning, expression, purification, crystallization and preliminary crystallographic analysis of NifH1 from Methanocaldococcus jannaschii.

    PubMed

    Wu, Hao; Yuan, Ye; Ma, Jinming; Gao, Yongxiang

    2011-05-01

    Nitrogen fixation is catalyzed by the nitrogenase complex in Azotobacter, which is composed of dinitrogenase and dinitrogenase reductase. Dinitrogenase is an α(2)β(2) heterotetramer of the proteins NifD and NifK. Dinitrogenase reductase is a homodimer of the protein NifH. The expression of NifD/K and NifH nitrogenase homologues (named NflD/K and NflH for Nif-like D and H, respectively) has been detected in the non-nitrogen-fixing hyperthermophilic methanogen Methanocaldococcus jannaschii. Solving the structure of MjNifH1 may help in better understanding its function and may supply some clues to understanding the evolution of nitrogenase. The full-length protein with an additional His(6) tag at the C-terminus was expressed, purified and crystallized by the hanging-drop vapour-diffusion method at 287 K. An X-ray diffraction data set was collected to a resolution of 3.3 Å. The crystal belonged to space group P4(1)32, with unit-cell parameters a = b = c = 139.45 Å, and was estimated to contain one protein molecule per asymmetric unit. PMID:21543862

  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. PMID:26625288

  1. MraZ from Escherichia coli: cloning, purification, crystallization and preliminary X-ray analysis

    SciTech Connect

    Adams, Melanie A.; Udell, Christian M.; Pal, Gour Pada; Jia, Zongchao

    2005-04-01

    The crystallization and preliminary X-ray diffraction analysis of MraZ, formerly known as hypothetical protein YabB, from Escherichia coli K-12 is presented. The MraZ family of proteins, also referred to as the UPF0040 family, are highly conserved in bacteria and are thought to play a role in cell-wall biosynthesis and cell division. The murein region A (mra) gene cluster encodes MraZ proteins along with a number of other proteins involved in this complex process. To date, there has been no clear functional assignment provided for MraZ proteins and the structure of a homologue from Mycoplasma pneumoniae, MPN314, failed to suggest a molecular function. The b0081 gene from Escherichia coli that encodes the MraZ protein was cloned and the protein was overexpressed, purified and crystallized. This data is presented along with evidence that the E. coli homologue exists in a different oligomeric state to the MPN314 protein.

  2. Recombinant production, purification and crystallization of the Toxoplasma gondii coronin WD40 domain

    PubMed Central

    Kallio, Juha Pekka; Kursula, Inari

    2014-01-01

    Toxoplasma gondii is one of the most widely spread parasitic organisms in the world. Together with other apicomplexan parasites, it utilizes a special actin–myosin motor for its cellular movement, called gliding motility. This actin-based process is regulated by a small set of actin-binding proteins, which in Apicomplexa comprises only 10–15 proteins, compared with >150 in higher eukaryotes. Coronin is a highly conserved regulator of the actin cytoskeleton, but its functions, especially in parasites, have remained enigmatic. Coronins consist of an N-terminal actin-binding β-propeller WD40 domain, followed by a conserved region, and a C-terminal coiled-coil domain implicated in oligomerization. Here, the WD40 domain and the conserved region of coronin from T. gondii were produced recombinantly and crystallized. A single-wavelength diffraction data set was collected to a resolution of 1.65 Å. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 55.13, b = 82.51, c = 156.98 Å. PMID:24699753

  3. Cloning, expression, purification, crystallization and initial crystallographic analysis of FleN from Pseudomonas aeruginosa.

    PubMed

    Jain, Deepti

    2016-02-01

    The assembly of bacterial flagella requires the coordinated expression of a large number of genes in a hierarchical manner. These genes code for structural components of flagella, regulatory components and components that are required for chemotaxis. Stringent spatial and numerical control of flagella biosynthesis is essential for promoting motility and pathogenesis in bacteria. These genes are regulated at the level of transcription. FleN, a P-loop-containing ATPase, plays an important role in maintaining flagellar number in Pseudomonas aeruginosa. FleN exhibits anti-activator activity against FleQ, the global transcriptional regulator of flagellar genes. In order to gain insights into the regulatory mechanism of flagella synthesis, full-length FleN was crystallized in complex with the nonhydrolyzable ATP analogue β,γ-imidoadenosine 5'-triphosphate (AMPPNP) in space group C2221, with unit-cell parameters a = 49.1, b = 206.9, c = 103.3 Å. The Matthews coefficient is 2.19 Å(3) Da(-1) assuming the presence of two molecules in the asymmetric unit, and the corresponding solvent content is 43.7%. X-ray diffraction data were collected to a minimum Bragg spacing of 2.21 Å and crystals of FleN-AMPPNP were prepared with selenomethionine-labelled FleN for ab initio phasing. PMID:26841764

  4. Overexpression, purification and crystallization of an archaeal DNA ligase from Pyrococcus furiosus

    SciTech Connect

    Nishida, Hirokazu; Tsuchiya, Daisuke; Ishino, Yoshizumi; Morikawa, Kosuke

    2005-12-01

    Crystals of the archaeal DNA ligase from Pyrococcus furiosus were obtained using 6.6%(v/v) ethanol as a precipitant and diffracted X-rays to 1.7 Å resolution. DNA ligases seal single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome during various aspects of DNA metabolism, such as replication, excision repair and recombination. DNA-strand breaks are frequently generated as reaction intermediates in these events and the sealing of these breaks depends solely on the proper function of DNA ligase. Crystals of the archaeal DNA ligase from Pyrococcus furiosus were obtained using 6.6%(v/v) ethanol as a precipitant and diffracted X-rays to 1.7 Å resolution. They belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 61.1, b = 88.3, c = 63.4 Å, β = 108.9°. The asymmetric unit contains one ligase molecule.

  5. Expression, purification, crystallization and preliminary crystallographic analysis of a stand-alone RAM domain with hydrolytic activity from the hyperthermophile Pyrococcus furiosus

    SciTech Connect

    Agapay, Ramelito C.; Savvides, Savvas N.; Van Driessche, Gonzalez; Devreese, Bart; Van Beeumen, Jozef; Jongejan, Jaap A. Hagen, Wilfred R.

    2005-10-01

    A P. furiosus stand-alone RAM domain with hydrolytic activity has been cloned and expressed in E. coli. The purified protein was crystallized alone and with EPNP and PMSF, producing crystals that yield diffraction data to resolutions of 2.8, 2.2 and 2.8 Å, respectively. The RAM domain is one of several ligand-binding modules present in prokaryotes that are presumed to regulate the transcription of specific genes. To date, no hydrolytic activity has been reported for such modules. Curiously, a stand-alone RAM domain in Pyrococcus furiosus was isolated during a screen for hydrolytic activity against chromogenic esters. The gene encoding this protein was cloned and expressed in Escherichia coli and crystallized after a single purification step. X-ray diffraction data from the crystals were obtained to a resolution of 2.8 Å using a conventional X-ray source. The cocrystallization of the recombinant protein with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNP) and phenylmethylsulfonyl fluoride (PMSF) produced crystals that yielded data to 2.2 and 2.8 Å, respectively, using synchrotron radiation. Both the untreated and EPNP-treated crystals crystallize isomorphously in space group C2 and contain three dimers in the asymmetric unit. The PMSF-treated crystals also belong to this space group and have almost identical packing density, but show dramatically different unit-cell parameters.

  6. Remedial Strategies in Structural Proteomics: Expression, Purification, And Crystallization of the Vav1/Rac1 Complex

    SciTech Connect

    Brooun, A.; Foster, S.A.; Chrencik, H.E.; Chien, E.Y.T.; Kolatkar, A.R.; Streiff, M.; Ramage, P.; Widmer, H.; Weckbecker, G.; Kuhn, P.

    2007-07-03

    The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface compared to other GEF/GTPase pairs. Here, we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein interaction with potential to inhibit lymphocyte activation.

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

    SciTech Connect

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

    2007-07-01

    Recombinant SOS3 and SOS2 regulatory domain from A. thaliana have been coexpressed in E. coli, purified and crystallized by the hanging-drop vapour-diffusion method. An X-ray data set has been collected at 2.0 Å resolution. 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 P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 Å.

  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. 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

  10. 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.

  11. Purification of materials for crystal growth and glass processing; Proceedings of the Second Workshop, Champion, PA, Sept. 28-Oct. 1, 1987

    NASA Astrophysics Data System (ADS)

    Hopkins, Richard H.; Klein, Phillip H.; Feigelson, Robert S.

    1988-06-01

    Papers are presented on the analysis of high-purity solids by resonance ionization spectrosopy, the glow discharge mass spectrometry for high-purity material analysis, the photoluminescence of bulk and epitaxial GaAs, purification techniques and analytical methods for gaseous and metallic impurities in high-purity silane, and purity considerations for amorphous silicon thin films. Attention is also given to the production of extreme-purity aluminum and silicon by fractional crystallization processing, the characterization of directionally solidified lead chloride, the composition control of the microstructure of Ba2YCu3O(6+x), the preparation of multistage zone-refined materials for thermochemical standards, and the purification of organic materials for electrical and optical studies. Other papers are on the IR measurements of interstitial oxygen in heavily doped silicon, the purity and the purification of source materials for III-V MOCVD, and a comparison of alternate As-sources to arsine in the MOCVD growth of GaAs.

  12. 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

  13. 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

  14. 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.

  15. 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. PMID:24637766

  16. 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.

  17. 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

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

    PubMed

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

    2013-05-01

    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

  19. Purification, crystallization and preliminary X-ray diffraction analysis of a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) from Coffea canephora involved in chlorogenic acid biosynthesis

    PubMed Central

    Lallemand, Laura A.; McCarthy, James G.; McSweeney, Sean; McCarthy, Andrew A.

    2012-01-01

    Chlorogenic acids (CGAs) are a group of soluble phenolic compounds that are produced by a variety of plants, including Coffea canephora (robusta coffee). The last step in CGA biosynthesis is generally catalysed by a specific hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase (HQT), but it can also be catalysed by the more widely distributed hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT). Here, the cloning and overexpression of HCT from C. canephora in Escherichia coli as well as its purification and crystallization are presented. Crystals were obtained by the sitting-drop technique at 293 K and X-ray diffraction data were collected on the microfocus beamline ID23-2 at the ESRF. The HCT crystals diffracted to better than 3.0 Å resolution, belonged to space group P42212 with unit-cell parameters a = b = 116.1, c = 158.9 Å and contained two molecules in the asymmetric unit. The structure was solved by molecular replacement and is currently under refinement. Such structural data are needed to decipher the molecular basis of the substrate specifities of this key enzyme, which belongs to the large plant acyl-CoA-dependent BAHD acyltransferase superfamily. PMID:22750875

  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. Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate

    PubMed Central

    Voss, Jarrod E.; Scally, Stephen W.; Taylor, Nicole L.; Dogovski, Con; Alderton, Malcolm R.; Hutton, Craig A.; Gerrard, Juliet A.; Parker, Michael W.; Dobson, Renwick C. J.; Perugini, Matthew A.

    2009-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 Å are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme. PMID:19194017

  2. 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.

  3. Purification, partial characterization, crystallization and preliminary X-ray diffraction of a novel cardiotoxin-like basic protein from Naja naja atra (South Anhui) venom

    SciTech Connect

    Rong, Hui; Li, Yan; Lou, Xiao-hua; Zhang, Xio; Gao, Yong-xiang; Teng, Mai-kun Niu, Li-wen

    2007-02-01

    A novel cardiotoxin-like basic protein from Naja naja atra was crystallized and diffraction data were collected to 2.35 Å resolution. A novel cardiotoxin-like basic protein was isolated from the venom of the Chinese cobra (Naja naja atra) from the south of Anhui in China. The protein inhibits the expression of vascular endothelial growth factor and basic fibroblast growth factor in human lung cancer cell line H1299 and induces the haemolysis of rabbit erythrocytes under low-lecithin conditions. After a two-step chromatographic purification, the resultant 7 kDa protein was crystallized by the hanging-drop vapour-diffusion method at room temperature. A complete data set was collected to 2.35 Å resolution using an in-house X-ray diffraction system. The crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 43.2, c = 147.9 Å. There are two molecules in the crystallographic asymmetric unit.

  4. Overexpression, purification and crystallization of PhzA, the first enzyme of the phenazine biosynthesis pathway of Pseudomonas fluorescens 2-79.

    PubMed

    Ahuja, Ekta G; Mavrodi, Dimitri V; Thomashow, Linda S; Blankenfeldt, Wulf

    2004-06-01

    Phenazines are broad-spectrum antibiotic metabolites produced by organisms such as Pseudomonas and Streptomyces. Phenazines have been shown to enhance microbial competitiveness and the pathogenic potential of the organisms that synthesize them. PhzA (163 residues, approximate molecular weight 18.7 kDa) is the product of the first of seven genes of the operon responsible for phenazine biosynthesis in P. fluorescens 2-79. This enzyme is thought to catalyse one of the final steps in the formation of phenazine-1-carboxylic acid, the end product of phenazine biosynthesis in P. fluorescens 2-79. Here, the purification and crystallization of recombinant PhzA are reported. Crystals diffracting to 2.1 angstroms were obtained using 1.6 M magnesium sulfate and 2-morpholinoethanesulfonic acid monohydrate (MES) buffer pH 5.2-5.6. Crystals of both native and seleno-L-methionine-labelled protein belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 66.8, b = 75.3, c = 84.5 angstroms. The asymmetric unit contains one dimer of PhzA. PMID:15159577

  5. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv2827c from Mycobacterium tuberculosis

    SciTech Connect

    Janowski, Robert

    2006-08-01

    M. tuberculosis hypothetical protein Rv2827c was cloned, expressed, purified and crystallized. Preliminary X-ray diffraction data were collected to a resolution of 1.93 Å. The hypothetical protein Rv2827c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and then crystallized. Preliminary X-ray diffraction data analysis suggests the presence of two translationally related molecules in the asymmetric unit of the orthorhombic crystals.

  6. 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.

  7. Purification, crystallization and preliminary crystallographic analysis of the biotin–protein ligase from Pyrococcus horikoshii OT3

    SciTech Connect

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-02-01

    The biotin–protein ligase from P. horikoshii OT3 was overexpressed, purified, crystallized and cocrystallized with biotin, ADP and biotinyl-5′-AMP. The crystals belong to space group P2{sub 1} and diffract to beyond 1.6 Å resolution.

  8. Cloning, expression, purification, crystallization and initial crystallographic analysis of the preprotein translocation ATPase SecA from Thermus thermophilus

    SciTech Connect

    Vassylyeva, Marina N.; Mori, Hiroyuki; Tsukazaki, Tomoya; Yokoyama, Shigeyuki; Tahirov, Tahir H.; Ito, Koreaki; Vassylyev, Dmitry G.

    2006-09-01

    The SecA ATPase from T. thermophilus was cloned, expressed, purified and crystallized. Complete diffraction data sets were collected for two crystal forms at 2.8 and 3.5 Å resolution, respectively. Determination of the structure is now in progress. The Thermus thermophilus gene encoding the preprotein translocation ATPase SecA was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups P3{sub 1(2)}21 (a = b = 168.6, c = 149.8 Å) and P6{sub 1(5)}22 (a = b = 130.9, c = 564.6 Å). The crystals, improved by macroseeding, diffracted to beyond 2.8 and 3.5 Å resolution for the trigonal and hexagonal crystal forms, respectively. Structure determination using the multiple isomorphous replacement method is in progress.

  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. Cloning, expression, purification, crystallization and preliminary X-ray analysis of human liver glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Warizaya, Masaichi; Kinoshita, Takayoshi; Kato, Akemi; Nakajima, Hidenori; Fujii, Takashi

    2004-03-01

    Human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified and crystallized using PEG 3350 as a precipitant. However, the crystals were extremely fragile towards osmotic shock. A 1% change in PEG 3350 content causes destruction of the crystals. After many trials for freezing the crystals, X-ray diffraction data from a native crystal were collected at 2.8 A resolution using as a cryoprotectant a mixture consisting of paraffin oil and Paratone-N in a 3:1 ratio and a cryoloop covered with Formver film. Crystals belong to space group P2(1), with unit-cell parameters a = 63.23, b = 97.84, c = 84.23 A, beta = 104.1 degrees. Molecular replacement with a starting model consisting of a homology model based on the low-resolution structure of human skeletal muscle GAPDH, which has 90% identical residues with the liver protein, led to a solution. Most of the current model was assigned properly in the electron-density map, but the map corresponding to some important regions containing the phosphate-binding loop was ambiguous. It is planned to crystallize human liver GAPDH in the presence of phosphate ions and/or some kind of inhibitor in order to fix the flexible region. PMID:14993695

  11. Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3

    SciTech Connect

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    An archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue from P. horikoshii OT3 was overexpressed as native and selenomethionine-substituted protein, purified and crystallized. The native and selenomethionine-derivative crystals are isomorphous and diffract X-rays to 2.1 and 2.9 Å resolution, respectively. 6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 Å resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 Å. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V{sub M} = 2.24 Å{sup 3} Da{sup −1}). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 Å.

  12. Purification, crystallization and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, a putative archaeal homologue of γ-AMPK

    PubMed Central

    Gómez García, Inmaculada; Kortázar, Danel; Oyenarte, Iker; Mato, José María; Martínez-Chantar, María Luz; Martínez-Cruz, Luis Alfonso

    2009-01-01

    In mammals, AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (α) and two regulatory subunits (β and γ). The γ subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. This work describes the purification and preliminary crystallographic analysis of protein MJ1225 from Methanocaldococcus jannaschii, an archaeal homologue of γ-AMPK. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Diffraction data for MJ1225 were collected to 2.3 Å resolution using synchrotron radiation. The crystals belonged to space group H32, with unit-cell parameters a = b = 108.95, c = 148.08 Å, α = β = 90.00, γ = 120.00°. Preliminary analysis of the X-ray data indicated that there was one molecule per asymmetric unit. PMID:19652347

  13. 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.

  14. 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.

  15. 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%.

  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. 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

  18. 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%.

  19. 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

  20. 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.

  1. Purification, crystallization and preliminary X-ray analysis of the dissimilatory sulfite reductase from Desulfovibrio vulgaris Miyazaki F.

    PubMed

    Ogata, Hideaki; Shomura, Yasuhito; Goenka Agrawal, Aruna; Kaur, Amrit Pal; Gärtner, Wolfgang; Higuchi, Yoshiki; Lubitz, Wolfgang

    2010-11-01

    Dissimilatory sulfite reductase (Dsr) plays an important role in sulfate respiration in many sulfate-reducing bacteria. Dsr from Desulfovibrio vulgaris Miyazaki F has been purified and crystallized at 277 K using the sitting-drop vapour-diffusion method with PEG 3350 and potassium thiocyanate as precipitants. A data set was collected to 3.7 Å resolution from a single crystal at 100 K using synchrotron radiation. The Dsr crystal belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 163.26, c = 435.32 Å. The crystal structure of Dsr was determined by the molecular-replacement method based on the three-dimensional structure of Dsr from D. vulgaris Hildenborough. The crystal contained three α(2)β(2)γ(2) units per asymmetric unit, with a Matthews coefficient (V(M)) of 2.35 Å(3) Da(-1); the solvent content was estimated to be 47.7%. PMID:21045297

  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 RecA superfamily ATPase PH0284 from Pyrococcus horikoshii OT3

    SciTech Connect

    Bagautdinov, Bagautdin; Kunishima, Naoki

    2006-04-01

    RecA superfamily ATPase PH0284 from P. horikoshii OT3 was overexpressed, purified, crystallized and cocrystallized with ATP. Both crystal forms belong to the trigonal space group P3{sub 2}21 and diffract X-rays to 2.0 and 2.3 Å resolution, respectively. Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 Å resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3{sub 2}21, with unit-cell parameters a = b = 96.06, c = 298.90 Å. Assuming the presence of one hexamer in the asymmetric unit gives a V{sub M} value of 2.36 Å{sup 3} Da{sup −1} and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 Å resolution.

  4. Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3.

    PubMed

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 A resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 A. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V(M) = 2.24 A3 Da(-1)). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 A. PMID:17183164

  5. Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3

    PubMed Central

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 Å resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 Å. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V M = 2.24 Å3 Da−1). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 Å. PMID:17183164

  6. Purification, crystallization and preliminary X-ray diffraction studies of N-acetylglucosamine-phosphate mutase from Candida albicans

    SciTech Connect

    Nishitani, Yuichi; Maruyama, Daisuke; Nonaka, Tsuyoshi; Kita, Akiko; Fukami, Takaaki A.; Mio, Toshiyuki; Yamada-Okabe, Hisafumi; Yamada-Okabe, Toshiko; Miki, Kunio

    2006-04-01

    Preliminary X-ray diffraction studies on N-acetylglucosamine-phosphate mutase from C. albicans are reported. N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) in eukaryotes and belongs to the α-d-phosphohexomutase superfamily. AGM1 from Candida albicans (CaAGM1) was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belong to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 60.2, b = 130.2, c = 78.0 Å, β = 106.7°. The crystals diffract X-rays to beyond 1.8 Å resolution using synchrotron radiation.

  7. Expression, purification, crystallization and preliminary crystallographic analysis of a thermostable DNA ligase from the archaeon Thermococcus sibiricus.

    PubMed

    Petrova, T E; Bezsudnova, E Y; Dorokhov, B D; Slutskaya, E S; Polyakov, K M; Dorovatovskiy, P V; Ravin, N V; Skryabin, K G; Kovalchuk, M V; Popov, V O

    2012-02-01

    DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential to maintain the integrity of the genome in DNA replication, recombination and repair. A recombinant ATP-dependent DNA ligase from the hyperthermophilic anaerobic archaeon Thermococcus sibiricus was expressed in Escherichia coli and purified. Crystals were grown by vapour diffusion using the hanging-drop method with 17%(w/v) PEG 4000 and 8.5%(v/v) 2-propanol as precipitants. A diffraction experiment was performed with a single crystal, which diffracted X-rays to 3.0 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 58.590, b = 87.540, c = 126.300 Å. PMID:22297989

  8. Purification, crystallization and preliminary X-ray analysis of CMS1MS2: a cysteine proteinase from Carica candamarcensis latex

    PubMed Central

    Gomes, Marco Túlio Ribeiro; Teixeira, Raphael Dias; Ribeiro, Henrique de Assis Lopes; Turchetti, Andréia Pereira; Junqueira, Caroline Furtado; Lopes, Míriam Tereza Paz; Salas, Carlos Edmundo; Nagem, Ronaldo Alves Pinto

    2008-01-01

    Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P21), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 Å, β = 96.81°, and diffract X-rays to 1.8 Å resolution. PMID:18540057

  9. Isolation, purification, crystallization and preliminary crystallographic studies of amaryllin, a plant pathogenesis-related protein from Amaryllis belladonna.

    PubMed

    Kumar, Sanjit; Singh, Nagendra; Sinha, Mau; Kaur, Punit; Srinivasan, A; Sharma, Sujata; Singh, T P

    2009-06-01

    A novel antifungal protein, amaryllin, has been isolated from the underground bulbs of Amaryllis belladonna, purified to homogeneity and crystallized. The protein was extracted using ammonium sulfate fractionation. The purified protein samples indicated a molecular weight of 15 kDa on SDS-PAGE. The protein showed antifungal activity against Aspergillus flavus and Fusarium oxysporum. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation and did not show significant sequence identity to any known protein. The protein was crystallized using the hanging-drop vapour-diffusion method with 30% PEG 8000 as precipitating agent. The crystals diffracted to 2.7 A resolution and belonged to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 48.6, b = 61.9, c = 79.6 A. The complete sequence and structure determination of amaryllin are in progress. PMID:19478451

  10. Isolation, purification, crystallization and preliminary crystallographic studies of amaryllin, a plant pathogenesis-related protein from Amaryllis belladonna

    PubMed Central

    Kumar, Sanjit; Singh, Nagendra; Sinha, Mau; Kaur, Punit; Srinivasan, A.; Sharma, Sujata; Singh, T. P.

    2009-01-01

    A novel antifungal protein, amaryllin, has been isolated from the underground bulbs of Amaryllis belladonna, purified to homogeneity and crystallized. The protein was extracted using ammonium sulfate fractionation. The purified protein samples indicated a molecular weight of 15 kDa on SDS–PAGE. The protein showed antifungal activity against Aspergillus flavus and Fusarium oxysporum. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation and did not show significant sequence identity to any known protein. The protein was crystallized using the hanging-drop vapour-diffusion method with 30% PEG 8000 as precipitating agent. The crystals diffracted to 2.7 Å resolution and belonged to the orthorhombic space group I222 or I212121, with unit-cell parameters a = 48.6, b = 61.9, c = 79.6 Å. The complete sequence and structure determination of amaryllin are in progress. PMID:19478451

  11. 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.

  12. 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

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

    SciTech Connect

    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

    2006-06-01

    Rice Bowman–Birk inhibitor was expressed and crystallized. 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 P2{sub 1}2{sub 1}2{sub 1}, 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%.

  14. Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

    SciTech Connect

    Meramveliotaki, Chrysi; Kotsifaki, Dina; Androulaki, Maria; Hountas, Athanasios; Eliopoulos, Elias; Kokkinidis, Michael

    2007-10-01

    PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4{sub 2}, with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement.

  15. 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.

  16. 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.

  17. Purification and crystallization of Vibrio fischeri CcdB and its complexes with fragments of gyrase and CcdA

    PubMed Central

    De Jonge, Natalie; Buts, Lieven; Vangelooven, Joris; Mine, Natacha; Van Melderen, Laurence; Wyns, Lode; Loris, Remy

    2007-01-01

    The ccd toxin–antitoxin module from the Escherichia coli F plasmid has a homologue on the Vibrio fischeri integron. The homologue of the toxin (CcdBVfi) was crystallized in two different crystal forms. The first form belongs to space group I23 or I213, with unit-cell parameter a = 84.5 Å, and diffracts to 1.5 Å resolution. The second crystal form belongs to space group C2, with unit-cell parameters a = 58.5, b = 43.6, c = 37.5 Å, β = 110.0°, and diffracts to 1.7 Å resolution. The complex of CcdBVfi with the GyrA14Vfi fragment of V. fischeri gyrase crystallizes in space group P212121, with unit-cell parameters a = 53.5, b = 94.6, c = 58.1 Å, and diffracts to 2.2 Å resolution. The corresponding mixed complex with E. coli GyrA14Ec crystallizes in space group C2, with unit-cell parameters a = 130.1, b = 90.8, c = 58.1 Å, β = 102.6°, and diffracts to 1.95 Å. Finally, a complex between CcdBVfi and part of the F-plasmid antitoxin CcdAF crystallizes in space group P212121, with unit-cell parameters a = 46.9, b = 62.6, c = 82.0 Å, and diffracts to 1.9 Å resolution. PMID:17401216

  18. 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)

  19. Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum

    PubMed Central

    Colussi, Francieli; Textor, Larissa C.; Serpa, Viviane; Maeda, Roberto Nobuyuki; Pereira, Nei; Polikarpov, Igor

    2010-01-01

    The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source. PMID:20823521

  20. 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.

  1. Purification, crystallization and preliminary X-ray diffraction studies of UDP-N-acetylglucosamine pyrophosphorylase from Candida albicans

    SciTech Connect

    Maruyama, Daisuke; Nishitani, Yuichi; Nonaka, Tsuyoshi; Kita, Akiko; Fukami, Takaaki A.; Mio, Toshiyuki; Yamada-Okabe, Hisafumi; Yamada-Okabe, Toshiko; Miki, Kunio

    2006-12-01

    UDP-N-acetylglucosamine pyrophosphorylase was purified and crystallized and X-ray diffraction data were collected to 2.3 Å resolution. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 Å resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 Å, α = 90.01, β = 97.72, γ = 92.88°, whereas those of the product complex belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 Å.

  2. 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

  3. Purification, solution properties and crystallization of SIV integrase containing a continuous core and C-terminal domain.

    PubMed

    Li, Y; Yan, Y; Zugay-Murphy, J; Xu, B; Cole, J L; Witmer, M; Felock, P; Wolfe, A; Hazuda, D; Sardana, M K; Chen, Z; Kuo, L C; Sardana, V V

    1999-11-01

    The C-terminal two-thirds segment of integrase derived from the simian immunodeficiency virus has been cloned, expressed in Escherichia coli, and purified to greater than 95% homogeneity. The protein encompasses amino-acid residues 50-293 and contains a F185H substitution to enhance solubility. In dilute solutions at concentrations below 1 mg ml(-1), the enzyme is predominantly dimeric. At the higher concentrations (>10 mg ml(-1)) required to enable crystallization, the enzyme self-associates to form species with molecular weights greater than 200 kDa. Despite the apparent high aggregation in solution, the enzyme crystallizes from a 8%(v/v) polyethylene glycol (molecular weight 6000) solution in a form suitable for X-ray diffraction studies. The resulting single crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 79.76, b = 99.98, c = 150.2 A, alpha = beta = gamma = 90 degrees and Z = 4. Under X-ray irradiation generated with a rotating-anode generator, the crystals diffract to 2.8 A resolution and allow collection of a native 3 A resolution diffraction data set. PMID:10531491

  4. 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.

  5. Heterodimeric nitrate reductase (NapAB) from Cupriavidus necator H16: purification, crystallization and preliminary X-ray analysis

    SciTech Connect

    Coelho, Catarina; González, Pablo J.; Trincão, José; Carvalho, Ana L.; Najmudin, Shabir; Moura, José J. G.; Moura, Isabel; Romão, Maria J.

    2007-06-01

    Crystals of the oxidized form of the periplasmic nitrate reductase from Cupriavidus necator were obtained using polyethylene glycol 3350 as precipitant The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe–4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 Å at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 Å, β = 100.7°, space group C2, and one heterodimer is present per asymmetric unit.

  6. 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. PMID:24192374

  7. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of chlorite dismutase: a detoxifying enzyme producing molecular oxygen

    SciTech Connect

    Geus, Daniël C. de Thomassen, Ellen A. J.; Feltz, Clarisse L. van der; Abrahams, Jan Pieter

    2008-08-01

    Preliminary X-ray data collection and analysis for crystals of chlorite dismutase, a haem-based enzyme that very effectively reduces chlorite to chloride while producing molecular oxygen, is reported to 2.1 Å resolution. Chlorite dismutase, a homotetrameric haem-based protein, is one of the key enzymes of (per)chlorate-reducing bacteria. It is highly active (< 2 kU mg{sup −1}) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction. The chlorite dismutase gene in Azospira oryzae strain GR-1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli. Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P2{sub 1}2{sub 1}2 and were most likely to contain six subunits in the asymmetric unit. The refined unit-cell parameters were a = 164.46, b = 169.34, c = 60.79 Å. The crystals diffracted X-rays to 2.1 Å resolution on a synchrotron-radiation source and a three-wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available.

  8. 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.

  9. 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.

  10. Expression, Purification, Crystallization and Preliminary X-ray Crystallographic Studies of a Novel Acetylcitrulline deacetylase from Xanthomonas Campestris

    SciTech Connect

    Shi,D.; Yu, X.; Roth, L.; Hiroki, M.; Hathout, Y.; Allewell, N.; Tuchman, M.

    2005-01-01

    A novel N-acetyl-{sub 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 Angstrom resolution, with unit-cell parameters a = 94.13, b = 95.23, c = 43.61 Angstroms, {beta} = 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.

  11. 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.

  12. Purification, crystallization and preliminary crystallographic analysis of the biotin-protein ligase from Pyrococcus horikoshii OT3.

    PubMed

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-02-01

    Biotin-protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin-protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 A resolution from a native crystal and to 1.55 A resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 38.601, b = 78.264, c = 70.147 A, beta = 101.48 degrees. Assuming a homodimer per asymmetric unit gives a VM value of 2.14 A3 Da(-1) and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5'-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 A resolution, respectively. PMID:16510991

  13. Purification, crystallization and preliminary crystallographic analysis of the biotin–protein ligase from Pyrococcus horikoshii OT3

    PubMed Central

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-01-01

    Biotin–protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin–protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 Å resolution from a native crystal and to 1.55 Å resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 38.601, b = 78.264, c  =  70.147 Å, β = 101.48°. Assuming a homodimer per asymmetric unit gives a V M value of 2.14 Å3 Da−1 and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5′-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 Å resolution, respectively. PMID:16510991

  14. 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}.

  15. 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

  16. Expression, purification, crystallization and preliminary X-ray analysis of the HER3–9E12 Fab complex

    PubMed Central

    He, Kecheng; Huang, Ang; Huang, Yong; Takeda, Hiroaki

    2014-01-01

    9E12 is a fully human immunoglobulin G1/κ monoclonal antibody that is specific for the epidermal growth factor receptor 3 (HER3), the overexpression of which has been detected in many tumour types and is associated with poor survival outcomes. To date, knowledge of the molecular mechanism for targeted antibodies that directly inhibit HER3 signalling is limited. Because knowledge of such therapeutic antibodies would help basic immunological therapeutics, structural insights into the HER3–9E12 Fab complex are important. Recombinant human HER3 and Fab fragments of the 9E12 antibody were cloned, expressed and crystallized, and crystallographic data sets were collected. The crystals belonged to space group P1, with unit-cell parameters a = 74.4, b = 98.6, c = 99.6 Å, α = 106.0, β = 95.0, γ = 102.5° and diffracted to a resolution of 2.1 Å. PMID:24915094

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

    PubMed Central

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

    2006-01-01

    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. PMID:16682782

  18. Cloning, expression, purification, crystallization and preliminary X-ray diffraction data of the Pyrococcus horikoshii RadA intein

    PubMed Central

    Lyskowski, Andrzej; Oeemig, Jesper S.; Jaakkonen, Anniina; Rommi, Katariina; DiMaio, Frank; Zhou, Dongwen; Kajander, Tommi; Baker, David; Wlodawer, Alexander; Goldman, Adrian; Iwaï, Hideo

    2011-01-01

    The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P212121, with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement. PMID:21543876

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

    PubMed

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

    2006-04-01

    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 alpha-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 angstroms 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. PMID:16582486

  20. 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}.

  1. Expression, purification, crystallization and preliminary crystallographic study of a potential metal-dependent hydrolase with cyclase activity from Thermoanaerobacter tengcongensis

    PubMed Central

    Liu, Sen; Wu, Guangteng; Huang, Qichen; Lai, Luhua; Tang, Youqi; Unno, Hideaki; Kusunoki, Masami

    2005-01-01

    The putative metal-dependent hydrolase gene TTE1006 from Thermoanaerobacter tengcongensis strain MB4T (T = type strain; Genbank accession No. AE008691) was heterologously expressed in Escherichia coli. The 205-amino-acid gene product was purified and crystallized. The crystal used for data collection belongs to space group P21, with unit-cell parameters a = 85.2, b = 62.1, c = 172.4 Å, β = 104.2°. Using a synchrotron-radiation source, the resolution limit of the data reached 1.87 Å. Eight molecules were estimated to be present in the asymmetric unit, with a solvent content of 48%. Structure determination is ongoing using the multiple-wavelength anomalous diffraction (MAD) method and also the molecular-replacement (MR) method. PMID:16508100

  2. 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%. PMID:19574654

  3. Purification and crystallization of yeast glycosylphosphatidylinositol transamidase subunit PIG-S (PIG-S71–467)

    PubMed Central

    Kamariah, Neelagandan; Eisenhaber, Frank; Adhikari, Sharmila; Eisenhaber, Birgit; Grüber, Gerhard

    2011-01-01

    The transfer of glycosylphosphatidylinositol (GPI) anchors onto eukaryotic proteins is catalyzed by the transamidase complex, which is composed of at least five subunits (PIG-K, PIG-S, PIG-T, PIG-U and GPAA1). Here, the recombinant protein PIG-S71–467 from Saccharomyces cerevisiae, including residues 71–467 of the entire 534-residue protein, was cloned, expressed and purified to homogeneity. The monodisperse protein was crystallized by the vapour-diffusion method. A diffraction data set was collected to 3.2 Å resolution with 91.6% completeness. The crystals belonged to space group C2, with unit-cell parameters a = 106.72, b = 59.33, c = 124.3 Å, β = 114.19°, and contained two molecules in the asymmetric unit. PMID:21821889

  4. 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

  5. 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

  6. Preparation, purification, crystallization and preliminary crystallographic analysis of dual-domain β-propeller phytase from Bacillus sp. HJB17.

    PubMed

    Lu, Fang; Guo, Gangxin; Li, Qianqian; Feng, Duo; Liu, Yong; Huang, Huoqing; Yang, Peilong; Gao, Wei; Yao, Bin

    2014-12-01

    β-Propeller phytases (BPPs) are abundant in nature. Recently, dual-domain BPPs have been found in which the typical BPP domain is responsible for phytate hydrolysis. The dual-domain BPP (PhyH) from Bacillus sp. HJB17 was obtained with an incomplete N-terminal BPP domain (PhyH-DI; residues 41-318) and a typical BPP domain (PhyH-DII; residues 319-644) at the C-terminus. PhyH-DI was found to act synergistically (with a 1.2-2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. The structure of PhyH was therefore studied with the aim of explaining these functions. PhyH with the secreted signal peptide of the first 40 amino acids deleted (PhyHT) was cloned and expressed in Escherichia coli. Purified and active PhyHT protein was obtained by refolding from the precipitant. PhyHT was crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl pH 9.5, 25%(w/v) polyethylene glycol 4000 using 1 mg ml(-1) protein solution at 289 K. A complete data set was collected from a crystal to 2.85 Å resolution using synchrotron radiation at 100 K. The crystal belonged to space group P1211, with unit-cell parameters a = 46.82, b = 140.19, c = 81.94 Å, α = 90.00, β = 92.00, γ = 90.00°. The asymmetric unit was estimated to contain one molecule of PhyHT. PMID:25484224

  7. 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.

  8. 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.

  9. Cloning, expression, purification, crystallization and preliminary X-ray characterization of allantoinase from Bacillus licheniformis ATCC 14580.

    PubMed

    Conejero-Muriel, Mayte; Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Gavira, Jose A

    2014-11-01

    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 Rmerge 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 (VM=2.34 Å3 Da(-1)). PMID:25372819

  10. 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.

  11. Purification, crystallization and preliminary X-ray analysis of the glucosamine-6-phosphate N-acetyltransferase from human liver

    SciTech Connect

    Wang, Juan; Zhou, Yan-Feng; Li, Lan-Fen; Liang, Yu-He Su, Xiao-Dong

    2006-11-01

    Glucosamine-6-phosphate N-acetyltransferase from human liver was expressed, purified and crystallized. Diffraction data have been collected to 2.6 Å resolution. Glucosamine-6-phosphate N-acetyltransferase from human liver, which catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to the primary amine of d-glucosamine 6-phosphate to form N-acetyl-d-glucosamine 6-phosphate, was expressed in a soluble form from Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity using Ni{sup 2+}-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.6 Å resolution. 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 = b = 50.08, c = 142.88 Å.

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

    PubMed Central

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

    2014-01-01

    The thermostable d-lactate dehydrogenase from Lactobacillus jensenii (Lj d-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 Lj d-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 (V M) is 2.58 Å3 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

  13. 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

  14. 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%.

  15. Purification, crystallization and preliminary crystallographic analysis of RecA superfamily ATPase PH0284 from Pyrococcus horikoshii OT3.

    PubMed

    Bagautdinov, Bagautdin; Kunishima, Naoki

    2006-04-01

    Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 angstroms resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3(2)21, with unit-cell parameters a = b = 96.06, c = 298.90 angstroms. Assuming the presence of one hexamer in the asymmetric unit gives a V(M) value of 2.36 angstroms3 Da(-1) and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 angstroms resolution. PMID:16582499

  16. Purification, crystallization and preliminary crystallographic analysis of RecA superfamily ATPase PH0284 from Pyrococcus horikoshii OT3

    PubMed Central

    Bagautdinov, Bagautdin; Kunishima, Naoki

    2006-01-01

    Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 Å resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3221, with unit-cell parameters a = b = 96.06, c = 298.90 Å. Assuming the presence of one hexamer in the asymmetric unit gives a V M value of 2.36 Å3 Da−1 and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 Å resolution. PMID:16582499

  17. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of chlorite dismutase: a detoxifying enzyme producing molecular oxygen

    PubMed Central

    de Geus, Daniël C.; Thomassen, Ellen A. J.; van der Feltz, Clarisse L.; Abrahams, Jan Pieter

    2008-01-01

    Chlorite dismutase, a homotetrameric haem-based protein, is one of the key enzymes of (per)chlorate-reducing bacteria. It is highly active (>2 kU mg−1) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction. The chlorite dismutase gene in Azospira oryzae strain GR-1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli. Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P21212 and were most likely to contain six subunits in the asymmetric unit. The refined unit-cell parameters were a = 164.46, b = 169.34, c = 60.79 Å. The crystals diffracted X-rays to 2.1 Å resolution on a synchrotron-radiation source and a three-wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available. PMID:18678943

  18. Expression, purification, crystallization and preliminary X-ray diffraction analysis of a mammalian type 10 adenylyl cyclase

    PubMed Central

    Kleinboelting, Silke; van den Heuvel, Joop; Kambach, Christian; Weyand, Michael; Leipelt, Martina; Steegborn, Clemens

    2014-01-01

    The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1–10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ∼2.0 Å resolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04 Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies. PMID:24699740

  19. 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.

  20. 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

  1. Expression, purification, crystallization and preliminary X-ray diffraction analysis of a lactococcal bacteriophage small terminase subunit

    PubMed Central

    Ren, Bin; Pham, Tam M.; Surjadi, Regina; Robinson, Christine P.; Le, Thien-Kim; Chandry, P. Scott; Peat, Thomas S.; McKinstry, William J.

    2013-01-01

    Terminases are enzymes that are required for the insertion of a single viral genome into the interior of a viral procapsid by a process referred to as ‘encapsulation or packaging’. Many double-stranded DNA viruses such as bacteriophages T3, T4, T7, λ and SPP1, as well as herpes viruses, utilize terminase enzymes for this purpose. All the terminase enzymes described to date require two subunits, a small subunit referred to as TerS and a large subunit referred to as TerL, for in vivo activity. The TerS and TerL subunits interact with each other to form a functional hetero-oligomeric enzyme complex; however the stoichiometry and oligomeric state have not been determined. We have cloned, expressed and purified recombinant small terminase TerS from a 936 lactococcal bacteriophage strain ASCC454, initially isolated from a dairy factory. The terminase was crystallized using a combination of nanolitre sitting drops and vapour diffusion using sodium malonate as the precipitant, and crystallization optimized using standard vapour-diffusion hanging drops set up in the presence of a nitrogen atmosphere. The crystals belong to the P2 space group, with unit-cell parameters a = 73.93, b = 158.48, c = 74.23 Å, and diffract to 2.42 Å resolution using synchrotron radiation. A self-rotation function calculation revealed that the terminase oligomerizes into an octamer in the asymmetric unit, although size-exclusion chromatography suggests that it is possible for it to form an oligomer of up to 13 subunits. PMID:23519803

  2. Expression, purification, crystallization and preliminary X-ray analysis of the EIICGlc domain of the Escherichia coli glucose transporter

    PubMed Central

    Zurbriggen, Andreas; Schneider, Philipp; Bähler, Priska; Baumann, Ulrich; Erni, Bernhard

    2010-01-01

    The glucose-import system of Escherichia coli consists of a hydrophilic EIIAGlc subunit and a transmembrane EIICBGlc subunit. EIICBGlc (UniProt P69786) contains two domains: the transmembrane EIICGlc domain (40.6 kDa) and the cytoplasmic EIIBGlc domain (8.0 kDa), which are fused by a linker that is strongly conserved among its orthologues. The EIICBGlc subunit can be split within this motif by trypsin. Here, the crystallization of the tryptic EIICGlc domain is described. A complete data set was collected to 4.5 Å resolution at 100 K. PMID:20516600

  3. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of variants of monoamine oxidase from Aspergillus niger

    SciTech Connect

    Atkin, Kate E.; Reiss, Renate; Turner, Nicholas J.; Brzozowski, Andrzej M.; Grogan, Gideon

    2008-03-01

    Crystals of A. niger monoamine oxidase variants display P2{sub 1} or P4{sub 1}2{sub 1}2/P4{sub 3}2{sub 1}2 symmetry, with eight or two molecules in the asymmetric unit, respectively. Monoamine oxidase from Aspergillus niger (MAO-N) is an FAD-dependent enzyme that catalyses the conversion of terminal amines to their corresponding aldehydes. Variants of MAO-N produced by directed evolution have been shown to possess altered substrate specificity. Crystals of two of these variants (MAO-N-3 and MAO-N-5) have been obtained; the former displays P2{sub 1} symmetry with eight molecules per asymmetric unit and the latter has P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2 symmetry and two molecules per asymmetric unit. Solution of these structures will help shed light on the molecular determinants of improved activity and high enantioselectivity towards a broad range of substrates.

  4. Expression, purification, crystallization and X-ray diffraction analysis of ChiL, a chitinase from Chitiniphilus shinanonensis.

    PubMed

    Ueda, Miruku; Shimosaka, Makoto; Arai, Ryoichi

    2015-12-01

    Chitin, a linear polysaccharide consisting of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc), is widely used because of its biochemical properties. GlcNAc oligomers prepared from chitin have useful biological activities, such as immunostimulation and the induction of plant defence responses. Microbial chitinolytic enzymes have been investigated extensively for their potential use in the eco-friendly enzymatic production of GlcNAc and its oligomers. Chitiniphilus shinanonensis SAY3(T) is a recently found bacterium with a strong chitinolytic activity. The chitinolytic enzymes from this strain are potentially useful for the efficient production of GlcNAc and its oligomers from chitin. ChiL from C. shinanonensis is an endo-type chitinase belonging to the family 18 glycoside hydrolases (GH18). To understand the enzymatic reaction mechanism of ChiL and utilize it for further enzyme engineering, the catalytic domain (41-406) of ChiL, the construct for which was carefully designed, was expressed, purified and crystallized by the vapour-diffusion method. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 69.19, b = 81.55, c = 130.01 Å, and diffracted to 1.25 Å resolution. The Matthews coefficient (VM = 2.2 Å(3) Da(-1)) suggested the presence of two monomers per asymmetric unit with a solvent content of 45%. PMID:26625294

  5. Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase from Pyrococcus horikoshii OT3

    PubMed Central

    Wang, Xiaoying; Akasaka, Ryogo; Takemoto, Chie; Morita, Satoshi; Yamaguchi, Machiko; Terada, Takaho; Shirozu, Mikako; Yokoyama, Shigeyuki; Chen, Shilin; Si, Shuyi; Xie, Yong

    2011-01-01

    Adenylosuccinate synthetase (AdSS) is a ubiquitous enzyme that catalyzes the first committed step in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the purine-biosynthetic pathway. Although AdSS from the vast majority of organisms is 430–457 amino acids in length, AdSS sequences isolated from thermophilic archaea are 90–120 amino acids shorter. In this study, crystallographic studies of a short AdSS sequence from Pyrococcus horikoshii OT3 (PhAdSS) were performed in order to reveal the unusual structure of AdSS from thermophilic archaea. Crystals of PhAdSS were obtained by the microbatch-under-oil method and X-ray diffraction data were collected to 2.50 Å resolution. The crystal belonged to the trigonal space group P3212, with unit-cell parameters a = b = 57.2, c = 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å3 Da−1 and an approximate solvent content of 43%. In contrast, the results of native polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that the recombinant PhAdSS formed a dimer in solution. PMID:22139164

  6. 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.

  7. Expression, purification and crystallization of a BH domain from the GTPase regulatory protein associated with focal adhesion kinase.

    PubMed

    Sheffield, P J; Derewenda, U; Taylor, J; Parsons, T J; Derewenda, Z S

    1999-01-01

    Signaling by small GTPases is down-regulated by GTPase activating proteins (GAPs) which enhance the rate of GTP hydrolysis. The activity of GAPs specific for Rho GTPases resides in the BH domain, many homologues of which are found in any mammalian genome. One of them was identified in the GTPase regulator associated with focal-adhesion kinase (GRAF). It shares approximately 20% sequence identity with p50RhoGAP. This GAP activates RhoA and Cdc42Hs, but not Rac. In order to dissect the molecular basis of this specificity, a 231-residue-long fragment corresponding to the BH domain of GRAF has been expressed, purified and crystallized. Trigonal crystals, of space group P3(1)21 or P3(2)21, with unit-cell dimensions a = b = 63.5, c = 90.38 A were grown from solutions of PEG 6000. Data to 2.15 A were collected from a flash-frozen sample on an R-AXIS IV imaging-plate detector mounted on a rotating anode X-ray generator. PMID:10232922

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Purification and crystallization of a putative transcriptional regulator of the benzoate oxidation pathway in Burkholderia xenovorans LB400

    PubMed Central

    Law, Adrienne M.; Bains, Jasleen; Boulanger, Martin J.

    2009-01-01

    Burkholderia xenovorans LB400 harbours two paralogous copies of the recently discovered benzoate oxidation (box) pathway. While both copies are functional, the paralogues are differentially regulated and flanked by putative transcriptional regulators from distinct families. The putative LysR-type transcriptional regulator (LTTR) adjacent to the megaplasmid-encoded box enzymes, Bxe_C0898, has been produced recombinantly in Escherichia coli and purified to homogeneity. Gel-filtration studies show that Bxe_C0898 is a tetramer in solution, consistent with previously characterized LTTRs. Bxe_C0898 crystallized with four molecules in the asymmetric unit of the P43212/P41212 unit cell with a solvent content of 61.19%, as indicated by processing of the X-ray diffraction data. DNA-protection assays are currently under way in order to identify potential operator regions for this LTTR and to define its role in regulation of the box pathway. PMID:19851006

  14. Purification, crystallization and preliminary X-ray analysis of TP0435 (Tp17) from the syphilis spirochete Treponema pallidum

    PubMed Central

    Brautigam, Chad A.; Deka, Ranjit K.; Norgard, Michael V.

    2013-01-01

    Syphilis, caused by the bacterial spirochete Treponema pallidum, remains a prominent sexually transmitted infection worldwide. Despite sequencing of the genome of this obligate human pathogen 15 years ago, the functions of a large number of the gene products of T. pallidum are still unknown, particularly with respect to those of the organism’s periplasmic lipoproteins. To better understand their functions, a structural biology approach has been pursued. To this end, the soluble portion of the T. pallidum TP0435 lipoprotein (also known as Tp17) was cloned, hyper-expressed in Escherichia coli and purified to apparent homogeneity. The protein crystals obtained from this preparation diffracted to 2.4 Å resolution and had the symmetry of space group R3. In the hexagonal setting, the unit-cell parameters were a = b = 85.7, c = 85.4 Å. PMID:23545658

  15. 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

  16. GigaDB: promoting data dissemination and reproducibility.

    PubMed

    Sneddon, Tam P; Zhe, Xiao Si; 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

  17. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of propionate kinase (TdcD) from Salmonella typhimurium.

    PubMed

    Simanshu, Dhirendra K; Murthy, M R N

    2005-01-01

    In the cell, propionate is mainly formed during beta-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(1)21 or P3(2)21, with unit-cell parameters a = b = 111.47, c = 66.52 A. A complete data set to 2.2 A resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator. PMID:16508089

  18. 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

  19. Streptomyces beta-alanine:alpha-ketoglutarate aminotransferase, a novel omega-amino acid transaminase. Purification, crystallization, and enzymologic properties.

    PubMed

    Yonaha, K; Suzuki, K; Toyama, S

    1985-03-25

    An enzyme which catalyzes the transamination of beta-alanine with alpha-ketoglutarate was purified to homogeneity from Streptomyces griseus IFO 3102 and crystallized. Molecular weight of the enzyme was found to be 185,000 +/- 10,000 by a gel-filtration method. The enzyme consists of four subunits identical in molecular weight (51,000 +/- 1,000). The transaminase is composed of 483 amino acids/subunit containing 7 and 8 residues of half-cystine and methionine, respectively. The enzyme exhibits absorption maxima at 278 and 415 nm. The pyridoxal 5'-phosphate content was determined to be 4 mol/mol of enzyme. The enzyme catalyzes transamination of omega-amino acids including taurine and hypotaurine. beta-Alanine and DL-beta-aminoisobutyrate served as a good amino donor; the Michaelis constants are 8.0 and 12.5 mM, respectively. alpha-Ketoglutarate is the only amino acceptor (Km = 4.0 mM); pyruvate and oxalacetate are inactive. Based on the substrate specificity, the terminology of beta-alanine:alpha-ketoglutarate transaminase is proposed for the enzyme. Carbonyl reagents, HgCl2,DL-gabaculine, and alpha-fluoro-beta-alanine strongly inhibited the enzyme. PMID:3972825

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. Updrafts, Downdrafts, Entrainment, and Detrainment in the Giga-LES

    NASA Astrophysics Data System (ADS)

    Krueger, S. K.; Glenn, I.

    2012-12-01

    We are investigating the properties of evolving three-dimensional updraft and downdraft "cores" in a model dataset from the Giga-LES, a large-domain LES (large-eddy simulation) of tropical oceanic deep convection (Khairoutdinov et al. 2009). We have also applied the analysis method developed by Kuang and Bretherton (2006) to investigate various aspects of the ensemble characteristics of cumulus convection in the Giga-LES. Our results agree with those of Kuang and Bretherton for the cumulus updraft properties. We have examined the relative merits of different entrainment and cloud-top-height assumptions in spectral plume models of cumulus updrafts, the characteristics of downdrafts, and the nature of a rapid transition from shallow to deep convection.isualization of cumulus clouds from the Giga-LES. The realistic structure is associated with entrainment.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. Development of the analysis of fecal stanols in the oyster Crassostrea gigas and identification of fecal contamination in shellfish harvesting areas.

    PubMed

    Harrault, Loïc; Jardé, Emilie; Jeanneau, Laurent; Petitjean, Patrice

    2014-06-01

    The objective of this work was to study the effects of washing and purification steps on qualitative and quantitative analysis of fecal stanols in the oyster Crassostrea gigas using either single or a combination of lipid purification steps on silica gel or aminopropyl bonded silica gel (NH2) or a washing step. Among the three analytical pathways compared, the two including water extraction or NH2 purification did not lead to higher recoveries and decreased repeatabilities of extractions compared to the single purification on silica gel. This latter led to similar recoveries (ca. 80%) and repeatabilities (ca. 10%) for both spiked standards (coprostanol and sitostanol). This analytical pathway has been applied to oysters collected in a harvesting area in Brittany (France) where fecal contaminations are important and allowed to quantify eight stanols in oysters. The relative proportions of fecal stanols of these oysters were combined with principal component analysis in order to investigate the usefulness of their stanol fingerprints to record a fecal contamination and to distinguish its source between human, porcine and bovine contaminations. Oysters non-fecally contaminated by Escherichia coli did not present specific stanol fingerprints while oysters fecally contaminated had a bovine fingerprint, suggesting a contamination of these samples by bovine sources. As a consequence, the method developed here allows the use of stanol fingerprints of oysters as a microbial source tracking tool that can be applied to shellfish harvesting areas subjected to fecal contaminations in order to identify the different sources of contamination and improve watershed management. PMID:24771549

  16. 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.

  17. Stochasticity of convection in Giga-LES data

    NASA Astrophysics Data System (ADS)

    De La Chevrotière, Michèle; Khouider, Boualem; Majda, Andrew J.

    2015-12-01

    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

  18. 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}.

  19. Expression, purification, crystallization and preliminary X-ray analysis of a C-terminal fragment of the Epstein–Barr virus ZEBRA protein

    SciTech Connect

    Morand, Patrice; Budayova-Spano, Monika; Perrissin, Monique; Müller, Christoph W. Petosa, Carlo

    2006-03-01

    A C-terminal fragment of the Epstein–Barr virus lytic switch protein ZEBRA has been crystallized in complex with DNA. A C-terminal fragment of the Epstein–Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 Å resolution using synchrotron radiation (λ = 0.976 Å). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 Å, β = 103.9°.

  20. Purification, crystallization and preliminary X-ray diffraction studies of the soluble domain of the oligosaccharyltransferase STT3 subunit from the thermophilic archaeon Pyrococcus furiosus

    SciTech Connect

    Igura, Mayumi; Maita, Nobuo; Obita, Takayuki; Kamishikiryo, Jun; Maenaka, Katsumi; Kohda, Daisuke

    2007-09-01

    The C-terminal soluble domain of the catalytic subunit (STT3) of the oligosaccharyltransferase from P. furiosus was purified and crystallized. A native crystal and a SeMet derivative have been analyzed using X-ray diffraction. Oligosaccharyltransferase catalyzes the transfer of preassembled oligosaccharides onto asparagine residues in nascent polypeptide chains. The STT3 subunit is thought to bear the catalytic site. The C-terminal domain of the STT3 protein of Pyrococcus furiosus was expressed in Escherichia coli cells. STT3 protein prepared from two different sources, the soluble fraction and the inclusion bodies, produced crystals that diffracted to 2.7 Å. During crystallization screening, cocrystals of P. furiosus STT3 with an E. coli 50S ribosomal protein, L7/L12, were accidentally obtained. This cross-species interaction is not biologically relevant, but may be used to design a built-in polypeptide substrate for the STT3 crystals.

  1. Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

    SciTech Connect

    Rohman, Ali; Oosterwijk, Niels van; Kralj, Slavko; Dijkhuizen, Lubbert; Dijkstra, Bauke W.; Puspaningsih, Ni Nyoman Tri

    2007-11-01

    The β-xylosidase was crystallized using PEG 6000 as precipitant. 5% PEG 6000 yielded bipyramid-shaped tetragonal crystals diffracting to 1.55 Å resolution, and 13% PEG 6000 gave rectangular monoclinic crystals diffracting to 1.80 Å resolution. The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-β-xylanase and β-xylosidase. β-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-β-xylanase into xylose monomers. The β-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 62.53, c = 277.4 Å diffracted to 1.55 Å resolution. The rectangular crystals belonged to space group P2{sub 1}, with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 Å, β = 90.5°, and diffracted to 1.80 Å resolution.

  2. 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

  3. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the structural domain of the nucleocapsid N protein from porcine reproductive and respiratory syndrome virus (PRRSV).

    PubMed

    Doan, Danny N P; Dokland, Terje

    2003-08-01

    The structural domain of the PRRSV nucleocapsid N protein was overexpressed in Escherichia coli and purified to homogeneity. Crystals of the expressed protein, designated His-Ndelta(57), were obtained by hanging-drop vapour diffusion using PEG 3350 as precipitant at pH 6.5. A native data set from a frozen crystal was collected to 2.7 A resolution using synchrotron radiation. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = 44.41, c = 125.05, and contain a dimer in the asymmetric unit. PMID:12876367

  4. 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

  5. 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

  6. Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor

    SciTech Connect

    Singh, Prem; Talawar, Ramappa K.; Krishna, P. D. V.; Varshney, Umesh; Vijayan, M.

    2006-12-01

    Uracil N-glycosylase from M. tuberculosis has been crystallized in complex with a proteinaceous inhibitor (Ugi) and X-ray diffraction data have been collected. Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 Å, β = 109.7°. X-ray data from the crystal have been collected for structure analysis.

  7. 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.

  8. 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

  9. Expression, purification, crystallization and preliminary X-ray analysis of the human RuvB-like protein RuvBL1

    SciTech Connect

    Gorynia, Sabine; Gonçalves, Susana; Coelho, Ricardo; Lopes, Gonçalo; Thomaz, Mónica; Huber, Martina; Donner, Peter

    2006-01-01

    Human RuvB-like protein RuvBL1 plays important roles in essential signaling pathways like c-Myc and Wnt, in transcription, and in DNA repair and apoptosis. Crystals of both native and a Se-Met derivative were obtained and characterized. SAD data leading to the structure solution at 2.2 Å were measured from the Se-Met crystals. RuvBL1, an evolutionary highly conserved protein related to the AAA{sup +} family of ATPases, has been crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystals are hexagonal and belong to space group P6, with unit-cell parameters a = b = 207.1, c = 60.7 Å and three molecules in the asymmetric unit.

  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. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of L-lactate dehydrogenase and its H171C mutant from Bacillus subtilis

    SciTech Connect

    Zhang, Yanfeng; Gao, Xiaoli

    2012-08-31

    L-Lactate dehydrogenase (LDH) is an important enzyme involved in the last step of glycolysis that catalyzes the reversible conversion of pyruvate to L-lactate with the simultaneous oxidation of NADH to NAD{sup +}. In this study, wild-type LDH from Bacillus subtilis (BsLDH-WT) and the H171C mutant (BsLDH-H171C) were expressed in Escherichia coli and purified to near-homogeneity. BsLDH-WT was crystallized in the presence of fructose 1,6-bisphosphate (FBP) and NAD{sup +} and the crystal diffracted to 2.38 {angstrom} resolution. The crystal belonged to space group P3, with unit-cell parameters a = b = 171.04, c = 96.27 {angstrom}. BsLDH-H171C was also crystallized as the apoenzyme and in complex with NAD{sup +}, and data sets were collected to 2.20 and 2.49 {angstrom} resolution, respectively. Both BsLDH-H171C crystals belonged to space group P3, with unit-cell parameters a = b = 133.41, c = 99.34 {angstrom} and a = b = 133.43, c = 99.09 {angstrom}, respectively. Tetramers were observed in the asymmetric units of all three crystals.

  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. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP5 2-K from Arabidopsis thaliana

    PubMed Central

    Baños-Sanz, Jose Ignacio; Sanz-Aparicio, Julia; Brearley, Charles A.; González, Beatriz

    2012-01-01

    Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5 2-K) is a key enzyme that catalyzes the synthesis of phytic acid (IP6) from inositol 1,3,4,5,6-pentakisphos­phate (IP5) and ATP. The first structure of IP5 2-K, that from Arabidopsis thaliana, has been solved previously; it only crystallized in the presence of inositol, either the substrate IP5 or the product IP6, and failed to crystallize in its free state (without inositol). Based on structural analysis, a point mutation of IP5 2-K (W129A) has been produced in order to overcome this limitation and obtain information about protein conformational changes upon substrate binding. Here, the production and crystallization of W129A IP5 2-K in its free state and with bound nucleotide is described. These crystals differed from the native crystals and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 66.00, b = 68.23, c = 105.80 Å and a = 63.06, b = 71.80, c = 100.23 Å, respectively. The crystals diffracted to resolutions of 2.22 Å (apo) and 2.05 Å (nucleotide bound) using synchrotron radiation and contained one molecule per asymmetric unit. The structures have been determined using the molecular-replacement method and refinement is being undertaken. PMID:22684075

  17. 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.

  18. 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.

  19. 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.

  20. Overexpression, purification and crystallization of the two C-terminal domains of the bifunctional cellulase ctCel9D-Cel44A from Clostridium thermocellum

    SciTech Connect

    Najmudin, Shabir; Guerreiro, Catarina I. P. D.; Ferreira, Luís M. A.; Romão, Maria J. C.; Fontes, Carlos M. G. A.; Prates, José A. M.

    2005-12-01

    The two C-terminal domains of the cellulase ctCel9D-Cel44A from C. thermocellum cellulosome have been crystallized in tetragonal space group P4{sub 3}2{sub 1}2 and X-ray diffraction data have been collected to 2.1 and 2.8 Å from native and seleno-l-methionine-derivative crystals, respectively. Clostridium thermocellum produces a highly organized multi-enzyme complex of cellulases and hemicellulases for the hydrolysis of plant cell-wall polysaccharides, which is termed the cellulosome. The bifunctional multi-modular cellulase ctCel9D-Cel44A is one of the largest components of the C. thermocellum cellulosome. The enzyme contains two internal catalytic domains belonging to glycoside hydrolase families 9 and 44. The C-terminus of this cellulase, comprising a polycystic kidney-disease module (PKD) and a carbohydrate-binding module (CBM44), has been crystallized. The crystals belong to the tetragonal space group P4{sub 3}2{sub 1}2, containing a single molecule in the asymmetric unit. Native and seleno-l-methionine-derivative crystals diffracted to 2.1 and 2.8 Å, respectively.

  1. Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-β-d-xylanase 10B in complex with xylohexaose

    SciTech Connect

    Najmudin, Shabir; Pinheiro, Benedita A.; Romão, Maria J.; Prates, José A. M.; Fontes, Carlos M. G. A.

    2008-08-01

    The N-terminal moiety of C. thermocellum endo-1,4-β-d-xylanase 10B, comprising a carbohydrate-binding module (CBM22-1) and a GH10 E337A mutant domain, has been crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3{sub 2}21, contain a dimer in the asymmetric unit and diffract to beyond 2.0 Å resolution. The cellulosome of Clostridium thermocellum is a highly organized multi-enzyme complex of cellulases and hemicellulases involved in the hydrolysis of plant cell-wall polysaccharides. The bifunctional multi-modular xylanase Xyn10B is one of the hemicellulase components of the C. thermocellum cellulosome. The enzyme contains an internal glycoside hydrolase family 10 catalytic domain (GH10) and a C-terminal family 1 carbohydrate esterase domain (CE1). The N-terminal moiety of Xyn10B (residues 32–551), comprising a carbohydrate-binding module (CBM22-1) and the GH10 E337A mutant, was crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3{sub 2}21 and contain a dimer in the asymmetric unit. The crystals diffracted to beyond 2.0 Å resolution.

  2. 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.

  3. Expression, purification, crystallization and preliminary crystallographic analysis of a GH20 β-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi.

    PubMed

    Meekrathok, Piyanat; Bürger, Marco; Porfetye, Arthur T; Vetter, Ingrid R; Suginta, Wipa

    2015-04-01

    Vibrio harveyi β-N-acetylglucosaminidase (VhGlcNAcase) is a new member of the GH20 glycoside hydrolase family responsible for the complete degradation of chitin fragments, with N-acetylglucosamine (GlcNAc) monomers as the final products. In this study, the crystallization and preliminary crystallographic data of wild-type VhGlcNAcase and its catalytically inactive mutant D437A in the absence and the presence of substrate are reported. Crystals of wild-type VhGlcNAcase were grown in 0.1 M sodium acetate pH 4.6, 1.4 M sodium malonate, while crystals of the D437A mutant were obtained in 0.1 M bis-tris pH 7.5, 0.1 M sodium acetate, 20% PEG 3350. X-ray data from the wild-type and the mutant crystals were collected at a synchrotron-radiation light source and were complete to a resolution of 2.5 Å. All crystals were composed of the same type of dimer, with the substrate N,N'-diacetylglucosamine (GlcNAc₂ or diNAG) used for soaking was cleaved by the active enzyme, leaving only a single GlcNAc molecule bound to the protein. PMID:25849504

  4. 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.

  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 crystallographic studies of the complex between Smc5 and the SUMO E3 ligase Mms21

    SciTech Connect

    Duan, Xinyuan; Ye, Hong

    2010-03-04

    Smc5/6, a protein complex that belongs to the structural maintenance of chromosome (SMC) family, plays a key role in DNA replication, sister chromatid recombination and DNA damage repair. The complex contains eight subunits, including a SUMO E3 ligase Mms21 (Nse2). The activity of Mms21 is important for regulation of Smc5/6 in the response to DNA damage. Mms21 and the Mms21-binding region of Smc5 were overexpressed and purified individually in Escherichia coli with a C-terminal LEHHHHHH tag. The Mms21-Smc5 protein complex was crystallized. The diffraction of the crystals was improved greatly by glutaraldehyde treatment. X-ray diffraction data sets were collected to resolutions of 2.3 and 3.9 {angstrom} from native and selenomethionine-derivative protein crystals, respectively. The crystals belonged to space group C2221, with unit-cell parameters a = 47.465, b = 97.574, c = 249.215 {angstrom} for the native crystals.

  7. 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.

  8. Purification, crystallization and preliminary X-ray crystallographic analysis of the UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF) from Acinetobacter baumannii.

    PubMed

    An, Young Jun; Jeong, Chang-Sook; Yu, Jeong Hee; Chung, Kyung Min; Cha, Sun-Shin

    2014-07-01

    The emergence and global spread of multidrug-resistant Acinetobacter baumannii strains are major threats to public health. Inhibition of peptidoglycan biosynthesis is an effective strategy for the development of antibiotics. The ATP-dependent UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF) that is responsible for the last step of peptidoglycan biosynthesis is a validated target for the development of antibiotics. Crystals of A. baumannii MurF in complex with ATP were grown by the microbatch crystallization method at 295 K. The crystals belonged to space group P322₁, with unit-cell parameters a=b=85.42, c=129.86 Å. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 54.32%. PMID:25005102

  9. 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.

  10. 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%.

  11. 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.

  12. 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.

  13. 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%.

  14. Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the putative NlpC/P60 endopeptidase, TTHA0266, from Thermus thermophilus HB8

    PubMed Central

    Wong, Jaslyn E. M. M.; Blaise, Mickael

    2013-01-01

    Autolysins belong to a protein family involved in peptidoglycan degradation and remodelling. Within this family, NlpC/P60 endopeptidases are involved in the hydrolysis of the peptide arm of peptidoglycan. In this work, the putative NlpC/P60 endopeptidase TTHA0266 from Thermus thermophilus HB8 was overexpressed, purified and crystallized. The crystals diffracted to 2.4 Å resolution and belonged to the hexagonal space group P61, with unit-cell parameters a = b = 71.19, c = 198.68 Å, γ = 120°. Selenomethionine-substituted protein was crystallized and the structure was solved by single-wavelength anomalous dispersion. PMID:24192372

  15. 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

  16. 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%.

  17. 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.

  18. 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}.

  19. 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

  20. 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

  1. 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%.

  2. Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus

    SciTech Connect

    Nishida, Hirokazu; Matsumiya, Shigeki; Tsuchiya, Daisuke; Ishino, Yoshizumi; Morikawa, Kosuke

    2006-03-01

    A stable stoichiometric complex of archaeal DNA polymerase with proliferating cell nuclear antigen (PCNA) was formed using a PCNA monomer mutant and the complex was successfully crystallized. Replicative DNA polymerase interacts with processivity factors, the β-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 Å resolution using synchrotron radiation. The crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 Å.

  3. Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of XC847, a 3′-5′ oligoribonuclease from Xanthomonas campestris

    SciTech Connect

    Wu, Yan-You; Chin, Ko-Hsin; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Gao, Fei Philip; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-10-01

    A DEDDh-type 3′-5′ oligoribonuclease from the plant pathogen X. campestris pv. campestris has been overexpressed in E. coli, purified and crystallized. The crystals diffracted to 2.1 Å with good quality. Oligoribonucleases are essential components of RNA and DNA metabolism and close homologues of genes encoding them are found not only in prokaryotes but also in a wide range of eukaryotes, including yeast and humans. Inactivation of the oligoribonuclease gene (orn) can result in cellular lethality. Despite their important biological function, they have been studied little from a structural point of view. In this report, the cloning, expression, crystallization and preliminary X-ray analysis of XC847, a DEDDh-type 3′-5′ oligoribonuclease from the plant pathogen Xanthomonas campestris pv. campestris, a Gram-negative bacterium causing major worldwide disease of cruciferous crops, is described. The XC847 crystals diffracted to a resolution of at least 2.1 Å. They are tetragonal and belong to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 67.5, c = 89.8 Å. One molecule is present per asymmetric unit.

  4. 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.

  5. 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.

  6. 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.

  7. Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of Ss-LrpB, a transcription regulator from Sulfolobus solfataricus

    SciTech Connect

    Peeters, Eveline; Hoa, Bach Thi Mai; Zegers, Ingrid; Charlier, Daniel; Maes, Dominique

    2005-11-01

    The C-terminal domain of the transcriptional regulator Ss-LrpB from S. solfataricus was purified by affinity chromatography and crystallized. Crystals belong to space group P2{sub 1}2{sub 1}2. A complete data set was collected to a resolution of 2 Å. Ss-LrpB from Sulfolobus solfataricus P2 belongs to the bacterial/archaeal superfamily of Lrp-like (leucine-responsive regulatory protein-like) transcription regulators. The N-terminal DNA-binding domain contains a HTH motif and the C-terminal domain contains an αβ-sandwich (βαββαβ fold). The C-terminal domain was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 59.35, b = 74.86, c = 38.08 Å and a data set was collected to 2.0 Å resolution. Structure determination using a selenomethionine derivative is under way.

  8. Purification, crystallization and preliminary X-ray analysis of the BseCI DNA methyltransferase from Bacillus stearothermophilus in complex with its cognate DNA

    SciTech Connect

    Kapetaniou, Evangelia G.; Kotsifaki, Dina; Providaki, Mary; Rina, Maria; Bouriotis, Vassilis; Kokkinidis, Michael

    2007-01-01

    The DNA methyltransferase M.BseCI from B. stearothermophilus was crystallized as a complex with its cognate DNA. Crystals belong to space group P6 and diffract to 2.5 Å resolution at a synchrotron source. The DNA methyltransferase M.BseCI from Bacillus stearothermophilus (EC 2.1.1.72), a 579-amino-acid enzyme, methylates the N6 atom of the 3′ adenine in the sequence 5′-ATCGAT-3′. M.BseCI was crystallized in complex with its cognate DNA. The crystals were found to belong to the hexagonal space group P6, with unit-cell parameters a = b = 87.0, c = 156.1 Å, β = 120.0° and one molecule in the asymmetric unit. Two complete data sets were collected at wavelengths of 1.1 and 2.0 Å to 2.5 and 2.8 Å resolution, respectively, using synchrotron radiation at 100 K.

  9. 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.

  10. Expression, purification, crystallization and preliminary X-ray analysis of the KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

    PubMed Central

    Kang, Hee-Jin; Kubota, Keiko; Miyazono, Ken-ichi; Tanokura, Masaru

    2011-01-01

    KaiC is the central protein in the circadian rhythm in cyanobacteria. The 28 kDa KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of PH0187 were obtained using a reservoir solution consisting of 1.0 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic pH 5.3 (the final pH value of the reservoir solution was 4.8) and diffracted X-rays to 2.75 Å resolution. The crystal of PH0187 belonged to space group P6322, with unit-cell parameters a = b = 239.1, c = 106.5 Å. The crystal contained four PH0187 molecules in the asymmetric unit. PMID:21206047

  11. Purification, crystallization and preliminary X-ray analysis of human mannose-binding lectin-associated serine protease-1 (MASP-1) catalytic region

    PubMed Central

    Dobó, József; Harmat, Veronika; Sebestyén, Edina; Beinrohr, László; Závodszky, Péter; Gál, Péter

    2008-01-01

    MASP-1, a multidomain serine protease, is a component of the lectin pathway of complement. Its precise function is unknown, although it seems to enhance the complement-activating capacity of MASP-2, a related enzyme. MASP-1 has also been implicated as playing a role in blood coagulation. It is mostly found associated with mannose-binding lectin (MBL) and ficolins. Early attempts to crystallize MASP-1 failed because of the inhomogeneity of the purified material. MASP-1 was shown by acidic nondenaturing PAGE to be composed of differently charged species, which are most likely to be the products of deamidation occurring during the refolding procedure. Sequential cation-exchange and anion-exchange chromatography resulted in a homogeneous material, which was successfully crystallized. The best crystal diffracted to 2.55 Å resolution and belonged to space group P212121, with unit-cell parameters a = 68.4, b = 70.4, c = 121.4 Å. The crystal structure of MASP-1 may help in understanding the function of this mysterious serine protease. PMID:18765903

  12. Purification, crystallization and preliminary X-ray analysis of human mannose-binding lectin-associated serine protease-1 (MASP-1) catalytic region.

    PubMed

    Dobó, József; Harmat, Veronika; Sebestyén, Edina; Beinrohr, László; Závodszky, Péter; Gál, Péter

    2008-09-01

    MASP-1, a multidomain serine protease, is a component of the lectin pathway of complement. Its precise function is unknown, although it seems to enhance the complement-activating capacity of MASP-2, a related enzyme. MASP-1 has also been implicated as playing a role in blood coagulation. It is mostly found associated with mannose-binding lectin (MBL) and ficolins. Early attempts to crystallize MASP-1 failed because of the inhomogeneity of the purified material. MASP-1 was shown by acidic nondenaturing PAGE to be composed of differently charged species, which are most likely to be the products of deamidation occurring during the refolding procedure. Sequential cation-exchange and anion-exchange chromatography resulted in a homogeneous material, which was successfully crystallized. The best crystal diffracted to 2.55 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 68.4, b = 70.4, c = 121.4 A. The crystal structure of MASP-1 may help in understanding the function of this mysterious serine protease. PMID:18765903

  13. Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

    PubMed Central

    Rohman, Ali; van Oosterwijk, Niels; Kralj, Slavko; Dijkhuizen, Lubbert; Dijkstra, Bauke W.; Puspaningsih, Ni Nyoman Tri

    2007-01-01

    The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-β-­xylanase and β-xylosidase. β-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-β-xylanase into xylose monomers. The β-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P43212, with unit-cell parameters a = b = 62.53, c = 277.4 Å diffracted to 1.55 Å resolution. The rectangular crystals belonged to space group P21, with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 Å, β = 90.5°, and diffracted to 1.80 Å resolution. PMID:18007043

  14. Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system

    SciTech Connect

    Roversi, Pietro; Johnson, Steven; Field, Terry; Deane, Janet E.; Galyov, Edouard E.; Lea, Susan M.

    2006-09-01

    A construct consisting of residues 10–310 of mature BipD, a component of the B. pseudomallei type III secretion system, has been crystallized. Native BipD crystals and SeMet and K{sub 2}PtCl{sub 4} derivative crystals have undergone preliminary crystallographic analysis. 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 Å{sup 3} Da{sup −1} (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å{sup 3} Da{sup −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 P2{sub 1}2{sub 1}2 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P2{sub 1}2{sub 1}2 crystals diffract to 3.3 Å resolution. A K{sub 2}PtCl{sub 4} derivative of the P2{sub 1}2{sub 1}2 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan

    PubMed Central

    Vieira, Diana; Figueiredo, Teresa A.; Verma, Anil; Sobral, Rita G.; Ludovice, Ana M.; de Lencastre, Hermínia; Trincao, Jose

    2014-01-01

    Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases. PMID:24817726

  20. 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. PMID:22102044

  1. Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the C-­terminal domain of Ss-LrpB, a transcription regulator from Sulfolobus solfataricus

    PubMed Central

    Peeters, Eveline; Hoa, Bach Thi Mai; Zegers, Ingrid; Charlier, Daniel; Maes, Dominique

    2005-01-01

    Ss-LrpB from Sulfolobus solfataricus P2 belongs to the bacterial/archaeal superfamily of Lrp-like (leucine-responsive regulatory protein-like) transcription regulators. The N-terminal DNA-binding domain contains a HTH motif and the C-terminal domain contains an αβ-sandwich (βαββαβ fold). The C-­terminal domain was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P21212, with unit-cell parameters a = 59.35, b = 74.86, c = 38.08 Å and a data set was collected to 2.0 Å resolution. Structure determination using a selenomethionine derivative is under way. PMID:16511214

  2. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) from Bacillus cereus

    PubMed Central

    Skråmo, Silje; Hersleth, Hans-Petter; Hammerstad, Marta; Andersson, K. Kristoffer; Røhr, Åsmund K.

    2014-01-01

    Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) are key enzymes involved in catalysing electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. In Bacillus cereus there are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallized in its NADPH/NADP+-free form. Diffraction data have been collected to 2.5 Å resolution from crystals belonging to the orthorhombic space group P21212, with unit-cell parameters a = 57.2, b = 164.3, c = 95.0 Å, containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has been solved by molecular replacement, and is a member of the homodimeric thioredoxin reductase-like class of FNRs. PMID:24915092

  3. 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

  4. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Aspergillus terreus endo-β-1,4-glucanase from glycoside hydrolase family 12.

    PubMed

    Segato, Fernando; Berto, Gabriela L; Ares de Araújo, Evandro; Muniz, João Renato; Polikarpov, Igor

    2014-02-01

    Endoglucanases are important enzymes that are involved in the modification and degradation of cellulose. Filamentous fungi such as Aspergillus terreus are effective biomass degraders in nature owing to their capacity to produce an enzymatic arsenal of glycoside hydrolases, including endoglucanase from glycoside hydrolase family 12 (GH12). The A. terreus GH12 endoglucanase was cloned and overexpressed in A. nidulans, purified and crystallized. A single crystal was obtained from a solution consisting of 2 M ammonium sulfate, 5%(v/v) 2-propanol. X-ray diffraction data were collected to a resolution of 1.85 Å using synchrotron radiation and a preliminary molecular-replacement solution was obtained in the trigonal space group P3(2)21. The unit-cell parameters were a = b = 103.24, c = 48.96 Å. PMID:24637772

  5. 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.

  6. 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

  7. Expression, purification, crystallization and preliminary X-ray diffraction studies of triosephosphate isomerase from methicillin-resistant Staphylococcus aureus (MRSA252)

    PubMed Central

    Mukherjee, Somnath; Dutta, Debajyoti; Saha, Baisakhee; Das, Amit Kumar

    2009-01-01

    Triosephosphate isomerase from methicillin-resistant Staphylococcus aureus (MRSA252) was cloned in pQE30 vector, overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized from 1.6 M trisodium citrate dihydrate pH 6.5 using the hanging-drop vapour-diffusion method. The crystals belonged to space group P43212, with unit-cell parameters a = b = 79.15, c = 174.27 Å. X-ray diffraction data were collected and processed to a maximum resolution of 1.9 Å. The presence of two molecules in the asymmetric unit gave a Matthews coefficient (V M) of 2.64 Å3 Da−1, with a solvent content of 53.63%. PMID:19342791

  8. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Aspergillus terreus endo-β-1,4-glucanase from glycoside hydrolase family 12

    PubMed Central

    Segato, Fernando; Berto, Gabriela L.; Ares de Araújo, Evandro; Muniz, João Renato; Polikarpov, Igor

    2014-01-01

    Endoglucanases are important enzymes that are involved in the modification and degradation of cellulose. Filamentous fungi such as Aspergillus terreus are effective biomass degraders in nature owing to their capacity to produce an enzymatic arsenal of glycoside hydrolases, including endoglucanase from glycoside hydrolase family 12 (GH12). The A. terreus GH12 endoglucanase was cloned and overexpressed in A. nidulans, purified and crystallized. A single crystal was obtained from a solution consisting of 2 M ammonium sulfate, 5%(v/v) 2-propanol. X-ray diffraction data were collected to a resolution of 1.85 Å using synchrotron radiation and a preliminary molecular-replacement solution was obtained in the trigonal space group P3221. The unit-cell parameters were a = b = 103.24, c = 48.96 Å. PMID:24637772

  9. 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.

  10. 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

  11. A thioredoxin fusion protein of VanH, a D-lactate dehydrogenase from Enterococcus faecium: cloning, expression, purification, kinetic analysis, and crystallization.

    PubMed Central

    Stoll, V. S.; Manohar, A. V.; Gillon, W.; MacFarlane, E. L.; Hynes, R. C.; Pai, E. F.

    1998-01-01

    The gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D-lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition site under a variety of conditions resulted in nonspecific proteolysis and inactivation of the protein. The intact fusion protein was, therefore, used for kinetic studies and crystallization trials. It has been purified to greater than 90% homogeneity by ammonium sulfate precipitation followed by phenyl Sepharose chromatography. Based on k(cat)/KM for pyruvate, it is 20% as active as native VanH. Michaelis constants for NADPH, NADH, and pyruvate, of approximately 3.5 microM, 19.0 microM, and 1.5 mM, respectively, were comparable to those reported for the native VanH (Bugg TDH et al., 1991, Biochemistry 30:10408-10415). Like native VanH, maximum activity of the fusion protein requires the presence of an anion (phosphate or acetate), however, in addition, a strongly reducing environment is needed for optimal efficacy. Competitive inhibition constants for ADP-ribose, NAD+, and oxamate have also been determined. Crystallization by hanging drop vapor diffusion produced two different crystal forms, one hexagonal and the other tetragonal. Flash-frozen crystals of the tetragonal form diffracted to 3.0 A resolution at a synchrotron radiation source. PMID:9605319

  12. 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.

  13. 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.

  14. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase from Streptococcus agalactiae NEM316.

    PubMed

    Nagarajan, Revathi; Ponnuraj, Karthe

    2014-07-01

    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. Purification, crystallization and preliminary X-ray analysis of the catalytic domain of the Escherichia coli tRNase colicin D

    SciTech Connect

    Takahashi, Kazutoshi; Ogawa, Tetsuhiro; Hidaka, Makoto; Ohsawa, Kanju; Masaki, Haruhiko; Yajima, Shunsuke

    2006-01-01

    The tRNase domain of colicin D, which is specific to tRNA{sup Arg}s, has been crystallized. A diffraction data set has been collected to a resolution of 1.05 Å. The tRNase domain of colicin D, which cleaves only tRNA{sup Arg}s at the 3′ side of their anticodon loops, has been expressed in Escherichia coli with its inhibitor protein and purified to a form free from the inhibitor using a low-pH buffer. Crystals were obtained by the hanging-drop vapour-diffusion method at 278 K from a buffer containing 100 mM Tris–HCl pH 8.5, 22% PEG MME 2000 and 1 mM nickel(II) chloride. Diffraction data to 1.05 Å resolution were collected at BL41XU, SPring-8. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 34.7, b = 65.5, c = 96.5 Å.

  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. PMID:25760706

  17. 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

  18. 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.

  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. 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

  1. Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction studies of histidinol phosphate aminotransferase (HisC2) from Mycobacterium tuberculosis

    PubMed Central

    Nasir, Nazia; Vyas, Rajan; Chugh, Chetna; Ahangar, Mohammad Syed; Biswal, Bichitra K.

    2012-01-01

    HisC2 from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel–nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion technique from a condition consisting of 7 mg ml−1 HisC2 (in 20 mM Tris pH 8.8, 50 mM NaCl and 5% glycerol), 1 M succinic acid pH 7.0, 0.1 M HEPES pH 7.0 and 1%(w/v) polyethylene glycol monomethyl ether 2000. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 255.98, b = 77.09, c = 117.97 Å. X-ray diffraction data were recorded to 2.45 Å resolution from a single crystal using the in-house X-ray facility. PMID:22232166

  2. 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.

  3. 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.

  4. 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.

  5. Purification, crystallization and preliminary X-ray diffraction analysis of the Fyn SH2 domain and its complex with a phosphotyrosine peptide

    PubMed Central

    Huculeci, Radu; Buts, Lieven; Lenaerts, Tom; van Nuland, Nico A. J.; Garcia-Pino, Abel

    2012-01-01

    SH2 domains are widespread protein-binding modules that recognize phosphotyrosines and play central roles in intracellular signalling pathways. The SH2 domain of the human protein tyrosine kinase Fyn has been expressed, purified and crystallized in the unbound state and in complex with a high-affinity phosphotyrosine peptide. X-ray data were collected to a resolution of 2.00 Å for the unbound form and 1.40 Å for the protein in complex with the phosphotyrosine peptide. PMID:22442244

  6. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine-6-phosphate 2-epimerase from methicillin-resistant Staphylococcus aureus.

    PubMed

    North, Rachel A; Kessans, Sarah A; Griffin, Michael D W; Watson, Andrew J A; Fairbanks, Antony J; Dobson, Renwick C J

    2014-05-01

    Sialic acids are one of the most important carbohydrate classes in biology. Some bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This sequestration and subsequent catabolism of sialic acid require a cluster of genes known as the `Nan-Nag' cluster. The enzymes coded by these genes are important for pathogen colonization and persistence. Importantly, the Nan-Nag genes have proven to be essential for Staphylococcus aureus growth on sialic acids, suggesting that the pathway is a viable antibiotic drug target. The enzyme N-acetylmannosamine-6-phosphate 2-epimerase is involved in the catabolism of sialic acid; specifically, the enzyme converts N-acetylmannosamine-6-phosphate into N-acetylglucosamine-6-phosphate. The gene was cloned into an appropriate expression vector, and recombinant protein was expressed in Escherichia coli BL21 (DE3) cells and purified via a three-step procedure. Purified N-acetylmannosamine-6-phosphate 2-epimerase was screened for crystallization. The best crystal diffracted to a resolution of beyond 1.84 Å in space group P21212. Understanding the structural nature of this enzyme from methicillin-resistant S. aureus will provide us with the insights necessary for the development of future antibiotics. PMID:24817730

  7. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the transcriptional repressor SirR from Mycobacterium tuberculosis H37Rv

    PubMed Central

    Saha, Baisakhee; Mukherjee, Somnath; Dutta, Debajyoti; Das, Amit Kumar

    2009-01-01

    SirR, a metal-dependent transcriptional repressor from Mycobacterium tuber­culosis (Rv2788), was cloned in pQE30 expression vector with an N-­terminal His6 tag for heterologous overexpression in Escherichia coli M15 (pREP4) cells and purified to homogeneity using chromatographic procedures. The purified protein was crystallized using the sitting-drop vapour-diffusion technique. The crystals belonged to the tetragonal space group P41212/P43212, with unit-cell parameters a = 105.21, b = 105.21, c = 144.85 Å. The X-ray diffraction data were processed to a maximum resolution of 2.5 Å. The Matthews coefficient suggests the presence of two (V M = 4.01 Å3 Da−1) to four (V M = 2.0 Å3 Da−1) molecules in the asymmetric unit. Calculation of the self-rotation function shows a crystallographic fourfold symmetry axis along the z axis (χ = 90°) and also a twofold symmetry axis around the z axis (χ = 180°). PMID:19194009

  8. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the transcriptional repressor SirR from Mycobacterium tuberculosis H37Rv.

    PubMed

    Saha, Baisakhee; Mukherjee, Somnath; Dutta, Debajyoti; Das, Amit Kumar

    2009-02-01

    SirR, a metal-dependent transcriptional repressor from Mycobacterium tuberculosis (Rv2788), was cloned in pQE30 expression vector with an N-terminal His(6) tag for heterologous overexpression in Escherichia coli M15 (pREP4) cells and purified to homogeneity using chromatographic procedures. The purified protein was crystallized using the sitting-drop vapour-diffusion technique. The crystals belonged to the tetragonal space group P4(1)2(1)2/P4(3)2(1)2, with unit-cell parameters a = 105.21, b = 105.21, c = 144.85 A. The X-ray diffraction data were processed to a maximum resolution of 2.5 A. The Matthews coefficient suggests the presence of two (V(M) = 4.01 A(3) Da(-1)) to four (V(M) = 2.0 A(3) Da(-1)) molecules in the asymmetric unit. Calculation of the self-rotation function shows a crystallographic fourfold symmetry axis along the z axis (chi = 90 degrees) and also a twofold symmetry axis around the z axis (chi = 180 degrees). PMID:19194009

  9. 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.

  10. 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

  11. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AHP2, a signal transmitter protein from Arabidopsis thaliana

    PubMed Central

    Degtjarik, Oksana; Dopitova, Radka; Puehringer, Sandra; Nejedla, Eliska; Kuty, Michal; Weiss, Manfred S.; Hejatko, Jan; Janda, Lubomir; Kuta Smatanova, Ivana

    2013-01-01

    Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1–5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution. PMID:23385758

  12. Cloning, purification, crystallization and preliminary X-ray diffraction studies of Escherichia coli PapD-like protein (EcpD).

    PubMed

    Pandey, Nishant Kumar; Pal, Ravi Kant; Kashyap, Maruthi; Bhavesh, Neel Sarovar

    2012-08-01

    Many Gram-negative bacteria are characterized by hair-like proteinaceous appendages on their surface known as fimbriae. In uropathogenic strains of Escherichia coli, fimbriae mediate attachment by binding to receptors on the host cell, often contributing to virulence and disease. E. coli PapD-like protein (EcpD) is a periplasmic chaperone that plays an important role in the proper folding and guiding of Yad fimbrial proteins to the outer membrane usher protein in a process known as pilus biogenesis. EcpD is essential for pilus biogenesis in uropathogenic E. coli and plays an important role in virulence. In the present study, EcpD was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 1.67 Å resolution and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 100.3, b = 127.6, c = 45.9 Å. There was a single molecule in the asymmetric unit and the corresponding Matthews coefficient was calculated to be 3.02 Å(3) Da(-1), with 59% solvent content. Initial phases were determined by molecular replacement. PMID:22869131

  13. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine kinase from methicillin-resistant Staphylococcus aureus.

    PubMed

    North, Rachel A; Seizova, Simona; Stampfli, Anja; Kessans, Sarah A; Suzuki, Hironori; Griffin, Michael D W; Kvansakul, Marc; Dobson, Renwick C J

    2014-05-01

    N-Acetylmannosamine kinase (EC 2.7.1.60) is involved in the catabolism of sialic acid for many bacterial pathogens implicated in human disease such as Escherichia coli, Staphylococcus aureus, Vibrio cholerae and V. vulnificus. Interestingly, some human commensals and bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This process requires a cluster of genes known as the `Nan-Nag cluster', which have proven to be essential for S. aureus growth on sialic acids, suggesting that the pathway is a viable antimicrobial drug target. The enzyme N-acetylmannosamine kinase is involved in the catabolism of sialic acid, transferring a phosphate group from adenosine-5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine-6-phosphate. The gene was cloned into an appropriate expression vector; recombinant protein was expressed in E. coli BL21 (DE3) cells and purified via anion-exchange chromatography, hydrophobic interaction chromatography and size-exclusion chromatography. Purified N-acetylmannosamine kinase was screened for crystallization. The best crystal diffracted to a resolution of beyond 2.6 Å in space group P2. Understanding the structural nature of this enzyme from methicillin-resistant S. aureus will provide insights necessary for the development of future antimicrobials. PMID:24817729

  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 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

  16. 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%.

  17. Purification, crystallization and preliminary X-ray diffraction studies of the three components of the toluene 2,3-dioxygenase enzyme system

    SciTech Connect

    Lee, Kyoung; Friemann, Rosmarie; Parales, Juan V.; Gibson, David T.; Ramaswamy, S.

    2005-07-01

    All three components of the toluene dioxygenase system have been expressed, purified and crystallized. Pseudomonas putida F1 can grow with toluene as its sole source of carbon and energy. The initial reaction of the degradation of toluene is catalyzed by a three-component toluene dioxygenase enzyme system consisting of a reductase (Reductase{sub TOL}), a ferredoxin (Ferredoxin{sub TOL}) and a Rieske non-heme iron dioxygenase (Oxygenase{sub TOL}). The three components and the apoenzyme of the dioxygenase (apo-Oxygenase{sub TOL}) were overexpressed, purified and crystallized. Reductase{sub TOL} diffracts to 1.8 Å and belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 77.1, c = 156.3 Å. Ferredoxin{sub TOL} diffracts to 1.2 Å and belongs to space group P2{sub 1}, with unit-cell parameters a = 30.5, b = 52.0, c = 30.95 Å, β = 113.7°. Apo-Oxygenase{sub TOL} and Oxygenase{sub TOL} diffract to 3.2 Å and belong to space group P4{sub 3}32, with unit-cell parameters a = 235.9 Å and a = 234.5 Å, respectively.

  18. Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome

    PubMed Central

    Campos, Bruna Medeia; Alvarez, Thabata Maria; Liberato, Marcelo Vizona; Polikarpov, Igor; Gilbert, Harry J.; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

    2014-01-01

    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

  19. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AHP2, a signal transmitter protein from Arabidopsis thaliana.

    PubMed

    Degtjarik, Oksana; Dopitova, Radka; Puehringer, Sandra; Nejedla, Eliska; Kuty, Michal; Weiss, Manfred S; Hejatko, Jan; Janda, Lubomir; Kuta Smatanova, Ivana

    2013-02-01

    Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1-5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution. PMID:23385758

  20. Purification, crystallization and preliminary X-ray analysis of the periplasmic haem-binding protein HutB from Vibrio cholerae.

    PubMed

    Agarwal, Shubhangi; Biswas, Maitree; Dasgupta, Jhimli

    2015-04-01

    The mechanism of haem transport across the inner membrane of pathogenic bacteria is currently insufficiently understood at the molecular level and no information is available for this process in Vibrio cholerae. To obtain structural insights into the periplasmic haem-binding protein HutB from V. cholerae (VcHutB), which is involved in haem transport through the HutBCD haem-transport system, at the atomic level, VcHutB was cloned, overexpressed and crystallized using 1.6 M ammonium sulfate as a precipitant at pH 7.0. X-ray diffraction data were collected to 2.4 Å resolution on the RRCAT PX-BL-21 beamline at the Indus-2 synchrotron, Indore, India. The crystals belonged to space group P4₃2₁2, with unit-cell parameters a = b = 62.88, c = 135.8 Å. Matthews coefficient calculations indicated the presence of one monomer in the asymmetric unit, with an approximate solvent content of 45.02%. Molecular-replacement calculations with Phaser confirmed the presence of a monomer in the asymmetric unit. PMID:25849499

  1. Purification, crystallization and preliminary X-ray diffraction analysis of the IL-20–IL-20R1–IL-20R2 complex

    PubMed Central

    Logsdon, Naomi J.; Allen, Christopher E.; Rajashankar, Kanagalaghatta R.; Walter, Mark R.

    2012-01-01

    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 P41212 or P43212, with unit-cell parameters a = 111, c = 135 Å, and diffracted X-­rays to 3 Å resolution. The crystallographic asymmetric unit contains one IL-­20–IL-20R1–IL-20R2 complex, corresponding to a solvent content of approximately 54%. PMID:22232181

  2. Purification, crystallization and preliminary X-ray diffraction analysis of RafE, a sugar-binding lipoprotein from Streptococcus pneumoniae

    PubMed Central

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

    2006-01-01

    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 P6122. Diffraction data have been successfully phased to 2.90 Å using Se SAD data and model building is in progress. PMID:16820692

  3. 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

  4. 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.

  5. Purification, crystallization and preliminary X-ray diffraction studies of the three components of the toluene 2,3-dioxygenase enzyme system

    SciTech Connect

    Lee, Kyoung; Friemann, Rosmarie; Parales, Juan V.; Gibsona, David T.; Ramaswamy, S.

    2010-07-19

    Pseudomonas putida F1 can grow with toluene as its sole source of carbon and energy. The initial reaction of the degradation of toluene is catalyzed by a three-component toluene dioxygenase enzyme system consisting of a reductase (Reductase{sub TOL}), a ferredoxin (Ferredoxin{sub TOL}) and a Rieske non-heme iron dioxygenase (Oxygenase{sub TOL}). The three components and the apoenzyme of the dioxygenase (apo-Oxygenase{sub TOL}) were overexpressed, purified and crystallized. Reductase{sub TOL} diffracts to 1.8 {angstrom} and belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 77.1, c = 156.3 {angstrom}. Ferredoxin{sub TOL} diffracts to 1.2 {angstrom} and belongs to space group P2{sub 1}, with unit-cell parameters a = 30.5, b = 52.0, c = 30.95 {angstrom}, {beta} = 113.7{sup o}. Apo-Oxygenase{sub TOL} and Oxygenase{sub TOL} diffract to 3.2 {angstrom} and belong to space group P4{sub 3}32, with unit-cell parameters a = 235.9 {angstrom} and a = 234.5 {angstrom}, respectively.

  6. 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

  7. Initial Data of Digital Correlation ECE with a Giga Hertz Sampling Digitizer

    NASA Astrophysics Data System (ADS)

    Tsuchiya, Hayato; Inagaki, Shigeru; Tokuzawa, Tokihiko; Nagayama, Yoshio

    2015-03-01

    The proposed Digital Correlation ECE (DCECE) technique is applied in Large Helical Device. DCECE is realized by the use of the Giga Hertz Sampling Digitizer. The waveform of intermediate frequency band of ECE, whose frequency is several giga hertz, can be discretized and saved directly. The discretized IF data can be used for the analysis of correlation ECE with arbitrary parameter of spatial resolution and temporal resolution. In this paper, the characteristic of DCECE and initial Data in LHD is introduced.

  8. Purification, N-terminal sequencing, crystallization and preliminary structural determination of atratoxin-b, a short-chain alpha-neurotoxin from Naja atra venom.

    PubMed

    Lou, Xiaohua; Tu, Xiongying; Pan, Guoqiang; Xu, Chaoyin; Fan, Rong; Lu, Wanhua; Deng, Wenhan; Rao, Pingfan; Teng, Maikun; Niu, Liwen

    2003-06-01

    Atratoxin-b, a short-chain alpha-neurotoxin purified from Naja atra (mainland Chinese cobra) venom using a three-step chromatography procedure, has an apparent molecular mass of 6950 Da with an alkaline pI value (>9.5) and consists of one single polypeptide chain as estimated by MALDI-TOF mass spectrometry and SDS-PAGE. The protein is toxic to mice, with an in vitro LD(50) of about 0.18 mg kg(-1). Its N-terminal amino-acid sequence, LECHNQQSSQTPTIT, displays a very high homology to those of other alpha-neurotoxins. The overall three-dimensional structure of atratoxin-b is very similar to that of the homologous erabutoxin-a, as shown by the crystallographic molecular replacement and preliminary refinement results, with an R factor and R(free) of 27 and 29%, respectively. The microcrystal slowly grew to dimensions of approximate 0.1 x 0.1 x 0.15 mm over eight months using hanging-drop vapour-diffusion method. It gave a set of diffraction data to 1.56 A resolution using X-rays of wavelength 1.1516 A generated by the X-ray Diffraction and Scattering Station of beamline U7B at the National Synchrotron Radiation Laboratory (Hefei, China); this is the first example of the use of this beamline in protein crystallography. The crystals belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 49.28, c = 44.80 A, corresponding to one molecule per asymmetric unit and a volume-to-mass ratio of 1.96 A(3) Da(-1). PMID:12777767

  9. Habitat suitability and conservation of the Giant Gartersnake (Thamnophis gigas) in the Sacramento Valley of California

    USGS Publications Warehouse

    Halstead, B.J.; Wylie, G.D.; Casazza, M.L.

    2010-01-01

    Resource managers often have little information regarding the habitat requirements and distribution of rare species. Factor analysis-based habitat suitability models describe the ecological niche of a species and identify locations where these conditions occur on the landscape using existing occurrence data. We used factor analyses to assess the suitability of habitats for Thamnophis gigas (Giant Gartersnake), a rare, threatened species endemic to the Central Valley of California, USA, and to map the locations of habitat suitable for T. gigas in the Sacramento Valley. Factor analyses indicated that the niche of T. gigas is composed of sites near rice agriculture with low stream densities. Sites with high canal densities and near wetlands also appeared suitable, but results for these variables were sensitive to potential sampling bias. In the Sacramento Valley, suitable habitats occur primarily in the central portion of the valley floor. Based upon the results of the factor analyses, recovery planning for T. gigas will require an on-the-ground assessment of the current distribution and abundance of T. gigas, maintaining the few remaining natural wetlands and the practice of rice agriculture in the Sacramento Valley, and studying the effects of agricultural practices and land use changes on populations of T. gigas. ?? 2010 by the American Society of Ichthyologists and Herpetologists.

  10. Habitat suitability and conservation of the giant gartersnake (Thamnophis gigas) in the Sacramento Valley of California

    USGS Publications Warehouse

    Halstead, Brian J.; Wylie, Glenn D.; Casazza, Michael L.

    2010-01-01

    Resource managers often have little information regarding the habitat requirements and distribution of rare species. Factor analysis-based habitat suitability models describe the ecological niche of a species and identify locations where these conditions occur on the landscape using existing occurrence data.We used factor analyses to assess the suitability of habitats for Thamnophis gigas (Giant Gartersnake), a rare, threatened species endemic to the Central Valley of California, USA, and to map the locations of habitat suitable for T. gigas in the Sacramento Valley. Factor analyses indicated that the niche of T. gigas is composed of sites near rice agriculture with low stream densities. Sites with high canal densities and near wetlands also appeared suitable, but results for these variables were sensitive to potential sampling bias. In the Sacramento Valley, suitable habitats occur primarily in the central portion of the valley floor. Based upon the results of the factor analyses, recovery planning for T. gigas will require an on-the-ground assessment of the current distribution and abundance of T. gigas, maintaining the few remaining natural wetlands and the practice of rice agriculture in the Sacramento Valley, and studying the effects of agricultural practices and land use changes on populations of T. gigas.

  11. 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

  12. Perkinsus marinus in Crassostrea gigas in the Gulf of California.

    PubMed

    Enríquez-Espinoza, T L; Grijalva-Chon, J M; Castro-Longoria, R; Ramos-Paredes, J

    2010-04-01

    To determine the agent responsible for the massive mortalities of the Pacific oyster Crassostrea gigas in northwest Mexico, 30 oysters were sampled after a severe mortality event in 2006 along the Sonoran coast. Histological analyses revealed the presence of a protozoan and Ray's fluid thioglycollate medium (RFTM) assays showed the presence of Perkinsus sp., identified as P. marinus from the DNA sequence of the internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) gene complex. PCR analyses for Marteilia refringens, M. sydneyi, and Haplosporidium costale were negative. P. marinus presence in the Pacific oyster may be responsible for massive mortalities of the oyster, along with other environmental factors in the Gulf of California. PMID:20481094

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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).

  3. Ribonucleic acid purification.

    PubMed

    Martins, R; Queiroz, J A; Sousa, F

    2014-08-15

    Research on RNA has led to many important biological discoveries and improvement of therapeutic technologies. From basic to applied research, many procedures employ pure and intact RNA molecules; however their isolation and purification are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality. The current techniques to isolate and purify RNA molecules still have several limitations and the requirement for new methods able to improve RNA quality to meet regulatory demands is growing. In fact, as basic research improves the understanding of biological roles of RNAs, the biopharmaceutical industry starts to focus on them as a biotherapeutic tools. Chromatographic bioseparation is a high selective unit operation and is the major option in the purification of biological compounds, requiring high purity degree. In addition, its application in biopharmaceutical manufacturing is well established. This paper discusses the importance and the progress of RNA isolation and purification, considering RNA applicability both in research and clinical fields. In particular and in view of the high specificity, affinity chromatography has been recently applied to RNA purification processes. Accordingly, recent chromatographic investigations based on biorecognition phenomena occurring between RNA and amino acids are focused. Histidine and arginine have been used as amino acid ligands, and their ability to isolate different RNA species demonstrated a multipurpose applicability in molecular biology analysis and RNA therapeutics preparation, highlighting the potential contribution of these methods to overcome the challenges of RNA purification. PMID:24951289

  4. 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. 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

  6. 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

  7. [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

  8. 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.

  9. Mechanical properties and the laminate structure of Arapaima gigas scales.

    PubMed

    Lin, Y S; Wei, C T; Olevsky, E A; Meyers, Marc A

    2011-10-01

    The Arapaima gigas scales play an important role in protecting this large Amazon basin fish against predators such as the piranha. They have a laminate composite structure composed of an external mineralized layer and internal lamellae with thickness of 50-60 μm each and composed of collagen fibers with ~1 μm diameter. The alignment of collagen fibers is consistent in each individual layer but varies from layer to layer, forming a non-orthogonal plywood structure, known as Bouligand stacking. X-ray diffraction revealed that the external surface of the scale contains calcium-deficient hydroxyapatite. EDS results confirm that the percentage of calcium is higher in the external layer. The micro-indentation hardness of the external layer (550 MPa) is considerably higher than that of the internal layer (200 MPa), consistent with its higher degree of mineralization. Tensile testing of the scales carried out in the dry and wet conditions shows that the strength and stiffness are hydration dependent. As is the case of most biological materials, the elastic modulus of the scale is strain-rate dependent. The strain-rate dependence of the elastic modulus, as expressed by the Ramberg-Osgood equation, is equal to 0.26, approximately ten times higher than that of bone. This is attributed to the higher fraction of collagen in the scales and to the high degree of hydration (30% H(2)O). Deproteinization of the scale reveals the structure of the mineral component consisting of an interconnected network of platelets with a thickness of ~50 nm and diameter of ~500 nm. PMID:21783123

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  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. Giga-LES of Hector the Convector keeping the tallest updrafts undiluted

    NASA Astrophysics Data System (ADS)

    Dauhut, Thibaut; Chaboureau, Jean-Pierre; Escobar, Juan; Mascart, Patrick

    2015-04-01

    The trend of stratospheric water vapour during the past decades is not correctly reproduced by current GCMs. This may be due to lack of representation of rapid water transfers from troposphere to stratosphere. Our modeling study focused on a particular case of tropical very deep convection which takes an active part in this transport. We aimed at understanding its dynamics and the stratosphere moistening processes. We selected a Hector thunderstorm observed on 30 November 2005 over Tiwi Islands, Australia, during the SCOUT-O3 field campaign. Plumes of ice particles reaching 19 km altitude were measured by lidar aboard the Geophysica stratospheric aircraft. We performed a Giga Large-Eddy Simulation of Hector (100 m horizontal resolution, more than 1 billion grid points) using cutting-edge computing resources, as well as a series of simulations with coarser and coarser horizontal resolutions, from 200 m to 1600 m. A strong morning sea breeze deviated boundary layer westerlies and led to intense convergence of humid air over Tiwi Islands. Deep convection triggered around 12:15 pm and quickly reached 14 km altitude. The associated cold pools organised and generated upward motions at the surface. The most intense upward transport started 1 hour later and lasted around 2 hours. As a result, a couple of updrafts overshot the tropopause carrying ice crystals in the stratosphere. Part of the ice particles precipitated then whereas the remainder sublimated in the lower stratosphere. The consequent vapour pockets were transported and diluted within the stratosphere by easterlies. While moistening appeared to be robust with respect to the grid spacing used, grid spacing on the order of 100 m may be necessary for a reliable estimate of hydration (Dauhut et al. ASL 2014, doi: 10.1002/asl2.534). A comprehensive analysis of individual updrafts and their properties once sorted by their height has been carried out. The couple of updrafts that reach the stratosphere presents a higher

  18. 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

  19. 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

  20. 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